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PLANT ENDOSOMES : methods and protocols. [2 ed.]
 9781071607664, 1071607669

Table of contents :
Preface
Contents
Contributors
Chapter 1: Analysis of Endocytosis and Intracellular Trafficking of Boric Acid/Borate Transport Proteins in Arabidopsis
1 Introduction
2 Materials
2.1 Modified MGRL Medium
2.2 Plant Materials
2.3 Chemicals
2.4 Confocal Microscopy
2.5 Root Sectioning
3 Methods
3.1 Preparation of MGRL Media
3.1.1 MGRL Solid Medium
3.1.2 MGRL Liquid/Soft-Gel Medium for Short-Term Treatments with B or Chemicals
3.1.3 Induction Medium with β-Estradiol
3.2 Germination and Growth of Arabidopsis thaliana Seedlings
3.3 Inhibition of Endocytosis
3.4 Imaging of B Transporters in Longitudinal Optical Sections
3.4.1 Image Analysis: Quantification of Polarity Index from Longitudinal Optical Sections
3.5 Imaging of B Transporters in Cross Sections
3.6 Time-Lapse Imaging and Analysis of B-Induced Vacuolar Sorting of BOR1-GFP
3.7 Analysis of Constitutive Endocytosis of BOR1-GFP Using BFA
4 Notes
References
Chapter 2: Analysis of Membrane Proteins Transport from Endosomal Compartments to Vacuoles
1 Introduction
2 Materials
2.1 Plant Materials and Growth Conditions
2.2 Buffers and Solutions
2.3 Equipment and Other Materials
3 Methods
3.1 Seed Sterilization and Germination
3.2 Drug Treatment and Confocal Imaging
3.2.1 FM 4-64 Dye Treatment
3.2.2 DEX Treatment on DEX-Inducible RNAi Arabidopsis Seedlings
3.3 Dark Treatment
3.4 Confocal Imaging
4 Notes
References
Chapter 3: Analysis of Endoplasmic Reticulum-Endosome Association Using Live-Cell Imaging in Plant Cells
1 Introduction
2 Materials
2.1 Plant Plasmids
2.2 Agrobacterium Culture and Media
2.3 Plant Material
2.4 Seeds Sterilization
2.5 Plant Growth
2.6 Microscopy Imaging
2.7 Software for Imaging Analysis
2.8 Other Materials Needed
3 Methods
3.1 Plant Transformation
3.1.1 Transient Transformation in Tobacco
3.1.2 Transient and Stable Transformation of Arabidopsis
3.2 Imaging Leaf Material
3.3 FRET (Förster Resonance Energy Transfer) Imaging for ER-Endosome Interactions
3.4 ER-Endosome Colocalization
4 Notes
References
Chapter 4: Degradation of Abscisic Acid Receptors Through the Endosomal Pathway
1 Introduction
2 Materials
2.1 Analysis of CME of ABA Receptors by co-IP Assays
2.1.1 In Vitro Culture Media and Plant Treatments
2.1.2 Protein Extraction
2.1.3 Protein Complex co-IP and Analysis by Western Blot (WB)
2.2 Analysis of the Delivery of PYL4-RSL1 Complexes into the Lumen of the Vacuole
2.3 Stock Solutions
3 Methods
3.1 Analysis of CME of ABA Receptors by co-IP Assays
3.1.1 Preparation of the Biological Material
3.1.2 Total Protein Extraction and Quantification
3.1.3 Performing the co-IPs
3.1.4 Analysis of the Inputs and co-IPs by WB
3.2 Analysis of the Delivery of PYL4-RSL1 Complexes into the Lumen of the Vacuole by BiFC
3.2.1 Agroinfiltration of N. benthamiana Leaves
3.2.2 Colocalization Analysis
4 Notes
References
Chapter 5: Biochemical and Imaging Analysis of ALIX Function in Endosomal Trafficking of Arabidopsis Protein Cargoes
1 Introduction
2 Materials
2.1 BiFC Assay
2.2 Confocal Imaging
2.3 Microsomal Fractionation
3 Methods
3.1 In Vivo Analysis of Physical Interactions by BiFC.
3.2 Confocal Imaging of ALIX Function in Cargo Trafficking
3.3 Microsomal Fractionation to Analyze the Function of ALIX in Cargo Trafficking.
4 Notes
References
Chapter 6: Correlative Light and Electron Microscopy Imaging of the Plant trans-Golgi Network
1 Introduction
2 Materials
2.1 Plant Material and Growth
2.2 Reagents and Antibodies
2.3 Equipment
2.4 Software
3 Methods
3.1 Seedling Growth
3.2 Selection and Processing of Seedlings by High-Pressure Freezing and Freeze Substitution
3.3 Sample Mounting, Sectioning, and Immunofluorescence Labeling
3.4 Fluorescence Microscopy Imaging
3.5 Recovery, Washing, and Poststaining
3.6 TEM Analysis and Cell Wall-Based Correlation
4 Notes
References
Chapter 7: Imaging Plant Cells by High-Pressure Freezing and Serial block-face scanning electron microscopy
1 Introduction
2 Materials
2.1 Plant material
2.2 High-Pressure Freezing and Freeze Substitution, and Resin Embedding
2.3 Serial Block-Face Imaging
2.4 Software
3 Methods
3.1 High-Pressure Freezing and Sample Preparation
3.2 Serial Block-Face Imaging
3.3 Data Segmentation, Visualization, and Analyses
3.3.1 The Analyses Routine Through TrakEM2
3.3.2 The Analyses Routine Through KNOSSOS
4 Notes
References
Chapter 8: Functional Analysis of Plant FYVE Domain Proteins in Endosomal Trafficking
1 Introduction
2 Materials
2.1 Purification of Epitope-Tagged FYVE-Domain Proteins from Escherichia coli
2.2 Lipid Binding Assay
2.3 Arabidopsis Transformation and Crossing
2.4 Wortmannin Treatment and Confocal Microscopy
2.5 Protein Extraction and Western Blotting Analysis
3 Methods
3.1 In Vitro Lipid Binding Assay Using Purified FYVE domain Proteins from E. coli
3.2 Arabidopsis Transformation and Crossing
3.3 Wortmannin Treatment and Confocal Imaging
3.4 Analysis of Seed Storage Proteins and Ubiquitinated by Western Blotting
3.4.1 Analysis of 12S Globulin Processing
3.4.2 Abundance of Ubiquitinated Proteins in Cell Soluble (CS) and Cell Membrane (CM) Fractions
4 Notes
References
Chapter 9: Assessing Extrinsic Membrane Protein Dependency to PI4P Using a Plasma Membrane to Endosome Relocalization Transien...
1 Introduction
2 Materials
2.1 Plants and Agrobacteria
2.2 Cell Culture
2.3 Supplies and Equipment
2.4 Plasmids
3 Methods
3.1 Growth of Nicotiana benthamiana
3.2 Transient Transformation of Nicotiana benthamiana
3.3 Imaging Using Confocal Microscopy
3.4 Quantification
4 Notes
References
Chapter 10: Subcellular Localization of PI3P in Arabidopsis
1 Introduction
2 Materials
2.1 Plant Media and Growth
2.2 Crossing Arabidopsis Mutants
2.3 Inhibitor Treatment and Confocal Microscopy
2.4 Image Analysis Software
3 Methods
3.1 Preparation of Arabidopsis Plants for Crossing
3.2 Crossing a Mutant with Citrine-2 x FYVE Line
3.3 Selection of F1 and F2 Plants Expressing Citrine-2 x FYVE Reporter
3.4 Inhibitor Treatment and Confocal Microscopy
4 Notes
References
Chapter 11: Immunopurification of Intact Endosomal Compartments for Lipid Analyses in Arabidopsis
1 Introduction
2 Materials
2.1 Plant Material and Growth
2.2 Membrane Isolation and Protein Quantification
2.3 Immunoprecipitation
2.4 SDS-PAGE and Western Blotting
2.5 Fatty Acid Profiling (Fatty Acid Methyl Esters: FAMEs)
2.6 Sterol Profiling
2.7 Lipid Standards
2.8 Consumables
2.9 Equipment
2.10 Software
3 Methods
3.1 Growing Arabidopsis Seedlings in Liquid Culture
3.2 Membrane Extraction and Fractionation
3.3 Protein Quantification in the Total Membrane Fraction
3.4 Immunoprecipitation
3.5 Controls for IPs
3.6 Enrichment and Purity Assessment of the IP Fraction
3.7 Lipid Extraction
3.7.1 Total Fatty Acid Extraction (Fatty Acid Methyl Esters: FAMEs)
3.7.2 Sterol Extraction
3.8 GC-MS Analysis
3.8.1 GC-MS Method
3.8.2 Identification of Peaks
3.8.3 Calculation
4 Notes
References
Chapter 12: Cell-Free Protein Translation System for Expression of Lipid-Binding Proteins Tagged with Small epitopes and Their...
1 Introduction
2 Materials
2.1 In Vitro Protein Synthesis
2.2 Dot Blot and Lipid Overlay Assay
2.3 Extraction of Phosphoinositides from Plant Tissues
2.4 Homemade Lipid Nitrocellulose Strips
3 Method
3.1 In Vitro Protein Synthesis
3.2 Protein Detection by Dot Blot
3.3 Analysis of Lipid Binding Capacity of Target Protein(s) Using PLOA
3.4 Extraction of Acidic Lipids
3.5 Blotting Lipids on Nitrocellulose Membranes
4 Notes
References
Chapter 13: Isolation and Glycomic Analysis of Trans-Golgi Network Vesicles in Plants
1 Introduction
2 Materials
2.1 Plant Material
2.2 Vesicle Fractionation Components
2.3 Vesicle Immunoisolation Components
2.4 Material for Enzyme-Linked Immunosorbent Assay
3 Methods
3.1 Plant Preparation
3.2 Golgi/TGN Fractionation by Sucrose Density Gradient Ultracentrifugation
3.3 Immunoisolation of SYP61 Vesicles
3.4 Proceed to the Following Steps if You Are Using Beads Covalently Bound to the Antibody
3.5 Glycome Analysis
4 Notes
References
Chapter 14: Detection of Phosphorylation on Immunoprecipitates from Total Protein Extracts of Arabidopsis thaliana Seedlings
1 Introduction
2 Materials
2.1 Plant-Related Material
2.2 Immunoprecipitation (IP)
2.3 Phosphatase Treatment
2.4 SDS-Polyacrylamide Gel Electrophoresis
2.5 SDS-Polyacrylamide Gel Electrophoresis with Mn2+-Phos-tagTM-Gels
2.6 Total Protein Staining with SYPRO Ruby Protein Gel Stain
2.7 Phosphostaining with Pro-Q Diamond Phosphoprotein Gel Stain
3 Methods
3.1 Plant-Related Material
3.2 Immunoprecipitation (IP)
3.3 Phosphatase Treatment
3.4 SDS-Polyacrylamide Gel Electrophoresis
3.5 SDS-Polyacrylamide Gel Electrophoresis with Mn2+-Phos-tagTM-Gels
3.6 Total Protein Staining with SYPRO Ruby Protein Gel Stain
3.7 Phosphostaining with Pro-Q Diamond Phosphoprotein Gel Stain
4 Notes
References
Chapter 15: Purification and Interaction Analysis of a Plant-Specific RAB5 Effector by In Vitro Pull-Down Assay
1 Introduction
2 Materials
2.1 PUF2 Cloning and Expression in Bacteria
2.2 Protein Purification
2.3 PUF2 and ARA6 Interaction Assay
2.4 Western Blotting Analysis to Detect Interaction Between PUF2 and ARA6
3 Methods
3.1 Plasmid Preparation
3.2 Protein Purification of PUF2
3.3 Purification of GST and GST-Tagged ARA6
3.4 In Vitro Pull-Down Assay
3.5 Western Blotting Analysis of Coprecipitated PUF2 Proteins
4 Notes
References
Index

Citation preview

Methods in Molecular Biology 2177

Marisa S. Otegui Editor

Plant Endosomes Methods and Protocols Second Edition

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

Plant Endosomes Methods and Protocols Second Edition

Edited by

Marisa S. Otegui Department of Botany and Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, WI, USA

Editor Marisa S. Otegui Department of Botany and Laboratory of Cell and Molecular Biology University of Wisconsin-Madison Madison, WI, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-0766-4 ISBN 978-1-0716-0767-1 (eBook) https://doi.org/10.1007/978-1-0716-0767-1 © Springer Science+Business Media, LLC, part of Springer Nature 2020 This work is subject to copyright. All rights are reserved by the Publisher, or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface Endosomes are critical sorting organelles in both the secretory and the endocytic pathways. Two main types of structurally and functionally different endosomes have been described in plants, the Trans Golgi Network (TGN) that acts as early endosome and mediates most of the recycling of endocytosed cargo back to the plasma membrane and the multivesicular endosomes, which sort endocytosed proteins into intraluminal vesicles for degradation at the vacuole. Newly synthesized secretory cargo is transported from the endoplasmic reticulum to the Golgi and the TGN for either their direct delivery to the plasma membrane or to the vacuole through multivesicular endosomes. Thus, endosomes are critical sorting stations of two important vesicle trafficking pathways. Understanding how endosomes and their associated endosomal protein complexes decode the sorting signatures on cargo and mediate membrane deformation and vesiculation is under active investigation. This second edition of Plant Endosomes: Methods and Protocols gathers a collection of techniques to image trafficking of cargo through endosomes, to study structural aspects of plant endosomes by electron microscopy, and combined biochemical, omics, and imaging approaches to study the dynamics and contents of endosomal compartments. Finally, this edition also contains a group of chapters dedicated to the analysis of lipids on endosomes and the identification and analysis of lipid-binding proteins and lipid-binding domains relevant for the study of plant endosomes. I would like to extend my most sincere gratitude to all authors and colleagues for having contributed detailed and useful protocols and great ideas to this book and to the National Science Foundation for supporting research on endosomal trafficking in my laboratory over the years. Madison, WI, USA

Marisa S. Otegui

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Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1 Analysis of Endocytosis and Intracellular Trafficking of Boric Acid/Borate Transport Proteins in Arabidopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Akira Yoshinari and Junpei Takano 2 Analysis of Membrane Proteins Transport from Endosomal Compartments to Vacuoles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Mengqian Luo, Ying Zhu, Zhiqi Liu, and Liwen Jiang 3 Analysis of Endoplasmic Reticulum–Endosome Association Using Live-Cell Imaging in Plant Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Giovanni Stefano and Federica Brandizzi 4 Degradation of Abscisic Acid Receptors Through the Endosomal Pathway. . . . . 35 Borja Belda-Palazo n and Pedro L. Rodriguez 5 Biochemical and Imaging Analysis of ALIX Function in Endosomal Trafficking of Arabidopsis Protein Cargoes . . . . . . . . . . . . . . . . . . . . 49 Marta Garcı´a-Leo n and Vicente Rubio 6 Correlative Light and Electron Microscopy Imaging of the Plant trans-Golgi Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Pengfei Wang and Byung-Ho Kang 7 Imaging Plant Cells by High-Pressure Freezing and Serial Block-Face Scanning Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Kirk Czymmek, Abhilash Sawant, Kaija Goodman, Janice Pennington, Pal Pedersen, Mrinalini Hoon, and Marisa S. Otegui 8 Functional Analysis of Plant FYVE Domain Proteins in Endosomal Trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Wenjin Shen, Juan Wei, and Caiji Gao 9 Assessing Extrinsic Membrane Protein Dependency to PI4P Using a Plasma Membrane to Endosome Relocalization Transient Assay in Nicotiana benthamiana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Mehdi Doumane and Marie-Ce´cile Caillaud 10 Subcellular Localization of PI3P in Arabidopsis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Han Nim Lee, Hyera Jung, and Taijoon Chung 11 Immunopurification of Intact Endosomal Compartments for Lipid Analyses in Arabidopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Yoko Ito, Magali Grison, Nicolas Esnay, Laetitia Fouillen, and Yohann Boutte´ 12 Cell-Free Protein Translation System for Expression of Lipid-Binding Proteins Tagged with Small Epitopes and Their Use in Protein–Lipid Overlay Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Julio Paez-Valencia and Marisa S. Otegui

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Contents

Isolation and Glycomic Analysis of Trans-Golgi Network Vesicles in Plants . . . . 153 Guangxi Ren, Michel Ruiz Rosquete, Angelo G. Peralta, Sivakumar Pattathil, Michael G. Hahn, Thomas Wilkop, and Georgia Drakakaki Detection of Phosphorylation on Immunoprecipitates from Total Protein Extracts of Arabidopsis thaliana Seedlings . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Karin Vogel and Erika Isono Purification and Interaction Analysis of a Plant-Specific RAB5 Effector by In Vitro Pull-Down Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Emi Ito, Seung-won Choi, and Takashi Ueda

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors BORJA BELDA-PALAZO´N • Instituto de Biologı´a Molecular y Celular de Plantas, Consejo Superior de Investigaciones Cientı´ficas-Universidad Polite´cnica de Valencia, Valencia, Spain YOHANN BOUTTE´ • Laboratoire de Biogene`se Membranaire, UMR 5200, CNRS, Universite´ de Bordeaux, Villenave d’Ornon, France FEDERICA BRANDIZZI • MSU-DOE Plant Research Lab, Plant Biology Department, Michigan State University, East Lansing, MI, USA MARIE-CE´CILE CAILLAUD • Laboratoire Reproduction et De´veloppement des Plantes, Universite´ de Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRA, Lyon, France SEUNG-WON CHOI • Department of Natural Sciences, International Christian University, Mitaka, Tokyo, Japan TAIJOON CHUNG • Department of Biological Sciences, Pusan National University, Busan, Republic of Korea KIRK CZYMMEK • Advanced Bioimaging Laboratory, Donald Danforth Plant Science Center, St. Louis, MO, USA MEHDI DOUMANE • Laboratoire Reproduction et De´veloppement des Plantes, Universite´ de Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRA, Lyon, France GEORGIA DRAKAKAKI • Department of Plant Sciences, University of California, Davis, CA, USA NICOLAS ESNAY • Laboratoire de Biogene`se Membranaire, UMR 5200, CNRS, Universite´ de Bordeaux, Villenave d’Ornon, France; Department of Biological Sciences, BioDiscovery Institute, University of North Texas, Denton, TX, USA LAETITIA FOUILLEN • Laboratoire de Biogene`se Membranaire, UMR 5200, CNRS, Universite´ de Bordeaux, Villenave d’Ornon, France; MetaboHub-Metabolome Facility of Bordeaux, Functional Genomics Center, Bordeaux, France CAIJI GAO • Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, China MARTA GARCI´A-LEO´N • Centro Nacional de Biotecnologı´a (CNB-CSIC), Madrid, Spain KAIJA GOODMAN • Department of Botany, University of Wisconsin-Madison, Madison, WI, USA; Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, WI, USA MAGALI GRISON • Laboratoire de Biogene`se Membranaire, UMR 5200, CNRS, Universite´ de Bordeaux, Villenave d’Ornon, France MICHAEL G. HAHN • Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA MRINALINI HOON • Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, USA; McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI, USA ERIKA ISONO • Department of Biology, University of Konstanz, Konstanz, Germany EMI ITO • Department of Natural Sciences, International Christian University, Tokyo, Japan; Institute for Global Leadership, Ochanomizu University, Tokyo, Japan YOKO ITO • Laboratoire de Biogene`se Membranaire, UMR 5200, CNRS, Universite´ de Bordeaux, Villenave d’Ornon, France

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Contributors

LIWEN JIANG • Centre for Cell & Developmental Biology, State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China HYERA JUNG • Department of Biological Sciences, Pusan National University, Busan, Republic of Korea BYUNG-HO KANG • Center for Cell and Developmental Biology, State Key Laboratory for Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China HAN NIM LEE • Laboratory of Cell and Molecular Biology and Department of Botany, University of Wisconsin-Madison, Madison, WI, USA ZHIQI LIU • Centre for Cell & Developmental Biology, State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China MENGQIAN LUO • Centre for Cell & Developmental Biology, State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China MARISA S. OTEGUI • Department of Botany, University of Wisconsin-Madison, Madison, WI, USA; Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, WI, USA JULIO PAEZ-VALENCIA • Laboratory of Cell and Molecular Biology, University of WisconsinMadison, Madison, WI, USA SIVAKUMAR PATTATHIL • Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA; Mascoma LLC (Lallemand Inc.), Lebanon, NH, USA PAL PEDERSEN • Carl Zeiss Microscopy, LLC, White Plains, NY, USA JANICE PENNINGTON • Laboratory of Cell and Molecular Biology, University of WisconsinMadison, Madison, WI, USA ANGELO G. PERALTA • Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA GUANGXI REN • Department of Plant Sciences, University of California, Davis, CA, USA; School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China PEDRO L. RODRIGUEZ • Instituto de Biologı´a Molecular y Celular de Plantas, Consejo Superior de Investigaciones Cientı´ficas-Universidad Polite´cnica de Valencia, Valencia, Spain MICHEL RUIZ ROSQUETE • Department of Plant Sciences, University of California, Davis, CA, USA; Plant Biology Lab., Salk Institute for Biological Studies, La Jolla, CA, USA VICENTE RUBIO • Centro Nacional de Biotecnologı´a (CNB-CSIC), Madrid, Spain ABHILASH SAWANT • Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, USA; Department of Neuroscience, University of Wisconsin-Madison, Madison, WI, USA WENJIN SHEN • Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, China GIOVANNI STEFANO • MSU-DOE Plant Research Lab, Plant Biology Department, Michigan State University, East Lansing, MI, USA JUNPEI TAKANO • Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka, Japan TAKASHI UEDA • Division of Cellular Dynamics, National Institute for Basic Biology, Okazaki, Aichi, Japan; Department of Basic Biology, SOKENDAI, Okazaki, Aichi, Japan KARIN VOGEL • Department of Biology, University of Konstanz, Konstanz, Germany

Contributors

xi

PENGFEI WANG • Center for Cell and Developmental Biology, State Key Laboratory for Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China JUAN WEI • Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, China THOMAS WILKOP • Light Microscopy Core, University of Kentucky, Lexington, KY, USA AKIRA YOSHINARI • Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka, Japan; Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan YING ZHU • Centre for Cell & Developmental Biology, State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

Chapter 1 Analysis of Endocytosis and Intracellular Trafficking of Boric Acid/Borate Transport Proteins in Arabidopsis Akira Yoshinari and Junpei Takano Abstract Plants take up inorganic nutrients from the soil by transport proteins located in the plasma membrane of root cells. Boron (B) is an essential element for plant growth; it taken up and translocated by boric acid channels such as NIP5;1 and borate exporters such as BOR1 in Arabidopsis. NIP5;1 and BOR1 are localized to the plasma membrane of various root cells in polar manners toward soil- and stele-side, respectively, for efficient transport of B. In response to elevated B concentration, BOR1 undergoes vacuolar sorting for degradation to avoid accumulation of B to a toxic level in tissues. The polar localization and vacuolar sorting of the transport proteins are regulated through differential mechanisms of endocytosis and intracellular trafficking. In this chapter, we describe methods for quantitative live-cell imaging of GFP-NIP5;1 and BOR1-GFP as markers for the polar and vacuolar trafficking. Key words BOR1, NIP5;1, Polar localization, Endocytosis, Vacuolar sorting

1

Introduction Membrane traffic defines subcellular localization and abundance of various membrane proteins such as transporters and transmembrane receptors in eukaryotic cells. In plant cells, several transport proteins have been shown to be localized to specific domains of the plasma membrane to define the direction of transport [1, 2]. In addition, several transporters have been shown to be transported from the plasma membrane to vacuoles in response to environmental cues to maintain homeostasis of nutrients [2–4]. Boron (B) is one of the essential micronutrients for plant and B deficiency inhibits plant development and growth [5]. B as boric acid/borate cross-links two pectin monomers at rhamno galacturonan-II regions [6]. In Arabidopsis thaliana, a boric acid channel NIP5;1 and a borate transporter BOR1 facilitate B uptake and translocation under low B conditions [7–9]. NIP5;1 and BOR1 are localized in the plasma membrane in polar fashions toward soiland stele-sides, respectively, for directional transport of B [7].

Marisa S. Otegui (ed.), Plant Endosomes: Methods and Protocols, Methods in Molecular Biology, vol. 2177, https://doi.org/10.1007/978-1-0716-0767-1_1, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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Akira Yoshinari and Junpei Takano

Although B is an essential nutrient, it can be toxic when it accumulates in plant cells. To avoid B toxicity, cellular abundance of NIP5;1 and BOR1 are decreased in response to elevated B conditions. NIP5;1 abundance is controlled by mRNA degradation dependent on the 50 untranslated region (50 UTR) [10]. BOR1 abundance is controlled by translational repression dependent on the 50 UTR [11] of the BOR1 transcript. In addition, BOR1 located in the plasma membrane undergoes rapid endocytic degradation upon sufficient-B supply. Under B-abundant conditions, BOR1 is transported from the plasma membrane to the trans-Golgi network/early endosomes (TGN/EE) and then into the intraluminal vesicles of the multivesicular bodies (MVBs)/late endosomes [3, 12, 13]. The MVBs fuse with the vacuolar membrane to release the intraluminal vesicles and their content into the vacuole for degradation [2]. Upon sufficient-B supply, BOR1 is ubiquitinated at the lysine-590 residue located in the C-terminal cytosolic domain and the ubiquitination triggers endocytosis and vacuolar sorting of BOR1 [14, 15]. Clathrin-mediated endocytosis is the major endocytic mechanism in plant cells [2]. To examine the contribution of clathrinmediated endocytosis to BOR1 localization and degradation, we developed an inducible expression system of the dominant-negative DYNAMIN-RELATED PROTEIN 1A (DRP1A) [16]. DRP1A function in scission of clathrin-coated vesicles at the plasma membrane in Arabidopsis cells. The expression of DRP1A(K47A)mRFP efficiently inhibited endocytosis visualized by FM1-43 and BOR1-GFP. We then investigated the dominant negative effect on the endocytosis of BOR1. The constitutive endocytosis of BOR1 and other plasma membrane proteins can be visualized by brefeldin A (BFA) treatment which causes aggregation of the TGN/EE and inhibits recycling to the plasma membrane. The accumulation of BOR1-GFP in the BFA-induced endosomal aggregation was clearly reduced in the cells expressing DRP1A(K47A)-mRFP. Furthermore, the expression decreased the rates of the polar localization and vacuolar trafficking of BOR1-GFP. These results indicated the contribution of DRP1-dependent endocytosis for the polar localization and vacuolar trafficking of BOR1. This controlled inhibition of endocytosis by the inducible expression of DRP1A (K47A) is a useful tool to study the contribution of endocytosis to various aspects of plant cell biology. We further analyzed the pathways of endocytosis with emphasis on the clathrin adaptor AP-2 complex [14, 17]. Upon BFA treatment, GFP-NIP5;1 and BOR1-GFP were accumulated in endosomal aggregates and the accumulation was reduced in mutants of AP-2 complex. Furthermore, the polar localization of GFP-NIP5;1 and BOR1-GFP were affected in the mutants of AP-2 complex. These results indicate that the AP-2-dependent endocytosis and subsequent recycling maintain the polar localization of these

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transport proteins to the different plasma membrane domains. In contrast, the lack of AP-2 complex did not affect B-induced endocytosis for vacuolar trafficking of BOR1. This result indicates that the ubiquitinated BOR1 is recognized by a different adaptor rather than AP-2 complex. NIP5;1 and BOR1 tagged with GFP or other fluorescence proteins serve as markers for polar trafficking and vacuolar sorting in plant cells. Especially, two different pathways of endocytosis can be examined with BOR1-GFP. In this chapter, we update a former protocol on membrane trafficking of BOR1 [18] with emphasis on fluorescent live imaging by confocal microscopy.

2

Materials

2.1 Modified MGRL Medium

Prepare all solutions using Milli-Q ultrapure water (see Note 1). 1. 200 modified MGRL nutrients stock solutions [3] (see Table 1). 2. 1 mM or 500 mM boric acid solution. 3. Sucrose. 4. Gellan gum (see Note 2). 5. 1 L polycarbonate bottles (see Note 3). 6. Sterile disposable (140  100  14.5 mm).

2.2

Plant Materials

rectangular

petri

dish

1. Arabidopsis thaliana transgenic line harboring BOR1 promoter::BOR1-sGFP::NOS terminator [8]. 2. Arabidopsis thaliana transgenic line harboring NIP5;1 promoter (w/o 50 UTR)::sGFP-NIP5;1::NOS terminator (pShW16: [17]). 3. Arabidopsis thaliana transgenic line harboring BOR1 promoter::BOR1-GFP [8] and/or XVE:: DRP1A(WT/K47A)mRFP [18]. These transgenes were introduced at the same time by a floral dip method with a mixture of cultures of Agrobacterium strain GV3101::pMP90 harboring different plasmids. 4. 70% (v/v) ethanol. 5. Sterilized Milli-Q ultrapure water. 6. 20 μL and 1000 μL plastic pipette tips and micropipettes. 7. Surgical tape. 8. 1.5 mL microcentrifuge tube. 9. Aluminum foil.

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Table 1. Constituents of stock solutions for modified MGRL medium Stock solutions

Ingredients

per 1 L

200 Pi

NaH2PO4·2H2O Na2HPO4·12H2O

47.2 18.4

g g

1.51 0.26

mM mM

200 Mg

MgSO4·7H2O

74.0

g

1.50

mM

200 Ca, K, N

Ca(NO3)2·4H2O KNO3

94.4 60.7

g g

2.00 3.00

mM mM

200 micro w/o Fe and B

MnSO4 ZnSO4·7H2O CuSO4·5H2O CoCl2·6H2O (NH4)6Mo7O24·4H2O

311 57.5 50.0 6.20 5.90

mg mg mg mg mg

10.3 1.00 1.00 130 24.0

μM μM μM nM nM

200 Fe

Fe(III)·EDTA·3H2O

4.21

g

50.0

μM

500 mM boric acid

H3BO3

30.85

g



2.3

Chemicals

Final conc. (1)

1. Dimethyl sulfoxide (DMSO) 2. Cycloheximide (CHX) stock: Dissolve CHX in Milli-Q water at 50 mM concentration. Store the stock solution at 30  C. 3. BFA stock: Dissolve BFA in DMSO at 50 mM concentration. Store the stock solution at 30  C. 4. β-estradiol stock: Dissolve β-estradiol in DMSO at 10 mM concentration. Store the stock solution at 30  C. 5. N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide (FM4-64) stock: Dissolve FM4-64 in water at 1 mM concentration. Store the stock solution at 4  C.

2.4 Confocal Microscopy

1. Confocal laser scanning microscope equipped with 488 nm excitation line. 2. Water-immersion 40 objective lens (numerical aperture ¼ 1.10). 3. Glycerol-immersion 63 objective lens (numerical aperture ¼ 1.30). 4. Slide glass. 5. Coverslip. 6. 3 cm diameter petri dish. 7. Glass-bottom chamber/dish. 8. Razor blade.

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9. Tweezers. 10. ImageJ (https://imagej.nih.gov/ij/) or Fiji (https://fiji.sc) software for image quantification. 2.5

Root Sectioning

1. Razor blade 0.1 mm in thickness. 2. Tweezers. 3. Sterilized acupuncture needles 100 μm in diameter. 4. Toothpicks.

3

Methods

3.1 Preparation of MGRL Media 3.1.1 MGRL Solid Medium

For ten plates of solid modified MGRL medium [3], we prepare 800 mL of medium in a 1 L polycarbonate bottle. Do not use borosilicate glass bottles (see Note 3). 1. Add 4 mL each of 200 MGRL nutrients stock solutions (200 Pi, 200 Mg, 200 Ca, K, N, 200 micro w/o Fe and B, and 200 Fe. see table) into about 700 mL of Milli-Q water. 2. Add boric acid at final concentration of 0.5 μM. 3. Add 8 g sucrose, stir with magnetic stirrer until fully dissolved, and then make up to 800 mL. 4. Slowly add 12 g of gellan gum to the medium and mix well. 5. Autoclave the medium for 15 min at 121  C. 6. Distribute approximately 75 mL to sterile disposable rectangular petri dish, wait until the medium solidifies. 7. The MGRL solid medium can be stored at room temperature for a month.

3.1.2 MGRL Liquid/SoftGel Medium for Short-Term Treatments with B or Chemicals

1. Add 1/200 volume of 200 MGRL nutrients stock solutions (200 Pi, 200 Mg, 200 Ca, K, N, 200 micro w/o Fe and B, and 200 Fe) into Milli-Q water, and supplement boric acid stock to be 0.5 or 100 μM. Do not add sucrose. 2. For MGRL soft-gel medium, add 0.8% (w/v) gellan gum and dissolve by heating in a microwave oven or an autoclave. 3. Pour 1 mL of medium into a 3 cm petri dish and let solidify at room temperature. 4. Adjust temperature of the medium to 22  C before use to reduce temperature stress.

3.1.3 Induction Medium with β-Estradiol

1. Add 1/200 volume of 200 MGRL nutrients stock solution (200 Pi, 200 Mg, 200 Ca, K, N, 200 micro w/o Fe and B, and 200 Fe) into Milli-Q water, and supplement with

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0.5 μM of boric acid, 1% (v/v) sucrose, and 1% (w/v) gellan gum. 2. Autoclave the medium for 15 min at 121  C. 3. Cool down the medium to ~50  C. 4. Add β-estradiol at 10 μM or DMSO and mix well. 5. Solidify the media with and without β-estradiol in a rectangular petri dishes at room temperature. 6. Use plates with media within a week after preparation. 3.2 Germination and Growth of Arabidopsis thaliana Seedlings

1. For surface sterilization, immerse seeds in 500 μL of 70% ethanol in a 1.5 mL microcentrifuge tube for 2 min, discard the ethanol by pipetting, and rinse with 700 μL of sterilized water for three times (see Note 4). 2. Sow seeds on the solid medium with a 20 μL micropipette by pulling and release one by one. 3. Seal the rim of the plates with surgical tape. 4. Wrap the plates with aluminum foil and incubate at 4  C for 2 days to synchronize germination. 5. Place the plates vertically in a growth chamber at 22  C under a 16 h light/8 h dark cycle for 4 or 5 days (see Note 5).

3.3 Inhibition of Endocytosis

Generally, mutant plants defective in clathrin-mediated endocytosis show poor growth. To examine contribution of the clathrinmediated endocytosis at a specific growth stage, we recommend using the inducible expression of a dominant negative DRP1A to minimize pleiotropic defects. In addition, the heterogeneous gene expression pattern resulting from estradiol induction in the root epidermis allows for direct comparisons between cells with high and low levels of the dominant negative protein DRP1A (K47A)mRFP [16]. 1. Grow transgenic Arabidopsis harboring BOR1-GFP and/or XVE:: DRP1A(WT/K47A)-mRFP on MGRL solid medium with 0.5 μM boric acid for 4 days. 2. Transfer seedlings onto induction medium with β-estradiol or control medium with DMSO with tweezers in a sterile condition. 3. Seal lid with surgical tape. 4. Place the plates in a vertical position inside a plant growth chamber for 16–24 h.

3.4 Imaging of B Transporters in Longitudinal Optical Sections

For analysis of polar localization of GFP-NIP5;1 and BOR1-GFP, we recommend using a 40 water immersion objective lens. For analyses of endocytic rate and intracellular colocalization, we recommend a 63 glycerol immersion objective lens (see Note 6). Laser excitation/spectral detection bandwidths are 488/500–530 nm for

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GFP, 488/650–700 nm for FM4-64, 488/650–750 nm for chlorophyll autofluorescence, 561/580–650 nm for mRFP. The polar localization of GFP-NIP5;1, BOR1-GFP, and other membrane proteins fused with a fluorescent protein can be easily examined in longitudinal optical sections of roots by a confocal microscopy. However, it should be noted that signal intensities from different positions in the longitudinal optical section are affected by the reduction of light penetration in the tissue. The signal intensities tend to be stronger in outer positions of roots. For quantification, comparison with FM4-64 signal is recommended. 1. Cut off approximately 1 cm of root tip of transgenic plants expressing GFP-fused membrane proteins using a razor blade. Place two-to-three root tips on a slide glass using a tweezers with 50 μL of MGRL liquid medium, and gently cover by a cover glass (see Note 7). 2. The plasma membrane can be stained with the fluorescent dye, FM4-64 (optional). For this, dilute 1 mM stock solution of FM4-64 in MGRL liquid medium to a final concentration of 1 μM. Incubate whole seedlings in 1 mL of the solution for 30 min using 24-well-plate and wash with Milli-Q ultrapure water three times. 3. To observe polar localization in various cell layers, find an optical section through the center of a root tip (Fig. 1a).

Fig. 1 Quantification of polarity index of BOR1-GFP. (a) A confocal micrograph of a primary root tip of Arabidopsis thaliana expressing BOR1-GFP. (b) An enlarged image of the area indicated in a. (c) Plots of fluorescence intensity along the indicated lines in b. Formula and representative values of polarity index are shown on the right

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3.4.1 Image Analysis: Quantification of Polarity Index from Longitudinal Optical Sections

1. Use images of cells from the meristematic to transition zone of the root tip for quantification of polarity index by ImageJ software. 2. Select plasma membrane at apical and basal domains by straight lines with 3-pixel-width (Fig. 1b). 3. Measure fluorescence intensity along the lines (Fig. 1c). 4. Calculate polarity index as follows: Integrated density (total fluorescence) in inner half/Integrated density in outer half.

3.5 Imaging of B Transporters in Cross Sections

This method uses relatively hard solid media with holes to guide straight growth of roots as described previously [19]. The protocol used a vibratome to cut out sections. Here we describe a simple method to produce hand-cut sections without using a vibratome or other costly equipment. 1. Prepare MGRL solid medium as described above but with 3% (w/v) gellan gum. 2. Vertically perforate the medium with sterilized acupuncture needles. 3. Make a small pit on the top of the perforations with a sterilized tooth pick. 4. Sow surface-sterilized seeds in the pits. 5. Wrap dishes with aluminum foil and keep them in a refrigerator for 2 days at 4  C to synchronize germination. 6. Place the plates horizontally at 22  C under a 16 h light–8 h dark cycle for 4–6 days. 7. Cut out a block containing medium (~1 cm  1 cm) and growing roots (Fig. 2a). Ideally, roots should be growing straight down along the perforations but roots growing in different directions can also be used. 8. Cut block and roots in cross section by hand with a razor blade attached to a scalpel and collect root sections (~1 mm thickness) by a tweezers. Layered razor blades may be helpful (see Note 8, Fig. 2b). 9. Place the root sections on a slide grass with a drop of water or liquid medium and cover them with a coverslip. 10. Collect confocal images at few cell layers under the cut surface (see Fig. 2c).

3.6 Time-Lapse Imaging and Analysis of B-Induced Vacuolar Sorting of BOR1-GFP

Upon sufficient-B supply, BOR1 is internalized from the plasma membrane to the TGN via AP-2-independent endocytosis, and sorted into intraluminal vesicles of MVBs destined to the vacuole. BOR1-GFP is useful as a marker for inducible endocytosis and MVB sorting. To detect GFP accumulated inside vacuole, seedlings can be kept under a dark condition for 2 h. Darkness delays the

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Fig. 2 Preparation and imaging of root cross sections. (a) An illustration of a root growing in solid medium and the position of at which cross sections should be taken. (b) A layered razor blade. (c) A confocal micrograph of a cross section of a primary root of Arabidopsis thaliana expressing GFP-NIP5;1

degradation of GFP and enhances detection of cleaved GFP derived from the partial proteolysis of GFP-fused membrane protein delivered to the vacuole [20]. 1. Transfer a 5-day-old seedling expressing BOR1-GFP from the plate to MGRL liquid medium containing either 0.5 or 100 μM boric acid and incubate for 1 min. Precisely measure time after shifting media. Transfer the seedling onto a glassbottom dish and gently put MGRL soft-gel medium containing 0.5 or 100 μM boric acid on top of the roots (Fig. 3a). 2. Collect confocal images of epidermal cells in the meristematic to the transition zone of the roots at 3 min after the shifting media. 3. Take confocal images at the same position with same microscopy settings at 10, 20, 30, 45, 60, 90, and 120 min. 4. Select apical and basal plasma membrane domains by segmented lines with 3-pixel-width in ImageJ software (Fig. 3b, c).

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Akira Yoshinari and Junpei Takano

Fig. 3 Quantification of B-induced endocytosis and degradation of BOR1-GFP. (a) Illustration of experimental procedure. Five-day-old seedlings grown on solid medium with 0.5 μM boric acid are incubated in liquid medium containing 0.5 μM or 100 μM boric acid for 1 min and then transferred onto glass-bottom dish with the corresponding liquid medium and soft medium. BOR1-GFP in the epidermal cells is imaged with an inverted microscope. (b) Representative confocal micrographs of BOR1-GFP at 10 and 60 min after 100 μM B-supply. Formulas and representative values of fluorescent intensities in the plasma membrane per a cell are shown on the bottom. (c) A representative result of time-course analysis of B-dependent endocytosis and degradation of BOR1-GFP (n ¼ 10 cells from one root, error bars ¼ SD)

5. Measure mean gray values (average fluorescence intensities) in the selected region-of-interests (ROIs). 6. Calculate fluorescence intensity in plasma membrane in each cell as follows: (Mean gray value in apical domain + Mean gray value in basal domain)/2. 3.7 Analysis of Constitutive Endocytosis of BOR1GFP Using BFA

This method is to visualize BOR1-GFP accumulation in endosomal aggregates caused by BFA treatment. Pretreatment with CHX blocks de novo protein synthesis and thus allows for visualization of BOR1-GFP internalized from the plasma membrane by the constitutive endocytosis. 1. Transfer 5-day-old seedlings to MGRL liquid medium containing 0.5 μM boric acid and 50 μM CHX for 30 min to block de novo protein synthesis. 2. Transfer the seedlings to MGRL liquid medium containing 0.5 μM boric acid, 50 μM CHX, and 50 μM BFA or equivalent concentration of DMSO as a control. 3. Collect confocal images of the root epidermal cells between 30 and 120 min after BFA treatment. 4. Select apical and basal plasma membrane domains by segmented lines with 3-pixel-width in ImageJ software (Fig. 4b). 5. Select intracellular regions with polygon selection (Fig. 4b). 6. Measure mean gray values (average fluorescence intensities) in the ROIs.

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Fig. 4 Quantification of internalization rates of BOR1-GFP. (a) An illustration of experimental procedure. Fiveday-old seedlings grown on solid medium with 0.5 μM boric acid are treated with CHX for 30 min followed by CHX and BFA for 60 min. Primary roots are cut and mounted on slide-glass with liquid medium containing CHX and BFA or DMSO. (b) Representative confocal micrograph of BOR1-GFP in root epidermal cells after the BFA treatment. Enlarged image is on the right. Formula of internalization rate and representative value are shown on the bottom

7. Calculate internalization rates as follows: Mean gray value of intracellular region/(Mean gray value in apical domain + Mean gray value in basal domain)/2.

4

Notes 1. We use Milli-Q Advantage equipped with Q-Pod Elements to reduce contamination of boric acid. 2. We use gellan gum because agar contains significant amount of boron. 3. We use plasticware, such as polycarbonate bottles, to prepare low-B medium. We avoid using borosilicate glass, which causes contamination of boric acid. 4. Seeds can be stored in water after rinsing for 2–3 days at 4  C. 5. Root caps of very young seedlings (~3 days after germination) have cuticles which prevent penetration of dyes [21]. Root tips of old seedlings (more than 6 days after germination) are thick and not optimal for imaging. 6. Generally, high magnification objectives (>60) are not optimal for imaging deep in tissues and not suitable for analysis of inner/outer polar-localizations observed in BOR1 and NIP5;1. 7. Whole seedlings with cotyledons can be used for imaging of longitudinal optical section. However, the thickness of

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hypocotyl often affects the horizontal position of root tips on a cover grass. Putting multiple cut roots on a slide glass keeps the horizontal position of the root tips and prevents them from breaking due to pressure. 8. Layered razor blades can be used to produce sections. Attach razor blades (0.1 mm thickness) to plastic or paper sheets (~1 mm thickness) using adhesive tape to place 4–6 blades 1 mm apart from each other (Fig. 2b).

Acknowledgments We thank Chinami Shinohara (Osaka Prefecture University) for providing a confocal micrograph of GFP-NIP5;1. This research was, in part, supported by Grant-in-Aid for Young Scientists (19K16164) to Akira Yoshinari and Grants-in-Aid (26712007 and 19H00934) to Junpei Takano from the Japan Society for the Promotion of Science. References 1. Nakamura M, Grebe M (2018) Outer, inner and planar polarity in the Arabidopsis root. Curr Opin Plant Biol 41:46–53 2. Paez Valencia J, Goodman K, Otegui MS (2016) Endocytosis and endosomal trafficking in plants. Annu Rev Plant Biol 67:309–335 3. Takano J, Miwa K, Yuan L, von Wiren N, Fujiwara T (2005) Endocytosis and degradation of BOR1, a boron transporter of Arabidopsis thaliana, regulated by boron availability. Proc Natl Acad Sci 102:12276–12281 4. Liu C, Shen W, Yang C, Zeng L, Gao C (2018) Knowns and unknowns of plasma membrane protein degradation in plants. Plant Sci 272:55–61 5. Yoshinari A, Takano J (2017) Insights into the mechanisms underlying boron homeostasis in plants. Front Plant Sci 8:1–8 6. Funakawa H, Miwa K (2015) Synthesis of borate cross-linked rhamnogalacturonan II. Front Plant Sci 6:1–8 7. Takano J et al (2010) Polar localization and degradation of Arabidopsis boron transporters through distinct trafficking pathways. Proc Natl Acad Sci U S A 107:5220–5225 8. Takano J (2006) The Arabidopsis major intrinsic protein NIP5;1 is essential for efficient boron uptake and plant development under boron limitation. Plant Cell 18:1498–1509 9. Takano J et al (2002) Arabidopsis boron transporter for xylem loading. Nature 420:337–340

10. Tanaka M et al (2016) The minimum open reading frame, AUG-stop, induces borondependent ribosome stalling and mRNA degradation. Plant Cell 28:2830–2849 11. Aibara I et al (2018) Boron-dependent translational suppression of the borate exporter BOR1 contributes to the avoidance of boron toxicity. Plant Physiol 177:759–774 12. Yoshinari A, Korbei B, Takano J (2018) TOL proteins mediate vacuolar sorting of the borate transporter BOR1 in Arabidopsis thaliana. Soil Sci Plant Nutr 64:598–605 13. Viotti C et al (2010) Endocytic and secretory traffic in Arabidopsis merge in the trans-golgi network/early endosome, an independent and highly dynamic organelle. Plant Cell 22:1344–1357 14. Yoshinari A et al (2019) Polar localization of the borate exporter BOR1 requires AP2-dependent endocytosis. Plant Physiol 179:1569–1580 15. Kasai K, Takano J, Miwa K, Toyoda A, Fujiwara T (2011) High boron-induced ubiquitination regulates vacuolar sorting of the BOR1 borate transporter in Arabidopsis thaliana. J Biol Chem 286:6175–6183 16. Yoshinari A et al (2016) DRP1-dependent endocytosis is essential for polar localization and boron-induced degradation of the borate transporter BOR1 in arabidopsis thaliana. Plant Cell Physiol 57:1985–2000

Analysis of Endocytosis and Intracellular Trafficking of Boric Acid/Borate. . . 17. Wang S et al (2017) Polar localization of the NIP5;1 boric acid channel is maintained by endocytosis and facilitates boron transport in Arabidopsis roots. Plant Cell 29:824–842 18. Kasai K, Takano J, Fujiwara T (2014) Analysis of endocytosis and ubiquitination of the BOR1 transporter. In: Otegui MS (ed) Plant endosomes: methods and protocols. Springer New York, New York, NY, pp 203–217

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19. Sotta N, Fujiwara T (2017) Preparing thin cross sections of Arabidopsis roots without embedding. Biotechniques 63:281–283 20. Tamura K et al (2003) Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants. Plant J 35:545–555 21. Berhin A et al (2019) The root cap cuticle: a cell wall structure for seedling establishment and lateral root formation. Cell 176:1367–1378

Chapter 2 Analysis of Membrane Proteins Transport from Endosomal Compartments to Vacuoles Mengqian Luo, Ying Zhu, Zhiqi Liu, and Liwen Jiang Abstract Endocytosis and endosomal trafficking to vacuoles play important roles in regulating the homeostasis of plasma membrane (PM) proteins in plant cells. FREE1 (FYVE domain protein required for endosomal sorting 1) is a plant-unique component of the ESCRT (endosomal sorting complex required for transport) machinery. In free1 mutant plants, PIN-FORMED 2 (PIN2)-GFP was found to mislocalize from the PM to the tonoplast. In this chapter, we describe a detailed protocol for studying vacuolar sorting and degradation of PIN2-GFP by using T-DNA insertional mutants, dexamethasone (DEX) inducible RNAi lines, and other tools, including Fei-Mao (FM) dye staining and dark treatment. By using these methods, we illustrate the endosomal trafficking and vacuolar degradation of PIN2-GFP in plants. Key words Prevacuolar compartment, Multivesicular body, Vacuolar degradation, FM dye, FREE1, Plasma membrane protein

1

Introduction Endocytosis of plasma membrane (PM) proteins is important for cell-to-cell or cell-to-environment communication in plants. Certain PM proteins are internalized and delivered to early endosomes (EE)/ trans-Golgi networks (TGN) and then recycled back to the PM or further transported to the vacuole via late endosome (LE) or prevacuolar compartment/multivesicular body (PVC/MVB) for degradation in plants [1, 2]. The membrane proteins destined to the vacuole are first ubiquitinated and then sorted into the intraluminal vesicles (ILVs) of PVC/MVB via the endosomal sorting complex required for transport (ESCRT) machinery. The PVC/MVB further fuses with the vacuole and releases the ILVs into the vacuole lumen for degradation [3–7]. In yeast and mammalian cells, the canonical ESCRT complexes includes ESCRT-0, -I, - II, -III and other accessory components [8]. Interestingly, most of the evolutionary conserved ESCRT components have been identified in plants, except those of ESCRT-0 and the

Marisa S. Otegui (ed.), Plant Endosomes: Methods and Protocols, Methods in Molecular Biology, vol. 2177, https://doi.org/10.1007/978-1-0716-0767-1_2, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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ESCRT-I subunit Mvb12 [8, 9]. We previously reported multiple functions of a unique plant ESCRT component FYVE domain protein required for endosomal sorting 1 (FREE1) in regulating organelle biogenesis, plant development and abscisic acid signaling in plants [10–13]. The T-DNA insertional free1 mutant and dexamethasone (DEX) inducible FREE1-RNAi plants are lethal, which is the typical phenotype among the ESCRT-related proteins [10, 14, 15]. The defects such as absence of ILVs of MVBs, fragmented vacuoles and membrane protein mis-sorting were also observed [10]. Thus, FREE1 deficient mutants are suitable materials to study endocytosis and vacuolar degradation pathway in plants. More recently, via a genetic suppressor screening, we identified a plant-specific Bro1-domain protein called BRAF. BRAF regulates FREE1 recruitment to PVC/MVB, thus functioning as an ESCRT regulator and mediating ILVs formation and membrane protein sorting in plants [16]. The PIN-FORMED auxin efflux carriers, especially PIN2, have been widely used as membrane protein markers to study endosomal trafficking [17, 18]. PIN2 shows polarized localization in the basal or apical domain of root cells to preserve polar auxin transport [19]. PIN2 is an ESCRT cargo which undergoes MVB-dependent vacuolar degradation [19]. Thus, PIN2-GFP is an ideal marker for visualizing the trafficking route from MVB to vacuole. Besides fluorescence-tagged cargo proteins, Fei-Mao (FM) styryl dyes (e.g., FM4-64) are also frequently used for tracking endocytosis in plants. Upon uptake by the PM, FM dye can reach tonoplast via PVC/MVB [20]. In addition, exposure of plants to the dark stabilizes GFP-based reporter proteins in lytic compartments (e.g., vacuoles), thus allowing for direct visualization of GFP fluorescence signal in the vacuole [10]. Whereas in wild-type (WT) root cells, PIN2-GFP showed polarized PM localization, in free1 the mutant, it abnormally accumulates at the tonoplast (Figs. 1 and 2a), a characteristic defect of ESCRT mutants. In this protocol, we describe the study of PIN2GFP traffic from MVB to vacuole. By expressing PIN2-GFP in the free1 mutant and in DEX-inducible FREE1-RNAi transgenic plants, together with FM dye staining and darkness treatment, we illustrate the useful methods to analyze ESCRT-dependent membrane protein sorting and vacuolar degradation in plants (Fig. 2).

2

Materials

2.1 Plant Materials and Growth Conditions

1. Arabidopsis thaliana (ecotype Columbia-0 also called Col-0). 2. Transgenic lines expressing PIN2-GFP in Col-0, free1 (Arabidopsis transposon insertion line (15-1960-1) obtained from RIKEN), DEX::FREE1-RNAi backgrounds (see Note 1).

Methods to Visualize Vacuolar Trafficking of Membrane Proteins

17

Fig. 1 Working model of endocytic trafficking of PIN2-GFP from PM to vacuole via PVC/MVB. During endocytosis, the plasma membrane (PM) protein PIN2 (i) is internalized and delivered to the TGN/EE. (ii) PIN2 proteins can be recycled back to the PM (iii) or be recruited to the PVC/MVB. (a) In WT plant cells, PIN2 proteins are sorted into the intra-luminal vesicles (ILVs) of PVC/MVB and released into the vacuole for degradation. When seedlings are subjected to dark treatment, PIN2-GFP can be observed inside the lytic vacuole (LV) due to the stabilization of GFP. (b) In FREE1 deficient mutants, plant cells form numerous fragmented vacuoles and lack normal ILVs. PIN2-GFP proteins cannot enter the LV for degradation and accumulate at the tonoplast. CW cell wall, PM plasma membrane, ER endoplasmic reticulum, TGN trans-Golgi network, EE early endosome, PVC prevacuolar compartment, MVB multivesicular body, LE late endosome, LV lytic vacuole

3. Arabidopsis seeds were surface-sterilized and kept under a longday (16 h light/8 h dark) photoperiod in a growth chamber at 22  C. 2.2 Buffers and Solutions

1. Murashige and Skoog (MS) solid and liquid medium: 1.515 g MS salt, 3.5 g sucrose, ddH2O up to 350 ml, pH 5.7 (adjusted with KOH). Solid medium is prepared by the addition of 0.8% agar (see Notes 2 and 3). 2. DEX stock solution: 10 mM DEX prepared with ethanol (see Notes 4 and 5). 3. FM4-64 dye stock solution: 1 mg of FM4-64 dissolved in 150 μl of ddH2O. Aliquot and keep at 20  C (see Note 5). 4. Washing buffer for seed sterilization: 33% bleach in ddH2O containing 0.05% Triton X-100.

2.3 Equipment and Other Materials

1. Confocal microscope (for example, Leica SP8). 2. Microscope glass slides and coverslips for imaging. 3. 6-well petri dishes. 4. Eppendorf Research®plus Pipette 0.5–10 μl. 5. Eppendorf Research®plus Pipette 10–100 μl.

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Fig. 2 PIN2-GFP molecules destined to vacuole for degradation are trapped at the tonoplast in endosomal trafficking-deficient mutants. (a) PIN2-GFP show PM localization in WT root meristematic cells, whereas in free1 mutant, PIN2-GFP localize to both PM localization and tonoplast. (b) After 6 h dark treatment, PIN2-GFP can be detected either at the vacuolar lumen (arrowheads) or at the tonoplast (arrows) in DEX::FREE1-RNAi plants without ( ) or with (+)DEX induction, respectively. The image regions enclosed by white boxes are shown on the right at higher magnification and with split channels (top: GFP, bottom: RFP). Scale bar, 10 μm

6. Eppendorf Research®plus Pipette 100–1000 μl. 7. Pipette tips (P10, P200, P1000). 8. Forceps. 9. Sterile single use plasticware. 10. Growth chamber.

3

Methods

3.1 Seed Sterilization and Germination

1. Sterilize Col-0 and free1 seeds by soaking them in washing buffer for 1 min followed by five rinses with ddH2O. 2. Transfer sterilized seeds onto the MS solid-medium plates and incubate for 2 days at 4  C (see Note 6). 3. Transfer the plates to the growth chamber for 5 days (see Note 7).

Methods to Visualize Vacuolar Trafficking of Membrane Proteins

3.2 Drug Treatment and Confocal Imaging 3.2.1 FM 4-64 Dye Treatment

3.2.2 DEX Treatment on DEX-Inducible RNAi Arabidopsis Seedlings

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1. Place 1–2 ml MS liquid medium into each well of a 6-well petri dish, with or without FM 4–64 dye (1:1000 dilution from stock solution) (see Note 8). 2. Add 3–5-day-old Arabidopsis Col-0 and free1 seedlings into each well and incubate at room temperature for 6 h. 1. Surface-sterilize DEX-inducible RNAi Arabidopsis seeds by incubation in washing buffer for 1–3 min followed by five rinses with ddH2O. 2. Sow the seeds on MS+DEX medium and keep the plates at 4  C for 2 days before transferring them to the growth chamber (see Note 9). Five-day-old WT Col-0, free1, and DEX-inducible RNAi Arabidopsis seedlings can be subjected to different observations.

3.3

Dark Treatment

3.4 Confocal Imaging

1. Transfer seedlings into MS liquid medium in the dark for 6 h. Dark treatment promotes the endocytic internalization and vacuolar degradation of PIN2-GFP. 1. Image PIN2-GFP and FM4-64 in Arabidopsis root cells with a 60 water-immersion lens. Several drops of ddH2O could be used to cover the surface of the seedlings before adding coverslip on. 2. Collect fluorescence signals selecting for the proper excitation and emission wavelengths (see Note 10).

4

Notes 1. Stable transgenic lines of Arabidopsis are preferred over protoplast transient expression systems, allowing for long-term phenotypic observations and genetic studies. The DEX inducible system compensates for the seedling lethality of T-DNA insertional free1 mutant, thus facilitating the analysis of PIN2-GFP trafficking in a FREE1-knock-down background [21]. 2. The ideal temperature range for pouring MS plates is 50–55  C. At this temperature, it is safe to hold the bottle without extra protection. Chemicals such as DEX should be added at this and not higher temperatures since excessive heat may affect its chemical integrity and activity. 3. Make sure that the chemicals are well-mixed in MS liquid medium before placing seedlings. 4. 10–30 μM is the proper concentration range for DEX treatment. We suggest 10 μM DEX for DEX-induced RNAi transgenic Arabidopsis lines.

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5. DEX and FM dyes should be aliquoted in small amounts (100 μl for DEX/10 μl for FM dyes) and kept at 20  C. Thaw a new aliquot for each experiment. 6. Seeds should be kept at 4  C for several days before transferring them to the growth chamber to achieve relatively uniform germination. 7. As confocal imaging is done on root cells, the MS plates should be place perpendicularly in the growth chamber to keep roots growing straight. 8. FM dyes such as green-fluorescent FM 1-43 and red-fluorescent FM 4-64 dyes are frequently used for tracking endocytosis in plant cells. The proper dye could be selected according to the fluorescence tag carried by the original plant material. 9. All the prepared MS plates containing DEX need to be kept at 4  C and should be used within a week. 10. It is important to ensure that the seedlings are healthy without any signs of stress prior to chemical treatment and confocal imaging. Otherwise, stressed seedlings may obscure the results.

Acknowledgments We are grateful to the current and previous members of Prof. Jiang’s Laboratory for their contributions in improving methods of endocytic protein study. This work was supported by grants from the Research Grants Council of Hong Kong (CUHK14130716, 14102417, 14100818, R4005-18F, C4012-16E, C4002-17G and AoE/M-05/12), the National Natural Science Foundation of China (31670179 and 91854201) and Research Committee of CUHK to LJ. References 1. Piper RC, Luzio JP (2007) Ubiquitindependent sorting of integral membrane proteins for degradation in lysosomes. Curr Opin Cell Biol 19(4):459–465 2. Robinson DG, Jiang L, Schumacher K (2008) The endosomal system of plants: charting new and familiar territories. Plant Physiol 147:1482–1492 3. Henne WM, Buchkovich NJ, Emr SD (2011) The ESCRT pathway. Dev Cell 21:77–91 4. Raiborg C, Stenmark H (2009) The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins. Nature 458:445–452

5. Shields SB, Piper RC (2011) How ubiquitin functions with ESCRTs. Traffic 12:1306–1317 6. Winter V, Hauser MT (2006) Exploring the ESCRTing machinery in eukaryotes. Trends Plant Sci 11:115–123 7. Cui Y, Shen J, Gao C et al (2016) Biogenesis of plant prevacuolar multivesicular bodies. Mol Plant 9(6):774–786 8. Leung KF, Dacks JB, Field MC (2008) Evolution of the multivesicular body ESCRT machinery; retention across the eukaryotic lineage. Traffic 9:1698–1716 9. Cai Y, Zhuang X, Gao C et al (2014) The Arabidopsis endosomal sorting complex

Methods to Visualize Vacuolar Trafficking of Membrane Proteins required for transport III regulates internal vesicle formation of the prevacuolar compartment and is required for plant development. Plant Physiol 165:1328–1343 10. Gao C, Luo M, Zhao Q et al (2014) A unique plant ESCRT component, FREE1, regulates multivesicular body protein sorting and plant growth. Curr Biol 24(21):2556–2563 11. Gao C, Zhuang X, Cui Y et al (2015) Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation. Proc Natl Acad Sci U S A 112(6):1886–1891 12. Belda-Palazon B, Rodriguez L, Fernandez MA et al (2016) FYVE1/FREE1 interacts with the PYL4 ABA receptor and mediates its delivery to the vacuolar degradation pathway. Plant Cell 28(9):2291–2311 13. Li H, Li Y, Zhao Q et al (2019) The plant ESCRT component FREE1 shuttles to the nucleus to attenuate abscisic acid signalling. Nat Plants 5(5):512–524 14. Shen J, Gao C, Zhao Q et al (2016) AtBRO1 functions in ESCRT-I complex to regulate multivesicular body protein sorting. Mol Plant 9(5):760–763 15. Katsiarimpa A, Anzenberger F, Schlager N et al (2011) The Arabidopsis deubiquitinating enzyme AMSH3 interacts with ESCRT-III

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subunits and regulates their localization. Plant Cell 23(8):3026–3040 16. Shen J, Zhao Q, Wang X et al (2018) A plant Bro1 domain protein BRAF regulates multivesicular body biogenesis and membrane protein homeostasis. Nat Commun 9(1):3784 17. Kleine-Vehn J, Leitner J, Zwiewka M et al (2008) Differential degradation of PIN2 auxin efflux carrier by retromer-dependent vacuolar targeting. Proc Natl Acad Sci U S A 105 (46):17812–17817 18. Abas L, Benjamins R, Malenica N et al (2006) Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism. Nat Cell Biol 8:249–256 19. Spitzer C, Reyes FC, Buono R et al (2009) The ESCRT-related CHMP1A and B proteins mediate multivesicular body sorting of auxin carriers in Arabidopsis and are required for plant development. Plant Cell 21:749–766 20. Bolte S, Talbot C, Boutte Y et al (2004) FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells. J Microsc 214:159–173 21. Aoyama T, Chua NH (1997) A glucocorticoidmediated transcriptional induction system in transgenic plants. Plant J 11(3):605–612

Chapter 3 Analysis of Endoplasmic Reticulum–Endosome Association Using Live-Cell Imaging in Plant Cells Giovanni Stefano and Federica Brandizzi Abstract The endoplasmic reticulum (ER) is one of the most abundant endomembrane compartments and is in close association with most of the other organelles. In mammalian and yeast cells, the physiological roles and the molecular machineries underlying such association have only recently begun to emerge. In plant cells, recent live-cell confocal imaging and electron microscopy studies have established that endosomes are associated with the ER [1]. Here, we describe confocal imaging methods and software to analyze ER–endosome association in plant cells. Key words Endoplasmic reticulum, endosomes, contact sites, FRET, correlation analyses

1

Introduction In recent years, mounting evidence has been accumulating that endomembrane compartments establish an exceptionally high number of contacts sites with each other and with ontogenetically distinct organelles [2–4]. Each of these contact sites has probably specific features with completely different physical and chemical properties. Particularly the ER, one of the most abundant endomembrane compartments of the cell, has been shown by electron microscopy [5] and live-cell imaging analyses [6, 7] to form numerous contact sites with the plasma membrane. The membrane associations occur at sites of close apposition of about 10–30 nm between the heterotypic membranes [6]. In mammalian cells, the ER forms contact sites with endosomes; this association increases as endosomes mature [4]. Furthermore, the association with the ER tubules seems critical to establish the site of the fission of the endosomes [4, 8]. In plant cells, it has been shown that endosomes associate with the ER and that a disruption of the spatial distribution of such association leads to an abnormal endocytic traffic [1].

Marisa S. Otegui (ed.), Plant Endosomes: Methods and Protocols, Methods in Molecular Biology, vol. 2177, https://doi.org/10.1007/978-1-0716-0767-1_3, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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Here, we describe how the association between ER and endosomes can be analyzed by fluorescence resonance energy transfer (FRET) and colocalization analysis. There are a number of suitable fluorescent markers to label ER and endosomes: (a) ER luminal markers: the ER can be visualized in vivo using several luminal markers, such as ER-XFP or XFP-HDEL (Table 1) [9, 10], where X is a fluorescent protein (e.g., cyan, yellow, red). To our knowledge, these soluble ER markers do not affect the overall structure of the ER keeping the normal reticulated shape and arrangement of tubules and cisternae. (b) ER membrane markers: several membrane markers are routinely used to mark the ER; it should be considered that in conditions of overexpression, some of these markers can modify the ER network architecture (see Note 1). Indeed, markers such as those based on fluorescent protein fusions to reticulons (e.g., RTNLB3) or calnexin protein domains have been shown to act as membrane modifiers when over-expressed, causing extensive remodeling of ER tubules and enlargement of ER cisternae [12, 13, 15] (Table 1). (c) Endosome markers: in plant cells, the term endosomes defines populations of post-Golgi membrane compartments. The first endosomal compartments at the exocytosis and endocytic nexus are the early endosomes (EEs)[16]. Successively, multivesicular bodies (MVBs), late endosomes (LEs)/prevacuolar compartments, and late prevacuolar compartments originate from the EEs [17, 18]. Some of the most commonly used endosomes markers are listed in Table 1 (see Notes 2 and 3)

2 2.1

Materials Plant Plasmids

1. Several plant binary vectors can be used for subcloning genes of ER or endosome proteins fused to fluorescent tags for imaging. The vectors can be used for transient transformation of Nicotiana tabacum or N. benthamiana or Arabidopsis thaliana seedlings or stable transformation of A. thaliana. Alternatively, binary vectors carrying the ER or endosome markers can be purchased through the ABRC (Arabidopsis Biological Resource Center) or NASC (Nottingham Arabidopsis Stock Centre). 2. Plasmids for FRET experiments; the following list of constructs are needed for the FRET experiments described in Subheading 3.4: (a) CHC-YFP; (b) VAP27-1-CFP; (c) cYFP (cytosolicYFP).

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Table 1 List of ER and endosome markers available as fluorescent protein fusions (see Note 2) Organelle

Marker name

Reference

ER

ER-XFP (lumenal marker)

Nelson et al. [9]

ER

XFP-HDEL (lumenal marker)

Brandizzi et al. [10]

ER

Calnexin-XFP (ER-modifier)

Irons et al. [11]

ER

XFP-RTNLB3 (ER-modifier)

Stefano et al. [1];Sparkes et al. [12]; Tolley et al. [13]

Late endosome/prevacuole XFP-RabF2a

Stefano et al. [1]; Geldner et al. [14]

Endosomal/recycling endosome

XFP-RabA1g

Stefano et al. [1]; Geldner et al. [14]

Late endosome/prevacuole XFP-RabF2b

Stefano et al. [1]; Geldner et al. [14]

2.2 Agrobacterium Culture and Media

1. Agrobacterium strain GV3101::mp90 transformed with a binary vector (pBin20, pEarleyGate, etc.) containing the gene coding for an ER or endosome marker. 2. Appropriate antibiotics for selection of transformed Agrobacterium (e.g., gentamycin (#GTA202, Bioshop) 25 mg/mL, rifampicin (#R0079, TCI—Tokyo Chemical Industry) 50 mg/mL + antibiotic for binary vector resistance) 3. LB medium: 5.0 g/L yeast extract (#212750, BD Biosciences), 10.0 g/L tryptone (#211705, BD Biosciences), 10.0 g/L sodium chloride (#S3014, Sigma), pH 7.0. 4. Infiltration medium: 0.5% D-glucose (#1916-01, J.T. Baker Chem. Co.), 50 mM MES (#M2933, Sigma), 2 mM Na3PO4·12H2O (#S7778, Sigma), 0.2 mM acetosyringone (#D134406, Sigma). 5. Cocultivation medium: LS (#LSP03-50LT, Caisson) Medium 0.25, 100 μM acetosyringone, 0.005% Silwet L-77 (#NC0628903; Fisher Scientific). 6. Wash solution: 10 mM MgCl2 (#M8266, Sigma), 100 μM acetosyringone.

2.3

Plant Material

1. 3–4-week-old N. tabacum or N. benthamiana plants, or A. thaliana young seedlings (4–5 days after germination) can be used for transient transformation. For stable transformation A. thaliana plants at bolting stage can be used for floral dipping.

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2. Stable A. thaliana seed lines carrying the ER or endosome markers can be purchased through the ABRC or NASC. Plants can alternatively be transformed with either marker through floral dipping (see below). Homozygous plants selected via antibiotic selection can be used for imaging. For dual imaging of both markers, a seed line carrying the ER marker should to be crossed with the seed line harboring the desired endosome marker. The F1 progeny can be already used for imaging at the confocal. Once the F2 progeny has been obtained, stable lines carrying both markers can be screened and selected. The selected plants can be transferred on soil. Their progeny can be used to grow plants that express both the markers for all the imaging analyses. 2.4 Seeds Sterilization

1. Sterile hood. 2. Sterile 1 mL pipette tips or sterile Pasteur pipettes. 3. 70% (v/v) ethanol. 4. 20% (v/v) bleach. 5. Sterile distilled water. 6. Seeds coexpressing ER and endosomal markers (see Table 1).

2.5

Plant Growth

1. Linsmaier and Skoog medium (LS) plates, with the pH buffered to 5.7  0.2 containing macronutrients, micronutrients, and vitamins. (a) For A. thaliana stable lines use the following medium: For 1 L of medium (here named 1/2 LS) use 2.35 g of LS, 1% (w/v) sucrose (#S-0389, Sigma), and 0.8% (w/v) agar (#7558A, Acumedia), and then autoclave the media for 25 min. (b) For transient expression of reporters in Arabidopsis lines use the following: For 1 L of medium use 1.18 g of LS (here named 0.25 LS), 1% (w/v) sucrose, and 0.8% (w/v) agar, and then autoclave the media 25 min. 2. Growth chambers: For growing A. thaliana plants set the chamber at 16 h light (100 μmol m2 s1)/8 h dark, 22  C and 55% humidity. For N. tabacum or N. benthamiana use a controlled chamber with a cycle of 23  C for 18 h light (360 μmol m2 s1) and 18  C for 6 h dark, with humidity set to 90%.

2.6 Microscopy Imaging

1. Razor blades. 2. Forceps. 3. Distilled water or half strength Linsmaier and Skoog (LS) liquid medium. 4. Glass slides.

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5. Glass coverslips. 6. Confocal microscope with high resolution (e.g., 60 or 63 water or oil immersion objectives) and with double channel detection (see Note 3). 7. 25 μM Latrunculin B (Latrunculin B; #428020 Calbiochem) in distilled water. 8. 10 μM oryzalin (Oryzalin; 36182 Sigma) in distilled water. 2.7 Software for Imaging Analysis

2.8 Other Materials Needed

Image J/Fiji or any advanced confocal suite software available for imaging analysis installed on any commercial confocal microscope can be used. 1. Tips (1 mL, 200 μL, 20 μL) 2. Eppendorf tubes (1.5 mL) 3. Syringe (without needle) 0.5 mL 4. Permanent marker 5. Plant Pots (3-½ in.) and soil 6. Nalgene™ PPCO Centrifuge Bottle (500 mL) with Sealing Closure (#3141-0500PK Thermofisher) 7. Micropore 3M 8. Centrifuge

3

Methods

3.1 Plant Transformation 3.1.1 Transient Transformation in Tobacco

Tobacco transient transformation can be performed using a modified method based on a previous published protocol [19, 20]: 1. Inoculate 5 mL of LB medium with the Agrobacterium culture carrying the binary vector of interest (one culture with the bacteria carrying ER marker and the second one carrying the endosome marker) and add the appropriate antibiotics for selection; 2. Grow culture overnight at 28  C and 200 rpm orbital shaking. 3. Transfer 1 mL of culture into a sterile 1.5 mL Eppendorf tube. 4. Pellet Agrobacterium cells at 2655  g, 3 min at room temperature (20-25  C). 5. Remove supernatant. 6. Add 1 mL of infiltration medium and gently resuspend the pellet. 7. Repeat steps 4–6.

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8. Measure the absorbance at 600 nm of a 1 into 5 dilutions of cells, and calculate the OD of the culture by multiplying the OD value reads by 5. 9. Dilute the culture to 0.05–0.1 OD (see Note 4) for both Agrobacterium cultures and mix them in the same 1.5 mL Eppendorf tube. 10. Take a 3–4-week-old plant from the growth chamber and choose a well expanded leaf to be infiltrated. 11. Gently puncture a region of the leaf to be infiltrated using a 200 μL sterile tip. 12. Aspirate the resuspended solution containing the Agrobacterium cells with a 1 mL syringe (without the needle). 13. Using one hand, flip the leaf gently (to have the abaxial side facing upward) while placing one finger on the adaxial side of the region selected for infiltration. 14. Place the tip of the syringe on the abaxial side of the selected area and press the plunger down gently while supporting the adaxial side of the leaf with your finger, until the solution containing the Agrobacterium cells diffuses into the selected region 15. Mark the infiltrated area with a permanent marker. 16. Place the infiltrated plant back in the growth chamber for 48 up to 72 h. 3.1.2 Transient and Stable Transformation of Arabidopsis

The transient transformation of Arabidopsis seedlings often results in lower frequencies of transformed cells when compared to those achieved in tobacco. We tested several conditions to increase transformation efficiency in Arabidopsis and found that seedling age is one of the most critical factors. Based on our experience, it is best to use seedlings not older than 4–5 days after germination. Transient transformation of Arabidopsis seedlings can be performed using various protocols that have been previously published [21– 24]. The protocol published by Li et al. (2009) has been the most successful for our experiments. 1. Sterilize around 200 seeds of A. thaliana in a 1.5 mL eppendorf tube, as follows: (a) Add 1 mL of ethanol 70%, mix, centrifuge at low speed (around 100 rpm) and discard the solution by pipette. (b) Add 1 mL of 20% bleach solution, mix, centrifuge as before and discard the liquid by pipette. (c) Wash at least two times with sterile water then stratify the seeds on plate (LS 0.25 medium) and vernalize at 4  C for at least 48 h. 2. Incubate the plates in the growth chamber.

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3. Prepare the cocultivation media. 4. Three days after the seeds have germinated, inoculate the Agrobacterium (GV3101) carrying the binary vector with the fluorescent marker of interest into LB medium with appropriate antibiotics, incubate at 28  C with shaking at 200 rpm, until the optical density (OD) read at 600 nm (OD600) reach a value which is greater than 1.5. 5. Centrifuge the Agrobacterium culture at low speed 2655  g for 3 min then discard the supernatant. 6. Resuspend the Agrobacterium in 10 mL of wash solution by vortex. Repeat the last two steps one more time. 7. Resuspend the Agrobacterium pellet in 1 mL of wash solution. 8. Add Agrobacterium to 10 mL of cocultivation media to reach a final OD600 of 0.5, and then pour the solution in the plate containing the seedlings. 9. Incubate the plates in the dark for about 24 h. 10. Open the plates remove the media, and wash at least twice with 10 mL of co cultivation media. 11. Seal the plate with micropore 3M and incubate in standard growth conditions. 12. Perform confocal microscopy on cotyledons or root tissue 2–3 days after transformation. Stable Arabidopsis transformation can be performed with the most used method described earlier [25], which is based on floral dipping. To have a high percentage of independent transformants using this method, the health and age of the plants used for the transformation are extremely important factors to consider. Based on our experience, we found that healthy plants that are 4–5 weeks old work best for a successful transformation. 1. Grow four Arabidopsis plants for each 3-½ in. pot (one plant in each corner of the pot) until flowering. 2. Grow Agrobacterium (carrying fluorescent marker of interest) in 250 mL of LB media with antibiotics and incubate at 28  C with shaking at 200 rpm, until OD600 ranging between 0.8 and 1.0 is reached. 3. Using a 500 mL Nalgene centrifuge bottle spin down Agrobacterium at 4225  g for 10 min then discard supernatant. 4. Add 250 mL of LB media supplemented with 5% sucrose (dipping solution) and resuspend. 5. For each 3-½ in. pot to be dipped use 250 mL of dipping solution. 6. Before dipping, add Silwet L-77 into dipping solution to a concentration of 0.05% (125 μL/250 mL) and mix.

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7. Dip flowering stems of plants in Agrobacterium solution for 10–30 s, with gentle agitation. 8. Place Agrobacterium-treated plants under a dome in a dark O/N to maintain high humidity (lay pots on their side). 9. Grow plants in normal growth chamber conditions. 10. Harvest dry seeds. 11. Select for transformants using antibiotic or herbicide selectable marker. 12. Transplant transformants to soil. 3.2 Imaging Leaf Material

For confocal microscopy, the use of oil or water immersion lenses of the highest possible numerical aperture is recommended (e.g., 60, 63 objectives). 1. Within the Agrobacterium-infiltrated zone, cut a square section (approximately 0.5 cm2 area) from tobacco leaf or Arabidopsis cotyledon tissue using a razor blade. 2. Place the excised tissue in a drop of water on a glass slide with the abaxial side oriented toward the objective lens, and cover it with a glass coverslip. 3. Image the sample using a confocal microscope.

3.3 FRET (Fo¨rster Resonance Energy Transfer) Imaging for ER–Endosome Interactions

FRET measurements can be used to characterize the association between the ER-plasma membrane contact sites and endosomes [7]. Here, as example, we describe the FRET imaging of a clathrin heavy chain fused to YFP (CHC-YFP) that labels early endosomes and VAP27-1-CFP, which localizes to the ER and ER-plasma membrane contact sites. Controls for this type of experiments should include combinations of VAP27-1-CFP with cytosolic free YFP and a CHC-YFP coexpressed with a CFP-based ER marker. 1. Incubate samples (Arabidopsis cotyledons or a 0.5 cm2 square section of a tobacco leaf) for 30 min in 25 μM Latrunculin B to depolymerize actin filaments and 10 μM oryzalin to depolymerize microtubules. This will stop any kind of cytoskeletondependent organelle movement [7]. 2. Mount samples on microscope slides and cover them with coverslips using the solution containing Latrunculin B and oryzalin. 3. To set up the confocal for FRET imaging, scan the sample, find and select the region of interest (ROI) using nonphotobleaching and nonsaturating imaging settings. Ensure that the microscope settings prevent signal cross talk between the YFP and CFP channels. 4. Using the line-switching mode of the microscope, capture at least 10 prebleach images using the CFP and YFP channels.

Endoplasmic Reticulum-Endosome Association

31

5. Bleach YFP (acceptor) only for 10 s using high laser power intensity (use laser line 514 nm, power 100%). 6. Collect at least 1 postbleach image of the CFP and YFP channels (postbleaching acquisition). 7. Perform at least 10 FRET imaging acquisitions as detailed above and repeat the entire image acquisition scheme three times for each combination of fluorescent protein fusions, including controls. 8. Calculate the energy transfer efficiency (EFRET) between the paired proteins based on the change in fluorescence intensity of the donor before (last prebleach image) and after photobleaching (first postbleach image) using the formula EFRET ¼ (CFPafter – CFPbefore)/CFPafter  100 [26]. The CFPafter image is the first one obtained after the photobleaching step. 3.4 ER-Endosome Colocalization

Generally, to evaluate the colocalization between two fluorescent markers the Pearson correlation coefficient (PCC), Manders’s overlap coefficient (MOC), and Manders’s colocalization coefficient (MCC) are used [27]. We found that to quantify the degree of colocalization between ER and endosome signals MOC or MCC are the most appropriate correlation coefficient measurements. MOC reaches value equal to 0 only when the two probes are completely reciprocally exclusive. The MCC measures specifically the fraction of one fluorophore that colocalizes with another fluorophore. These coefficients are provided in almost any image analysis software, such as Nikon AR elements, Imaris, Volocity, and ImageJ via the JACoP plugin. MOC or MCC can be calculated using ImageJ and JACoP plugin [28, 29] as follows: 1. Having acquired the colocalization images with two fluorochromes (X1FP and-X2FP) in nonsaturating conditions, select an ROI in the merged image. 2. Open the X1FP channel image and X2FP channel independently in ImageJ via JACoP plugin. 3. Select automatically the thresholds for X1FP and-X2FP to remove background and signal noise, then calculate the colocalization levels [28, 29] (see Note 5).

4

Notes 1. Use low OD600 if transient transformation is performed (e.g., 0.005-0.01 OD600). 2. For additional markers of the ER and other secretory organelles, see refs. 9, 14.

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3. Choose fluorescent protein combinations that do not favor fluorescence signal cross talk. 4. In case of very low transformation efficiency, we recommend infiltrating Agrobacterium at different OD600 starting from 0.05 to 0.5. 5. It is possible to measure colocalization over time by acquiring time-lapse images with a narrow temporal interval between frames. The colocalization analysis for ER-endosomes should be performed manually on the ROI of interest over the desired time frame. References 1. Stefano G et al (2015) ER network homeostasis is critical for plant endosome streaming and endocytosis. Cell Discov 1:15033 2. Stefano G, Hawes C, Brandizzi F (2014) ER the key to the highway. Curr Opin Plant Biol 22:30–38 3. English AR, Voeltz GK (2013) Endoplasmic reticulum structure and interconnections with other organelles. Cold Spring Harb Perspect Biol 5:a013227 4. Friedman JR et al (2013) Endoplasmic reticulum-endosome contact increases as endosomes traffic and mature. Mol Biol Cell 24:1030–1040 5. Staehelin LA (1997) The plant ER: a dynamic organelle composed of a large number of discrete functional domains. Plant J 11:1151–1165 6. Wang P et al (2014) The plant cytoskeleton, NET3C, and VAP27 mediate the link between the plasma membrane and endoplasmic reticulum. Curr Biol 24:1397–1405 7. Stefano G et al (2018) Plant endocytosis requires the ER membrane-anchored proteins VAP27-1 and VAP27-3. Cell Rep 23:2299–2307 8. Rowland AA et al (2014) ER contact sites define the position and timing of endosome fission. Cell 159:1027–1041 9. Nelson BK, Cai X, Nebenfuhr A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51:1126–1136 10. Brandizzi F et al (2003) ER quality control can lead to retrograde transport from the ER lumen to the cytosol and the nucleoplasm in plants. Plant J 34:269–281 11. Irons SL, Evans DE, Brandizzi F (2003) The first 238 amino acids of the human lamin B

receptor are targeted to the nuclear envelope in plants. J Exp Bot 54:943–950 12. Sparkes I et al (2010) Five Arabidopsis reticulon isoforms share endoplasmic reticulum location, topology, and membrane-shaping properties. Plant Cell 22:1333–1343 13. Tolley N et al (2010) Transmembrane domain length is responsible for the ability of a plant reticulon to shape endoplasmic reticulum tubules in vivo. Plant J 64:411–418 14. Geldner N et al (2009) Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J 59:169–178 15. Stefano G, Renna L, Brandizzi F (2014) The endoplasmic reticulum exerts control over organelle streaming during cell expansion. J Cell Sci 127:947–953 16. Viotti C et al (2010) Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle. Plant Cell 22:1344–1357 17. Tse YC et al (2004) Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells. Plant Cell 16:672–693 18. De Marcos Lousa C, Gershlick DC, Denecke J (2012) Mechanisms and concepts paving the way towards a complete transport cycle of plant vacuolar sorting receptors. Plant Cell 24:1714–1732 19. Sparkes IA et al (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1:2019–2025 20. Batoko H et al (2000) A rab1 GTPase is required for transport between the endoplasmic reticulum and golgi apparatus and for

Endoplasmic Reticulum-Endosome Association normal golgi movement in plants. Plant Cell 12:2201–2218 21. Campanoni P et al (2007) A generalized method for transfecting root epidermis uncovers endosomal dynamics in Arabidopsis root hairs. Plant J 51:322–330 22. Li JF, Nebenfuhr A (2010) FAST technique for Agrobacterium-mediated transient gene expression in seedlings of Arabidopsis and other plant species. Cold Spring Harbor Protoc 2010. https://doi.org/10.1101/pdb.prot5428. 23. Li JF et al (2009) The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods 5:6 24. Marion J et al (2008) Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings. Plant J 56:169–179

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25. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743 26. Bleckmann A et al (2010) Stem cell signaling in Arabidopsis requires CRN to localize CLV2 to the plasma membrane. Plant Physiol 152:166–176 27. Dunn KW, Kamocka MM, McDonald JH (2011) A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol 300:C723–C742 28. Manders EMM, Verbeek FJ, Aten JA (1993) Measurement of colocalization of objects in dual-color confocal images. J Microsc 169:375–382 29. Bolte S, Cordelieres FP (2006) A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224:213–232

Chapter 4 Degradation of Abscisic Acid Receptors Through the Endosomal Pathway Borja Belda-Palazo´n and Pedro L. Rodriguez Abstract Turnover of membrane proteins or soluble proteins associated to plasma membrane involves clathrinmediated endocytosis (CME), endosomal trafficking, and vacuolar degradation. Thus, endocytic and endosomal trafficking regulate numerous physiological processes, including mineral transport, hormone signaling, and pathogen response. Abscisic acid (ABA) signaling is triggered upon ABA perception by PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR), which are soluble proteins that can associate to membrane by interaction with members of the C2-domain ABA-related (CAR) protein family and the RING finger of seed longevity (RSL1) E3 ubiquitin ligase. Half-life of PYR/PYL/RCAR ABA receptors is regulated by ubiquitination and degradation in different subcellular compartments. In particular, pharmacological, genetic, and cell biology approaches have been used to study the different steps that encompass from CME to receptor degradation in the vacuole. In this chapter, we will focus on (1) coimmunoprecipitation (co-IP) assays of clathrin heavy chain (CHC) subunits together with HA-tagged PYL4 ABA receptor and (2) analysis of PYL4 delivery to the vacuole using the TMD23-Ub marker. Key words Absicisic acid receptors, PYR/PYL/RCAR, CHC, RSL1, Clathrin-mediated endocytosis, Endosomal trafficking, Vacuolar degradation, ESCRT, Vacuolar marker

1

Introduction Ubiquitination of membrane proteins via K63-linked chains is a signal that triggers endocytosis and cargo sorting through the endosomal sorting complex required for transport (ESCRT) followed by vacuolar degradation, and therefore controls turnover of different integral membrane proteins, such as BOR1, PIN2, BRI1, IRT1, PHT1, FLS2, or proteins transiently associated to plasma membrane as the PYR/PYL/RCAR ABA receptors [1–3]. Cargo internalization into intralumenal vesicles of multivesicular bodies (MVBs) is catalyzed by ESCRT complexes, which deform the endosomal membrane and promote endosomal lumen-directed scission, resulting in cargo concentration and engulfment inside intralumenal vesicles [2]. Specific deubiquitinases carry out

Marisa S. Otegui (ed.), Plant Endosomes: Methods and Protocols, Methods in Molecular Biology, vol. 2177, https://doi.org/10.1007/978-1-0716-0767-1_4, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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Borja Belda-Palazo´n and Pedro L. Rodriguez

deubiquitination of cargo proteins prior to MVB lumenal sequestration [4]. Finally, the fusion of MVBs to the vacuole leads to cargo degradation [2]. ABA receptors are soluble proteins that can be associated to plasma membrane via auxiliary proteins [5, 6]. In 2014, it was reported the ubiquitination of ABA receptors at plasma membrane by the RING between RING fingers (RBR)-type E3 ubiquitin (Ub) ligase RSL1, suggesting that ubiquitinated receptors could follow internalization via the endocytic route [7]. In 2016, ABA receptors were reported to follow clathrin-mediated endocytosis (CME), endosomal trafficking, sorting through the ESCRT machinery and vacuolar degradation [8, 9]. This was demonstrated through different studies boosted by the finding that FYVE DOMAIN-CONTAINING PROTEIN 1 (FYVE1)/FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1) interacts with and recruits PYL4 to endosomal compartments [8]. FYVE1/FREE1 and VPS23A proteins interact in the ESCRT-I complex and VPS23A also recognizes ABA receptors for endosomal degradation [9, 10]. Recently, ALG2-2INTERACTING PROTEIN (ALIX), an ESCRT-III associated protein, was reported to bind ABA receptors in late endosomes and regulate their turnover [3]. Thus, ABA receptors are trafficked from the plasma membrane to the endosomal/vacuolar degradation pathway, which depends on FYVE1/VPS23A/ALIX and receptor ubiquitination by RSL1. Complexes formed by ABA receptors and the E3 Ub ligase RSL1 have been detected both in plasma membrane and vacuole, suggesting that RSL1 travels with the ubiquitinated receptor until their delivery to the vacuole [8]. Initial evidence for the endocytic internalization of ABA receptors came from proteomic studies aimed to identify interacting proteins of HA-tagged PYR1, PYL4, and PYL8 [8]. Protein extracts were immunoprecipitated with anti-HA-coated magnetic beads and analyzed by LC-MS/MS. As a result, we found components of the clathrin-dependent coating and scission machinery that coimmunoprecipitated with HA-tagged ABA receptors [8]. Direct evidence for CME of ABA receptors was obtained by coimmunoprecipitation (co-IP) assays of CHC subunits together with HA-PYL4, which was abolished by the TyrA23 inhibitor of CME [8]. In this chapter we provide detailed protocols for the analysis of CME of ABA receptors in Arabidopsis thaliana as well as the delivery of the PYL4 receptor-RSL1 complex to the vacuole of Nicotiana benthamiana leaf cells (Fig. 1). This latter assay takes advantage of a fluorescent ubiquitin-tagged plasma membrane reporter, RFP-TMD23-Ub, which additionally localizes to endosomes and the lumen of the lytic vacuole [11]. In addition, we present a protocol for testing interactions between RSL1 and PYL4 by bimolecular fluorescence complementation (BiFC) in N. benthamiana leaf cells.

Turnover of ABA Receptors

Clathrinmediated endocytosis

PYL4 Ub RSL1

Endocytic vesicle RSL1

37

Plasma membrane

TyrA23

1

PYL4 Ub

Endosome recycling

RSL1

PYL4

Vacuole

TGN/EE

2 Ub

DUBs

Degradation PY

Endosomal sorting

L1 RS

RS

ESC RT

L1

MVB/PVC

Ub

L4

PY

PYL4 RSL1

L4

L1

RS

Ub

L4

PY

L1

L4

RS

PY

L4

PY L1

RS

ES

L1

RS

3A s2 I Vp FYVE Ub L4 PY

CR

T

Fig. 1 CME of ABA receptors and delivery to vacuole for degradation. The E3 Ub ligase RSL1 ubiquitinates PYL4 at the plasma membrane and promotes CME of PYL4. FYVE1/FREE1 and VPS23A proteins interact and recognize ubiquitinated PYL4 to internalize the PYL4-RSL1 complex into intralumenal vesicles of multivesicular bodies (MVBs), which leads to vacuolar degradation. This chapter focuses on protocols for the analysis of CME of ABA receptors (1) and the delivery of PYL4-RSL1 complexes into the lumen of the vacuole (2). Trans-Golgi network (TGN), early endosomes (EE), deubiquitinating enzymes (DUBs), prevacuolar compartment (PVC). (Figure was adapted from [8]. Copyright American Society of Plant Biologists)

2

Materials Unless otherwise indicated, all solutions should be prepared with sterile ultrapure water (Milli-Q, 18 megOhm); store all reagents at room temperature.

2.1 Analysis of CME of ABA Receptors by co-IP Assays

1. Homozygous Arabidopsis transgenic seeds.

2.1.1 In Vitro Culture Media and Plant Treatments

3. mL glass Erlenmeyer flasks (base diameter 64 mm).

thaliana

CaMV35S:HA-PYL4

2. 120  120 mm square petri dishes. 4. Controlled-environment growth chamber. 5. pH meter. 6. Autoclave. 7. Forceps.

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Borja Belda-Palazo´n and Pedro L. Rodriguez

8. 1 Murashige and Skoog (1MS) medium plates supplemented with 1% sucrose. To prepare 1 L of solid 1MS + 1% sucrose, dissolve 4 g of Murashige and Skoog including vitamins and MES buffer and 10 g of sucrose in 700 mL of water. Adjust pH to 5.7 with a 1 M KOH solution by using a pH meter, and bring the volume to 1 L with water. Add 10 g of Phytoagar and sterilize the medium at 120  C, 1 atm, for 20 min by autoclaving. In a laminar flow hood, pour 50 mL of medium into 120  120 mm square petri dishes and let the medium solidify. Store the plates at 4  C before use. 9. Glass Erlenmeyer flasks containing 3 mL of sterile 1MS + 1% sucrose. To prepare 1 L of liquid 1MS + 1% sucrose, dissolve 4 g of Murashige and Skoog including vitamins and MES buffer and 10 g of sucrose in 700 mL of water. Adjust pH to 5.7 with a 1 M KOH solution by using a pH meter, and bring the volume to 1 L with water. Add 3 mL of liquid 1MS + 1% sucrose medium into 100 mL glass Erlenmeyer flasks. Sterilize them at 120  C, 1 atm of pressure, for 20 min in an autoclave. Store the flasks at room temperature before use. 10. 50 mM Tyrphostin 23 (TyrA23) stock solution: add 537 μL of DMSO to 5 mg of TyrA23 (MW¼186.17) and mix well. Store at 20  C (see Note 1). 11. 5 mM MG132 stock solution: add 10.5 mL of DMSO to 25 mg of MG132 (MW ¼ 475.62 Da), mix well and store at 20  C. 12. Mortar. 13. 1.5 mL polypropylene tubes and tube adapters. 2.1.2 Protein Extraction

1. Lysis Buffer: 50 mM Tris–HCl pH 8.0, 150 mM NaCl, 3 mM dithiothreitol (DTT), 0.5% Triton X-100, anti-proteases cocktail (Complete mini EDTA free, Roche), 50 μM MG132, 10 nM ubiquitin aldehyde and 10 mM N-ethylmaleimide (NEM). To prepare 10 mL of Lysis Buffer, mix 500 μL of 1 M Tris–HCl pH 8.0, 300 μL of 5 M NaCl, 30 μL of 1 M DTT, 50 μL of 100% Triton X-100, 1 tablet of Complete mini EDTA free (Roche), 100 μL of 5 mM MG132, 1 μL of 100 μM ubiquitin aldehyde, and 250 μL of 400 mM NEM. Bring the volume to 10 mL with water, dissolve well and store at 4  C. In the case of the TyrA23-treated sample the Lysis Buffer should contain 50 μM TyrA23. Add 10 μL of 50 mM TyrA23 to 10 mL Lysis Buffer. 5Laemmli Buffer: 0.25 M Tris–HCl pH 6.8, 10% SDS, 0.5 M DTT, 30% (w/v) sucrose, 0.5% (w/v) bromophenol blue (BPB). To prepare 1 mL of 5LB, mix 250 μL of 1 M Tris–HCl pH 6.8, 300 mg of sucrose, 5 mg of BPB, 76 mg of DTT (MW ¼ 154.25), and 100 mg of SDS.

Turnover of ABA Receptors

39

Bring the volume to 1 mL with water and dissolve well. Store at 20  C. 2. Unrefrigerated benchtop centrifuge. 3. Refrigerated centrifuge with fixed-angle rotor for 1.5 mL tubes. 4. Thermoblock for 1.5 mL tubes. 2.1.3 Protein Complex co-IP and Analysis by Western Blot (WB)

1. 1 μg/μL anti-clathrin heavy-chain 1,2 (CHC1,2) antibody (e.g., AS10690 from Agrisera). 2. 30 mg/mL Dynabeads™ Protein G. 3. DynaMag-2™ magnetic rack. 4. End-over-end mixer. 5. Mini-gel and electrophoresis system. 6. Mini-gel wet protein transfer system 7. Polyvinylidene difluoride (PVDF) membrane. 8. Chemoluminescence detection substrate 9. 25 U/mL of anti-HA high affinity antibodies conjugated to horseradish peroxidase (HRP). 10. Anti-IgG(rabbit)-HRP antibody.

2.2 Analysis of the Delivery of PYL4-RSL1 Complexes into the Lumen of the Vacuole

1. Agrobacterium tumefaciens strains containing the appropriate vectors (Table 1). 2. Luria-Bertani medium (LB): 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl. For 1 L dissolve 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 700 mL of water. Bring

Table 1 A. tumefaciens strains used in this work and the corresponding proteins expressed from each binary vector A. tumefaciens strain

Protein expression/vector

Reference

Bacterial selection

YFPN-RSL1 (pYFPN43)

Bueso et al. (2014)

Kanamycin (50 μg/mL) Rifampicin (25 μg/mL)

C58C1/pGV2260

YFPC-PYL4 (pYFPC43)

Bueso et al. (2014)

Kanamycin (50 μg/mL) Rifampicin (25 μg/mL)

C58C1/pGV2260

CAR1-YFPN (pSPYNE-35SGW)

Diaz et al. (2016)

Kanamycin (50 μg/mL) Rifampicin (25 μg/mL)

C58C1/pGV2260

RFP-TM23-Ub (pPP11)

Scheuring et al. (2012)

Kanamycin (50 μg/mL) Rifampicin (25 μg/mL)

C58C1/pSoup

Kanamycin (50 μg/mL) Rifampicin (25 μg/mL)

C58C1/pCH32

p19 (pCB301)

Belda-Palazon et al. (2012)

40

Borja Belda-Palazo´n and Pedro L. Rodriguez

the volume to 1 L with water, autoclave it for 20 min at 120  C and 1 atm of pressure and stored at room temperature. To supplement 5 mL of LB with 50 μg/mL kanamycin and 25 μg/mL rifampicin add 5 μL of 50 mg/mL kanamycin and 5 μL of 25 mg/mL rifampicin. 3. 50 mL polypropylene tubes and tube adapters. 4. Orbital shaker. 5. 1.5 mL methacrylate cuvettes. 6. Cell density meter. 7. Refrigerated centrifuge with a fixed-angle rotor for 50 mL tubes. 8. MES (2-(N-morpholino)ethanesulfonic acid) Buffer: 10 mM MgCl2, 10 mM MES pH 5.6, 200 μM acetosyringone. To prepare 100 mL of MES Buffer dissolve 0.095 g of MgCl2 (MW 95.21) and 0.195 g of MES (MW 195.24) in 80 mL of water. Adjust the pH to 5.6 with KOH, add 100 μL of 200 mM acetosyringone, and bring the volume to 100 mL with water. Prepare it fresh just before resuspending the A. tumefaciens strains. 9. 1 mL needleless syringes. 10. Four-week-old N. benthamiana plants. 11. Microscope slides and glass coverslips. 12. Confocal laser scanning microscope. 13. ImageJ software ((http://rsb.info.gov/ij/) and its PSC (Pearson-Spearman Correlation) Colocalization plug-in (https://nottingham.ac.uk/research/groups/cvl/software/ psccolocalisation.aspx). 2.3

Stock Solutions

Prepare the following stock solutions in order to facilitate the elaboration of all the buffers and reagents depicted in this chapter. Unless otherwise indicated all the solutions should be autoclaved for 20 min at 120  C and 1 atm of pressure and stored at room temperature: 1. 1 M KOH (100 mL): 5.61 g of KOH (MW 56.11) dissolved in 100 mL of water. 2. 1 M Tris–HCl pH 6.8 or 8.0 (500 mL): 60.57 g of Tris base (MW ¼ 121.14) in 500 mL of water, pH to 6.8 or 8.0 adjusted with HCl. 3. 5 M NaCl (500 mL): 146.10 g of NaCl (MW ¼ 58.44) in 500 mL of water. 4. 1 M DTT (10 mL): 1.54 g of DTT (MW 154.25) in 10 mL of water. Do not autoclave. Store at 20  C.

Turnover of ABA Receptors

41

5. 100 μM ubiquitin aldehyde stock solution: 50 μg of ubiquitin aldehyde (MW 8.5) in 58.8 μL of water. Mix well and store at 20  C. Do not autoclave. 6. 400 mM NEM solution: 12.5 mg of NEM (MW ¼ 125.13) in 250 μL of absolute ethanol. Prepare it fresh before adding to the Lysis Buffer. Do not autoclave.

3

Methods

3.1 Analysis of CME of ABA Receptors by co-IP Assays

In this protocol we describe the co-IP of HA-PYL4 with CHC1,2 proteins, which can be inhibited by TyrA23 when endocytosis is impaired (Fig. 2). As an example, we use A. thaliana CaMV35S: HA-PYL4 transgenic seedlings [8, 12]. Other HA-tagged receptor lines could be used for the analysis of additional ABA receptors [9].

3.1.1 Preparation of the Biological Material

1. Sow 50 surface-sterilized seeds on solid 1MS plates supplemented with 1% sucrose. Conduct stratification in the dark at 4  C for 3 days and then, grow the seedlings vertically for 4 days (see Note 2). 2. Transfer carefully 10 seedlings using sterile forceps to each glass Erlenmeyer flask containing 3 mL of liquid 1MS + 1% sucrose and grow them for 10 additional days (see Note 3). 3. Then, remove medium and add 3 mL of fresh liquid 1MS + 1% sucrose. Next day treat the seedlings with 50 μM MG132 and mock solution or 50 μM TyrA23, by adding directly the compounds into the fresh medium 2 h after the onset of the daily light period. For the mock sample lacking TyrA23, add 30 μL of 5 mM MG132 and 3 μL of DMSO. For the TyrA23

35S:HA-PYL4 kDa 35-

250-

INPUT -

+

IP α-CHC1,2 -

+

50 μM TyrA23 α-HA

α-CHC1,2

Fig. 2 Co-IP of HA-PYL4 with CHC1,2 is inhibited by TyrA23. Anti-CHC1,2 antibodies were used to immunoprecipitate CHC1,2 as described in Subheading 3. Arabidopsis protein extracts were prepared from plants that were treated with 50 mM MG132 or 50 mM MG132 + 50 mM TyrA23 for 6 h. The immunoprecipitate was probed with anti-CHC1,2 and rat anti-HA-HRP antibodies to detect HA-PYL4.

42

Borja Belda-Palazo´n and Pedro L. Rodriguez

sample, add 30 μL of 5 mM MG132 and 3 μL of 50 mM TyrA23. Incubate seedlings for 6 h. 4. Collect the seedlings carefully using forceps and eliminate the excess of liquid medium with filter paper. Ground the material with liquid N2 using mortar and pestle until reduced to fine powder. Store the samples in 1.5 mL tubes at 80  C. 3.1.2 Total Protein Extraction and Quantification

1. Add 2 volumes (700 μL) of chilled Lysis Buffer to 350 mg of each of the powdered samples and mix gently by vortexing. Incubate the homogenates for 30 min on ice, vortexing every 5 min. 2. Clear the homogenates by centrifugation at 12,000  g for 15 min at 4  C. Collect separately the supernatants containing the total protein extracts into new 1.5 mL tubes and keep them on ice. 3. Quantify protein content in each extract using the Bradford assay [13] (see Note 4). 4. Take 100 μL of each extract and mix it with 25 μL of 5Laemmli buffer. Boil the extracts at 95  C for 10 min, cool them on ice, and centrifuge the samples (inputs) at 12,000  g for 5 min. Store them at 80  C before analysis.

3.1.3 Performing the co-IPs

The same amount of total protein should be used for each sample (mock- and TyrA23-treated samples). 1. For each sample, mix 4 mg (approximately 700 μL) of total protein with 5 μL of 1 μg/μL anti-CHC1,2 antibody and incubate the samples for 2 h in an end-over-end mixer at 4  C. 2. Add to each sample 50 μL of Dynabeads™ Protein G previously washed 3 times with Lysis Buffer by using the DynaMag2™ magnetic rack, and incubate for at least 12 h in the endover-end mixer at 4  C. 3. Using the DynaMag-2™ magnetic rack, separate the proteins bound to the magnetic beads from the not bound proteins present in the supernatant. 4. Wash the magnetic beads 4 times with 500 μL of Lysis Buffer in the end-over-end mixer at 4  C for 5 min and discard the Lysis Buffer using the DynaMag-2™ magnetic rack. 5. Elute coimmunoprecipitates from the magnetic beads by adding 60 μL of prewarmed 2Laemmli buffer to the samples (see Note 5) and incubating at 95  C for 5 min. Cool on ice. 6. Collect the eluted coimmunoprecipitates by using the DynaMag-2™ magnetic rack. Store samples at 80  C before analysis.

Turnover of ABA Receptors 3.1.4 Analysis of the Inputs and co-IPs by WB

43

1. Run the inputs and the co-IPs in an 8% SDS-PAGE. Table 2 shows the amount of each sample loaded in the SDS-PAGE for the appropriate immunodetection of the target proteins. 2. Transfer the proteins in wet conditions from the SDS-PAGE gel to a PVDF membrane. 3. Immunodetect the proteins of interest by chemiluminescence. Table 3 shows the antibodies and the conditions used for this case (see Note 6). 4. After immunodetection, check that the treatment with 50 μM TyrA23 has no effect on the input levels of CHC1,2 and HA-PYL4 proteins in comparison with the control sample (Fig. 2, left panel). Confirm that, for an equal immunoprecipitated quantity of CHC1,2 the co-IP of HA-PYL4 is abolished in the TyrA23-treated sample due to inhibition of CME (Fig. 2, right panel).

Table 2 Total protein and volume of immunoprecipitated sample that are loaded for immunodetection of CHC and HA-PYL4 proteins Sample

Immunodetection

Amount of protein

INPUT

CHC1,2 HA-PYL4

50 μg 15 μg

co-IP

CHC1,2 HA-PYL4

2 μL out of 60 μL 15 μL out of 60 μL

Table 3 Antibodies used in this work Primary antibody

Immunodetection Reference

Host

Working Incubation dilution

Rabbit

2 μg/mL

Anti-clathrin heavyCHC1,2 chain 1,2 (CHC1,2)

Agrisera AS10690

Anti-HA-HRP, High Affinity (3F10)

HA

Roche Cat. Rat No. 12013819001

Secondary antibody

Immunodetection Reference

Host

Amersham ECL antirabbit IgG HRP-linked whole Ab

IgG rabbit

Donkey 1:5000

GE Healthcare Life Sciences, NA9341ML

Overnight at 4 C

12.5 mU/ 1 h at room mL temperature Working Incubation dilution 1 h at room temperature

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Borja Belda-Palazo´n and Pedro L. Rodriguez

Fig. 3 Colocalization of reconstituted YFP signal from PYL4-RSL1 complexes with RFP-TMD23-Ub in the lumen of the vacuole. Confocal three-dimensional projection of a full z-series of confocal images obtained from transiently transformed N. benthamiana leaf cells coexpressing either YFPN-RSL1 or CAR1-YFPN and YFPCPYL4, in both cases plus RFP-TMD23-Ub. The RFP-TMD23-Ub marker localizes to the vacuolar lumen and reveals the presence of PYL4-RSL1 complexes in the central vacuole (Rp ¼ 0.60, Rs ¼ 0.63), whereas CAR1PYL4 complexes were not found in the vacuole when coexpressed with RFP-TMD23-Ub (Rp ¼ 0.23, Rs ¼ 0.04). Bars ¼ 30 μm 3.2 Analysis of the Delivery of PYL4-RSL1 Complexes into the Lumen of the Vacuole by BiFC

In this protocol we analyze the colocalization of reconstituted YFP signal coming from the PYL4-RSL1 BiFC pair and the vacuolar marker RFP-TMD23-Ub (Fig. 3).

3.2.1 Agroinfiltration of N. benthamiana Leaves

1. Obtain saturated A. tumefaciens cultures (3 mL) containing the appropriate vector as shown in Table 1. CAR1-YFPN serves as a negative control in this experiment. 2. In a 50 mL tube, inoculate 25 μL of each saturated culture into 5 mL of LB containing the appropriate antibiotics and incubate at 28  C in an orbital shaker (180 rpm) for 14 h. 3. Perform 1:10 dilutions for each culture by mixing 100 μL of each culture with 900 μL of LB.

Turnover of ABA Receptors

45

4. Pipette the dilutions into 1.5 mL methacrylate cuvettes and measure the optical density at 600 nm (OD600nm) of each culture using a Cell Density Meter or spectrophotometer. Multiply by 10 the value of OD600nm to obtain the real cellular density of each bacterial culture (see Note 7). 5. Centrifuge the cultures at 3000  g for 15 min and discard the supernatant. 6. Resuspend the cells in MES buffer to an OD600nm of 1.0 (see Note 8), and incubate for at least 3–4 h in the rocking shaker. 7. Combine cultures of the different A. tumefaciens strains as indicated in the Table 4, and infiltrate each culture mixture into N. benthamiana leaves by applying a needleless syringe onto the abaxial side. 8. Incubate the infiltrated plants in a controlled-environment growth chamber under long day photoperiod conditions for 72 h. 1. Incubate the infiltrated plants in darkness for 4 h in order to promote stabilization of the fluorescently tagged vacuolar marker and receptor-RSL1/CAR1 complexes.

3.2.2 Colocalization Analysis

2. Remove a piece 1.5 cm in diameter of infiltrated leaf using a hole puncher, and mount it with water onto a microscope slide with the abaxial side up and the adaxial side down. Cover the leaf samples with coverslips. 3. Image the RFP and YFP (YFPCPYL4-YFPNRSL1 BiFC) [14] signals with a confocal laser scanning microscopy (see Note 9). Table 5 describes the conditions used for imaging. Table 4 Composition of the A. tumefaciens mixtures (A and B) infiltrated into N. benthamiana leaves A. tumefaciens encoding (Final OD600nm ¼ 0.25 each) Mixture

YFPN-RSL1 (mL)

CAR1-YFPN (mL)

YFPC-PYL4 (mL)

RFP-TM23-Ub (mL)

p19 (mL)

A

1



1

1

1

B



1

1

1

1

Table 5 Confocal imaging conditions for the visualization of YFP and RFP fluorescent proteins Fluorescent protein

Excitation laser (nm)

MBS ¼ dichroic

Spectral detection (nm)

YFP

Argon 488

488

495–550

RFP

Argon 561

488/561

580–670

MBS main beam splitter

46

Borja Belda-Palazo´n and Pedro L. Rodriguez

4. Collect a full z-series of confocal images for a total thickness of approximately 50 μm, compiling 20 slices with an interval of 2.50 μm, and render them as a three-dimensional projection. 5. Analyze the colocalization of the vacuolar marker RFP-TMD23-Ub and the YFP signal generated by the PYL4RSL1 interaction, by the ImageJ software and its PSC colocalization plug-in. 6. Obtain the Pearson’s and Spearman’s correlation values (Rp and RS respectively) [15]. Values in the range +0.4 to 1 indicate colocalization, whereas lower values or negative values indicate lack of colocalization. 7. In this example, whereas PYL4-CAR1 complexes are not targeted to the vacuole, colocalization of PYL4-RSL1 complexes with the vacuolar marker RFP-TMD23-Ub indicates delivery of these complexes to the vacuole (Fig. 3)

4

Notes 1. TyrA23 has been used as an inhibitor of CME and therefore mock- and TyrA23-treated samples were obtained to analyze the endocytosis of PYL4 [8, 12]. As a result, we observed TyrA23-mediated inhibition of CME; however, TyrA23 has been described as a protonophore that inhibits endocytosis through nonspecific cytoplasmic acidification [16]. To avoid the undesirable effects, potent specific inhibitors of endocytosis named endosidin9 (ES9) and a chemically improved derivative (ES9-17) have been developed [17], and should replace TyrA23 in future experiments. 2. Grow seedlings in a controlled-environment growth chamber at 22  C under long day photoperiod conditions (16-h-light/ 8-h-dark photoperiod) at 80–100 μmol m2s1 of light intensity. 3. Do not let the medium dry. Check the medium every 4–5 days to ensure actively growing seedlings. Replenish with 3 mL of fresh 1MS + 1% sucrose medium if necessary. 4. For protein quantification, we recommend the Bio-Rad Protein assay following manufacturer’s instructions. 5. To prepare 1 mL of 2Laemmli buffer dilute 400 μL of 5Laemmli buffer with 600 μL of water. 6. We use rabbit anti-CHC1,2 antibodies for IP and immunoblot detection of the 193 kDa CHC1,2 band, and rat HRP-conjugated anti-HA antibodies for detecting HA-PYL4. Since HRP is conjugated directly to the primary rat antibody, we avoid using secondary anti-rabbit HRP-conjugated

Turnover of ABA Receptors

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antibodies that would recognize rabbit IgGs used for IP and therefore would mask the HA-PYL4 band. If necessary, use immunodetection reagents that only recognize native antibodies as for example VeriBlot Detection Reagent (HRP). 7. The growth of the A. tumefaciens strains should reach an OD600nm of around 2.0. 8. For an A. tumefaciens culture of 5 mL and OD600nm¼2.0, resuspend the bacterial pellet with 10 mL of MES Buffer in order to bring the OD600nm to 1.0. 9. In this example, confocal imaging was performed using a C-Apochromat 403/1.20 W corrective water immersion objective lens. For multicolor detection of two fluorescent proteins, we perform sequential channel acquisition mode. Pinholes were adjusted to 1 air unit for each wavelength.

Acknowledgments We acknowledge Dr. Peter Pimpl for the vacuolar marker RFP-TMD23-Ub. Work in Dr. Rodriguez’s laboratory was supported by the Ministerio de Ciencia, Innovacion y Universidades, Fondo Europeo de Desarrollo Regional and Consejo Superior de Investigaciones Cientificas (grant BIO2017-82503-R to P.L.R.). B.B-P. was funded by Programa VALi+d GVA APOSTD/2017/ 039. References 1. Paez Valencia J, Goodman K, Otegui MS (2016) Endocytosis and endosomal trafficking in plants. Annu Rev Plant Biol 67:309–335 2. Gao C et al (2017) Plant ESCRT complexes: moving beyond endosomal sorting. Trends Plant Sci 22:986–998 3. Garcı´a-Leo´n M et al (2019) Arabidopsis ALIX regulates stomatal aperture and turnover of abscisic acid receptors. Plant Cell 31:2411–2429 4. Kalinowska K et al (2015) Arabidopsis ALIX is required for the endosomal localization of the deubiquitinating enzyme AMSH3. Proc Natl Acad Sci U S A 112:E5543–E5551 5. Rodriguez L et al (2014) C2-domain abscisic acid-related proteins mediate the interaction of PYR/PYL/RCAR abscisic acid receptors with the plasma membrane and regulate abscisic acid sensitivity in Arabidopsis. Plant Cell 26:4802–4820 6. Diaz M et al (2016) Calcium-dependent oligomerization of CAR proteins at cell membrane

modulates ABA signaling. Proc Natl Acad Sci U S A 113:E396–E405 7. Bueso E et al (2014) The single-subunit RING-type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling. Plant J 80:1057–1071 8. Belda-Palazon B et al (2016) FYVE1/FREE1 Interacts with the PYL4 ABA receptor and mediates its delivery to the vacuolar degradation pathway. Plant Cell 28:2291–2311 9. Yu F et al (2016) ESCRT-I component VPS23A affects ABA signaling by recognizing ABA receptors for endosomal degradation. Mol Plant 9:1570–1582 10. Shen J et al (2016) AtBRO1 functions in ESCRT-I complex to regulate multivesicular body protein sorting. Mol Plant 9:760–763 11. Scheuring D et al (2012) Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway. BMC Plant Biol 12:164

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12. Dhonukshe P et al (2007) Clathrin-mediated constitutive endocytosis of PIN auxin efflux carriers in Arabidopsis. Curr Biol 17:520–527 13. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 14. Belda-Palazo´n B et al (2012) Aminopropyltransferases involved in polyamine biosynthesis localize preferentially in the nucleus of plant cells. PLoS One 7:e46907

15. French AP et al (2008) Colocalization of fluorescent markers in confocal microscope images of plant cells. Nat Protoc 3:619–628 16. Dejonghe W et al (2016) Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification. Nat Commun 7:11710 17. Dejonghe W et al (2019) Disruption of endocytosis through chemical inhibition of clathrin heavy chain function. Nat Chem Biol 15:641–649

Chapter 5 Biochemical and Imaging Analysis of ALIX Function in Endosomal Trafficking of Arabidopsis Protein Cargoes Marta Garcı´a-Leo´n and Vicente Rubio Abstract ALIX/Bro1 proteins are conserved in eukaryotes where they enable targeted trafficking of membraneassociated proteins through the late endosome route to the vacuole. For this, ALIX/Bro1 proteins associate with the endosomal sorting complex required for transport (ESCRT) machinery acting as ubiquitin receptors that recognize and sort protein cargoes by binding to ubiquitin–cargo conjugates. However, recent findings show direct interaction of ALIX and protein cargoes, pointing to the existence of different mechanisms for specific target recognition by ALIX. The catalogue of proteins that interact with the Arabidopsis homologue of ALIX is increasing, including both protein cargoes and regulatory proteins that mediate or modulate ALIX function. In this context, we describe a toolkit of techniques to analyze the effect of ALIX function in the endosomal trafficking of specific cargoes, which could be easily extended to other components of the plant ESCRT machinery. Key words ALIX , Late endosome route, Multivesicular body (MVB), ESCRT , Protein cargoes, Protein trafficking, Bimolecular fluorescent complementation (BiFC), Confocal imaging, Microsomal fractionation, Arabidopsis

1

Introduction ALG-2 INTERACTING PROTEIN-X (ALIX)/BCK1 (bypass of C kinase)-like resistance to osmotic shock (Bro1) are evolutionary conserved multifunctional proteins that mediate protein cargo trafficking through the endosomal system in eukaryotes [1]. In Arabidopsis thaliana, among different functions, ALIX (also termed AtBRO1) is critical to maintain phosphate homeostasis by ensuring targeted destabilization at the vacuole of PHT1 high affinity phosphate transporters under phosphate sufficient conditions. For this, ALIX associates with the ESCRT-III complex and enables internalization of PHT1 proteins into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs; also known as late endosomes or prevacuolar compartments) and their release in the vacuole lumen to be degraded [2]. This function is coordinated with the activity of

Marisa S. Otegui (ed.), Plant Endosomes: Methods and Protocols, Methods in Molecular Biology, vol. 2177, https://doi.org/10.1007/978-1-0716-0767-1_5, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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AMSH deubiquitinases that remove ubiquitin from numerous protein cargoes during formation of ILVs [3]. According to a general role for ALIX in cargo trafficking through the MVB pathway, ALIX mediates trafficking of additional membrane proteins, including the brassinosteroid receptor BRI1, although using different types of vesicles [2]. Our latest findings show that ALIX also mediates sorting of membrane-associated soluble cargoes, as shown for abscisic acid (ABA) receptors of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR) family [4]. In this case, there is direct interaction of ALIX with the PYR/PYL/ RCAR proteins, which might be enough to recruit the ABA receptors as cargoes for internalization into ILVs. Indeed, it has been reported that sorting of cargoes into the MVB pathway can occur in the absence of cargo ubiquitination, just by physical binding of cargoes to components of any ESCRT complex [5]. How ALIX specifies the sorting pathway depending on the cargo and the molecular mechanisms it uses for recognition of different cargoes is currently under study. In this context, recent studies indicate that components of the ESCRT-I and –III complexes participate, by physically interacting with ALIX, in sorting of specific ALIXtargeted protein cargoes. This is the case of ESCRT-I complex components FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FYVE1) and VACUOLAR PROTEIN SORTING 23A (VPS23A), and of ESCRT-III subunit VPS32 [2, 6–8]. The number of proteins contributing to ALIX function in Arabidopsis very likely includes additional yet-to-be described regulatory proteins. The same is likely for ALIX-targeted protein cargoes. Indeed, recently, we have reported a list of 62 proteins, belonging to different functional classes, identified as potential ALIX interactors in yeast two hybrids screens [4]. Evaluation and characterization of the physiological relevance of such interactions will require extensive future studies. To facilitate the latter, we have set up methods for different techniques that enable analysis of the effect of ALIX function in the endosomal trafficking of potential protein cargoes, and of the functional relationship between ALIX and ESCRT components or regulatory proteins. The aforementioned techniques aim (1) to confirm in vivo physical interactions between ALIX and candidate cargoes (and partners) by using Bimolecular Fluorescent Complementation (BiFC) assays; (2) to characterize, by confocal imaging, the effect of ALIX function in trafficking of a specific cargo, therefore allowing for detection of defects in protein cargo localization at different membrane compartments (e.g., plasma membrane, early endosomes/trans-Golgi network, late endosomes/MVB, and/or the vacuole) due to loss of ALIX function when compared to the wild type; and (3) analyze the effect of ALIX function in cargo

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trafficking by means of microsomal fractionation and immunoblotting, to test whether loss of ALIX function leads to altered accumulation of a certain cargo protein at a specific cell compartment. Although the methods provided here have been optimized for the characterization of Arabidopsis ALIX, they could be easily applied to the functional analysis of other ESCRT components from plants.

2

Materials All solutions should be prepared with ultrapure water and stored at room temperature, unless otherwise stated.

2.1

BiFC Assay

1. Nicotiana benthamiana plants grown from seeds during 3–4 weeks in a greenhouse with 16-h-light/8-h-dark long day photoperiod, and a temperature of 21  C (see Note 1). 2. Gateway-compatible destination vectors for BiFC of the proteins of interest to be tested (see Note 2). 3. p19-expressing Agrobacterium tumefaciens strain for gene silencing suppressor purposes [9]. 4. Fluorescent subcellular marker constructs (e.g., Wave constructs described by [10], including MVB markers such as fluorescently tagged ARA7 (Wave 2 construct). 5. Competent cells of Agrobacterium tumefaciens C58C1 strain. 6. Luria-Bertani (LB) liquid and solid media containing appropriate antibiotics. 7. Infiltration buffer: 10 mM MES-KOH (pH¼5.5), 10 mM MgCl2, 150 μM acetosyringone. 8. 1 mL syringes without needles. 9. Tabletop centrifuge. 10. Vortex. 11. Confocal fluorescence microscope (e.g., Leica TCS SP8 microscope). 12. Digital image processing and analysis software (e.g., LAS X Life Science Microscope software; Leica Microsystems).

2.2 Confocal Imaging

1. 0.5 Murashige–Skoog (MS) plates containing 1% sucrose and 1% agar. 2. Growth chamber. 3. 4-day-old Arabidopsis thaliana stable transgenic lines expressing the protein of interest tagged with a fluorescent marker such as GFP or YFP in the wild-type and alix-1 mutant backgrounds. 4. 100 mM Brefeldin A (BFA) stock solution. Store at 20  C.

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5. 1 mM Concanamycin A (ConA) stock solution. Store at 20  C. 6. 50 mM Wortmannin (WM) stock solution. Store at 20  C. 7. 1 mM LysoTracker Red DND-99 stock solution. Store at 20  C in darkness. 8. 0.5 Murashige and Skoog liquid medium. 2.3 Microsomal Fractionation

1. 200–300 mg of 7-day-old Arabidopsis thaliana stable transgenic lines expressing the protein of interest fused to the epitope of choice in the wild-type and the alix-1 mutant backgrounds (see Note 3). 2. Extraction buffer: 50mM HEPES pH¼7.9, 300 mM sucrose, 150 mM NaCl, 10 mM potassium acetate, 5 mM EDTA, 1 mM PMSF, 1 complete protease inhibitor (Roche). Adjust to pH ¼ 7.8 (see Note 4). 3. Resuspension buffer: Extraction buffer + 0.5% Triton X-100 (see Note 4). 4. Loading buffer 3: 167mM Tris–HCl (pH 6.8), 33% glycerol, 6.6% (w/v) SDS, and 0.01% (w/v) bromophenol blue. Add 5% of fresh β-mercaptoethanol prior to use. 5. Ultracentrifuge. 6. Refrigerated tabletop centrifuge.

3

Methods

3.1 In Vivo Analysis of Physical Interactions by BiFC.

Day 1

1. Grow 10 mL A. tumefaciens cultures for all needed constructs (i.e., pairs of BiFC vectors, subcellular markers, and suppressor of silencing p19) in LB liquid medium with the corresponding antibiotics at 28  C with constant shaking overnight. Appropriate negative controls should include mutated versions of one of the proteins to be tested carrying a defect in the interaction domain, or a totally unrelated protein, rather than using empty BiFC constructs. Day 2

1. Centrifuge the cultures in a tabletop centrifuge at 3220  g for 5 min. Discard the supernatant and resuspend the pellet in 5 mL infiltration buffer. Incubate in darkness at room temperature for 2.5 h. 2. Prepare 1:10 dilutions of the bacteria suspensions to measure the optical density at 600 nm (OD600) in a spectrophotometer.

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3. Prepare the infiltration mixtures. The standard OD600 for infiltration is 0.3, however, since ALIX forms homodimers, it is recommended to reduce the final OD to 0.1 in the infiltration mixture to avoid any artifacts caused by ALIX overexpression (see Note 5). To avoid silencing of the constructs, a p19-expressing A. tumefaciens culture should be included in the mixture at final OD ¼ 0.1 [9]. 4. Infiltrate the bacterial preparations in the abaxial surface of 3-week-old N. benthamiana leaves with 1-mL syringes [11]. Use one plant per each infiltration mixture (see Note 6). 5. Grow the infiltrated N. benthamiana plants 2-3 days at the greenhouse under a 16-h-light/8-h-dark long day photoperiod at 21  C. Day 4

6. Prepare the samples for confocal imaging. Cut the infiltrated leaves in approximately 1 cm  1 cm squares and place them on glass slides with water (see Note 7). 7. Observe infiltrated leaves in a confocal microscope with the appropriate settings. Colocalization of the fluorescent signals from the reconstitution of the BiFC reporter and that of specific cell compartment markers will provide functional information about the potential role of ALIX in the sorting of a specific protein cargo, as shown in Fig. 1 for the interaction of ALIX and an ABA receptor occurring at MVBs (see Note 8). Colocalization of fluorescent signals can be assessed using LAS X software to analyze the ROI (Region of Interest) curves of cosignaling. To do so, select at least 10 fields of the same size per sample and trace the ROIs in all the vesicles in each field; then quantify the number of vesicles exhibiting BiFC signal,

Fig. 1 Interaction of ALIX with an ABA receptor assessed by BiFC. Confocal images were taken of N. benthamiana leaf epidermal cells expressing different BiFC construct combinations together with the MVB marker mCherry-ARA7. Reconstitution of YFP fluorescence shows that ALIX and the ABA receptor constructs directly interact in mCherry-ARA7-labeled MVBs (i.e., punctae indicated by arrows in Overlay image). Bars ¼ 20 μm

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cell compartment marker signal alone or both signals at the same time. 3.2 Confocal Imaging of ALIX Function in Cargo Trafficking

1. Grow wild type and alix-1 mutant plants expressing GFP/YFPtagged proteins of interest vertically on MS plates for 4 days, in climatic chambers at 21  C under a 16-h-light/8-h-dark photoperiod using cool white fluorescent light conditions. 2. Transfer seedlings to 12-well culture plates with 3 mL MS liquid medium in each well. 3. For BFA treatment, add BFA stock solution to a final concentration of 50 μM, or an equal volume of DMSO as a mock control. Incubate during 90 min in darkness at room temperature. 4. For ConA treatment, add ConA stock solution to a final concentration of 1 μM and incubate for 6 h at room temperature. 5. For WM treatment, add WM stock solution to a final concentration of 33 μM and incubate seedlings for 30 min in darkness at room temperature. 6. To label lytic compartments, add LysoTracker Red DND-99 stock solution to a final concentration of 1 μM, together with the drugs, and incubate at room temperature for 30 min. 7. Image roots in a confocal microscope with the appropriate settings. We recommend imaging cells close to the root meristem where vacuoles are small and endosomal events can be better visualized.

3.3 Microsomal Fractionation to Analyze the Function of ALIX in Cargo Trafficking.

A schematic representation of the microsomal fractionation procedure is provided in Fig. 2. Day 1 1. Grow wild type and alix-1 mutant seedlings mutant plants expressing protein cargo of interest, in horizontal position on MS plates kept in a climatic chamber at 21  C under a 16-hlight/8-h-dark cycle using cool white fluorescent light conditions for 4 days. Day 5 2. Collect fresh seedlings (approximately 150–200) and grind them in 2 volumes of extraction buffer (approximately 400–600 μL; see Note 9). Never freeze the material, otherwise membrane compartments will collapse (CRITICAL STEP).

3. Transfer the homogenized material to precooled 1.5 mLeppendorf tubes with precooled wide bore or cut pipette tips and centrifuge at 200  g at 4  C to remove debris. If necessary, repeat twice.

+ 2 vol of EB

4˚C

DO NOT FREEZE!

200 g 5min 4˚C

Collect INPUT control

Discard pellet

S1 fraction ULTRACENTRIFUGE 100.000 xg 75min 4˚C

S2 cytosolic fraction

P2 pellet

RESUSPEND AVOIDING BUBBLES!

+ 1 vol of EB + Triton X-100

16.100 g 10min 4˚C

P2 microsomal proteins fraction

Discard pellet

Fig. 2 Schematic representation of the microsomal fractionation procedure. Three centrifugation steps allow for separation of the different fractions to be analyzed for the enrichment of specific proteins: (1) A low-speed centrifugation step allows for clarification of plant extracts. The resulting supernatant corresponds to the S1 fraction used as Input control. (2) An ultracentrifugation step separates the Cytosolic fractions S2 (supernatant) from the membrane compartment-enriched fraction (pellet). The latter is resuspended in a detergent-containing buffer to solubilize membranes and release proteins from membranes. (3) Upon a last centrifugation step, the resulting supernatant containing solubilized microsomal proteins is collected (P2 microsomal proteins fraction) whereas the pellet is discarded

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4. Collect supernatant (S1 fraction) and determine the protein concentration (e.g., using Bradford assay). Adjust volumes and concentration to normalize protein concentration in all samples. Save 10 μL as the input control, add loading buffer, and boil for 5 min at 95  C. 5. Precool the ultracentrifuge and the rotor and ultracentrifuge at 4  C; centrifuge the S1 fraction at 100,000  g during 75 min. 6. Collect the supernatants (S2 cytosolic fraction) that correspond to cytosolic proteins. Save 10 μL aliquot per sample, add loading buffer, and boil 5 min at 95  C. 7. Resuspend carefully the microsomal pellet (P2 pellet) in 1 volume (with regard to the starting fresh material volume) of resuspension buffer avoiding bubbles to release proteins from the microsomes. 8. Centrifuge for 5 min at 16,100  g at 4  C and collect the supernatant with the solubilized microsome-associated proteins (P2 microsomal proteins fraction). Prepare 30 μL aliquots, add loading buffer, and boil for 5 min at 95  C. Day 6 9. Load the samples corresponding to the input controls and the separated fractions (S2 and P2) for each genotype into SDS-PAGE gels and carry out immunoblots using specific antibodies against proteins (or protein fusions) of interest. Impaired function of ALIX in alix-1 mutants should lead to accumulation of its protein cargoes in the microsomal fraction compared to wild-type controls. For those cargoes corresponding to membrane-associated soluble proteins, accumulation could also occur in the cytosolic fraction of alix-1 mutants, as reported for ABA receptors [6].

4

Notes 1. Use 3–4-week-old plants that have not started to bloom yet. Use fully expanded young leaves. 2. Plasmids used in our studies are derived from the EYPF vectors from Clontech generated by [12], where pBIFP-1 and pBIFP2 vectors are used to fuse the N-terminal portion of the EYFP to the C- or N-terminus of the gene of interest, respectively; pBIFP-3 and pBIFP-4 vectors are used to fuse the C-terminal portion of the EYFP to the N- or C-terminus of the other gene of interest, respectively. 3. To better analyze the effect of ALIX on protein cargo trafficking and accumulation under physiological conditions, use

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specific antibodies against the endogenous protein cargo of interest whenever possible. 4. Hormones or nutrients such as phosphate can be supplemented to the buffer to test their effect in cargo trafficking. 5. The use of a mutant version of ALIX (e.g., ALIX-1; [2]) as a negative control is recommended. 6. To reach optimal construct expression, choose young leaves since they are more tender, which minimizes the damage caused during agroinfiltration. Try to infiltrate a whole leaf with a single injection. 7. Do not put too many leaf squares in the same glass slide since preparations will be too thick to be properly visualized. 8. To perform colocalization assays, it is essential to keep stringent parameters in the confocal microscope equipment. The most important settings to take into account are: – Keep a pinhole aperture below 1 Airy unit (it may vary depending on the manufacturer’s specifications for each system) – When designing the experiment choose, if possible, fluorophores with quite different and narrow emission spectra to avoid signal crossover and bleed-through. – Avoid signal saturation. – Intensity signals between the two fluorophores used should be comparable. 9. It is essential to keep all the material cold, including the tips.

Acknowledgments Research in our lab was supported by Grant BIO2016-80551-R funded by MINECO and AEI/FEDER/EU. M.G.-L. was supported by a FPI fellowship from the “Severo Ochoa” (BES2015071820; Spanish Ministry of Education) and EMBO Short-Term (ASTF 7678-2016) programs. We are very thankful to the CNB-CSIC Confocal Microscopy facility for comments on the imaging parameters and procedures. References 1. Odorizzi G (2006) The multiple personalities of Alix. J Cell Sci 119:3025–3032 2. Cardona-Lo´pez X, Cuyas L, Marı´n E, Rajulu C, Irigoyen ML, Gil E, Puga MI, Bligny R, Nussaume L, Geldner N, Paz-Ares J, Rubio V (2015) ESCRT-III-associated protein ALIX mediates high-affinity

phosphate transporter trafficking to maintain phosphate homeostasis in Arabidopsis. Plant Cell 27:2560–2581 3. Kalinowska K, Nagel MK, Goodman K, Cuyas L, Anzenberger F, Alkofer A, Paz-Ares J, Braun P, Rubio V, Otegui MS, Isono E (2015) Arabidopsis ALIX is required

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for the endosomal localization of the deubiquitinating enzyme AMSH3. Proc Natl Acad Sci U S A 112:E5543–E5551 4. Garcı´a-Leo´n M, Cuyas L, Abd El-Moneim D, Rodriguez L, Belda-Palazon B, Sa´nchez˜o Quant E, Ferna´ndez Y, Roux B, Zamarren AM, Garcia-Mina JM, Nussaume L, Rodriguez PL, Paz-Ares J, Leonhardt N, Rubio V (2019) Stomatal aperture and turnover of ABA receptors are regulated by Arabidopsis ALIX. Plant Cell 31(10). https://doi.org/10.1105/tpc. 19.00399; pii: tpc.00399.2019 5. Mageswaran SK, Dixon MG, Curtiss M, Keener JP, Babst M (2014) Binding to any ESCRT can mediate ubiquitin-independent cargo sorting. Traffic 15:212–229 6. Belda-Palazon B, Rodriguez L, Fernandez MA, Castillo MC, Anderson EA, Gao C, Gonza´lez-Guzma´n M, Peirats-Llobet M, Zhao Q, De Winne N, Gevaert K, De Jaeger G, Jiang L, Leon J, Mullen RT, Rodriguez PL (2016) FYVE1/FREE1 Interacts with the PYL4 ABA Receptor and Mediates its Delivery to the Vacuolar Degradation Pathway. Plant Cell 28:2291–2311 7. Shen J, Gao C, Zhao Q, Lin Y, Wang X, Zhuang X, Jiang L (2016) AtBRO1 functions

in ESCRT-I complex to regulate multivesicular body protein sorting. Mol Plant 9:760–763 8. Yu F, Lou L, Tian M, Li Q, Ding Y, Cao X, Wu Y, Belda-Palazon B, Rodriguez PL, Yang S, Xie Q (2016) ESCRT-I component VPS23A affects ABA signaling by recognizing ABA receptors for endosomal degradation. Mol Plant 9:1570–1582 9. Voinnet O (2003) RNA silencing bridging the gaps in wheat extracts. Trends Plant Sci 8:307–309 10. Geldner N, De´nervaud-Tendon V, Hyman DL, Mayer U, Stierhof YD, Chory J (2009) Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J 59:169–178 11. Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1:2019–2025 12. Azimzadeh J, Nacry P, Christodoulidou A, Drevensek S, Camilleri C, Amiour N, Parcy F, Pastuglia M, Bouchez D (2008) Arabidopsis TONNEAU1 proteins are essential for preprophase band formation and interact with centrin. Plant Cell 20: 2146–2159

Chapter 6 Correlative Light and Electron Microscopy Imaging of the Plant trans-Golgi Network Pengfei Wang and Byung-Ho Kang Abstract The plant trans-Golgi network (TGN) is a multifunctional organelle derived from the Golgi. It consists of tubulovesicular compartments scattered in the cytosol. They produce secretory vesicles delivering proteins and polysaccharides to the cell wall. They also serve as early endosomal compartments, receiving endocytic cargos from the plasma membrane. This versatility is thought to originate from functional variations among individual TGN compartments. Correlative light and electron microscopy (CLEM) combines the imaging capability of light microscopy and electron microscopy (EM) to determine the location of macromolecules in EM images in the cellular context. It is possible to identify organelles associated with specific fluorescent markers and examine their membrane architectures at nanometer-level resolutions using CLEM. In this chapter, we will explain the CLEM method that our lab uses to investigate functional and structural heterogeneity among individual TGN compartments in plant cells. Key words Correlative light and electron microscopy, Trans-Golgi network, Early endosome, Transmission electron microscopy, GFP, Immunofluorescence microscopy

1

Introduction The trans-Golgi network (TGN) serves as a hub of membrane trafficking in the plant cell, contributing to both secretory and endocytic pathways [1]. TGN cisternae mediate the sorting and packaging of cargo molecules in the secretory pathway for transport to the plasma membrane (PM) or vacuoles [2, 3]. The plant TGN is distinct from TGNs in other eukaryotes in that it also functions as the early endosome [4, 5]. It has been demonstrated that molecules internalized from the PM first reach the TGN in plant cells and that the TGN is involved in recycling endocytosed PM proteins [6, 7]. TGNs are derived from the trans-Golgi cisternae and eventually released from the Golgi to become free TGNs [8]. Plant cells, therefore, have TGN associated with the Golgi as well as free TGNs dispersed throughout the cytosol [9].

Marisa S. Otegui (ed.), Plant Endosomes: Methods and Protocols, Methods in Molecular Biology, vol. 2177, https://doi.org/10.1007/978-1-0716-0767-1_6, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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The convergence of the secretory and endocytic pathways at the TGN raises a question as to how they are organized in individual TGN compartments. Because TGNs are scattered throughout the cytosol, it is possible that plant cells have functionally distinct TGN subpopulations, some for secretion and others for endocytosis. It is also possible that TGN compartments are capable of handling trafficking in both directions. Plant TGNs are seen as tubulovesicular compartments of 300–700 nm in diameter when observed by transmission electron microscopy (TEM)[8, 10]. Electron tomography (ET) analysis of plant TGN cisternae revealed that the 3D structure of individual TGN cisternae differ from each other significantly and suggest that the ultrastructural diversity may reflect functional plasticity of the plant TGN [2, 8]. A drawback in the functional characterization of the plant TGN is the gap between TEM and fluorescence microscopy in TGN identification [11]. TGNs are identified in TEM by their morphological features while TGNs can only be identified by fluorescence microscopy based on their association with fluorescent markers. If fluorescent staining is for either endocytic or exocytic, fluorescent puncta in FM may correspond to subsets of tubulovesicular compartments recognized as TGNs in TEM. Furthermore, TGNs are often associated with Golgi stacks or multivesicular bodies [12] but the organelles are ignored under the black background in FM unless they are fluorescently labeled. Correlative light and electron microscopy (CLEM) is a microscopy method in which the same objects are observed by light and electron microscopy platforms [13, 14]. In general, organelles or proteins of interest are identified by fluorescence microscopy (FM) and the cellular space encompassing the organelles or proteins in the same sample is examined by TEM/ET. In most CLEM approaches, FM is performed to locate rare events or specific organelles and subsequent TEM imaging determines the ultrastructure associated with the events or organelles. Immunostaining with fluorescently labeled antibodies or biosynthetic fluorescent proteins (e.g., GFP) are utilized for FM. CLEM methods have been used widely for studying membrane trafficking and organelle dynamics in Saccharomyces cerevisiae and cultured mammalian cells [15, 16]. We adapted CLEM protocols from such studies to analyze TGN subpopulations and their cellwide organizations in Arabidopsis root tip cells. Using the CLEM method, we locate individual TGN compartments labeled by fluorescent markers of specific trafficking routes in TEM sections and examine the same sections to determine their ultrastructure.

Correlative Light and Electron Microscopy Imaging of the Plant trans...

2

61

Materials

2.1 Plant Material and Growth

1. Arabidopsis seeds expressing a trans-Golgi network (TGN) marker (e.g., vacuolar-type H+-ATPase subunit a1 fused to GFP (VHAa1-GFP)). 2. 70% (v/v) ethanol. 3. Absolute ethanol. 4. Plates containing agar-solidified nutrient medium (1/2 Murashige and Skoog salts).

2.2 Reagents and Antibodies

1. 1PBS Buffer (0.137 M sodium chloride, 0.0027 M potassium chloride, 0.008M sodium phosphate dibasic, 0.002 M potassium phosphate monobasic). 2. Distilled water pH 7.5 (see Note 1). 3. Primary antibody: CCRC-M1 (mouse monoclonal antibody, CarboSource, Complex Carbohydrate Research Center, University of Georgia). 4. Secondary antibody: Goat anti-mouse IgG conjugated to Alexa Fluor 568. 5. Cyanoacrylate glue (e.g., superglue). 6. Cryoprotectant for HPF: 0.15 M sucrose solution 7. Cryosubstitution medium: 0.25% glutaraldehyde and 0.1% uranyl acetate 8. Uranyl acetate solution: 0.75g Uranyl acetate dissolved in 20 ml 70% MeOH 9. Lead citrate solution: 0.665 g lead (II) nitrate and 0.880 g sodium citrate (Na3(C6H5O7)·2H2O) dissolved in 24 ml degassed distilled water and add 1 ml CO2 free 4N NaOH to facilitate precipitate dissolution.

2.3

Equipment

1. Stereomicroscope equipped with a mercury lamp and a GFP filter set. 2. High pressure freezer (e.g., Leica HPM100 HPF machine), type B aluminum planchettes for HPF, and freeze-substitution device (e.g., Leica EM AFS2). 3. Fluorescence microscope equipped with FITC filter (e.g., excitation 450–490 nm, band pass emission filter 500–550 nm), Rhodamine filter (e.g., excitation 510–560 nm, long pass filter emission 590 nm), 40 Plan-Neofluar objective. 4. Transmission electron microscope. 5. Ultramicrotome and Diamond knife. 6. Formvar film-coated slot grids (2 mm  1 mm slot Nickle grids).

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7. Microscope slides. 8. Cover glasses 22  22 mm. 2.4

3 3.1

Software

Adobe Photoshop (Adobe Creative Cloud)

Methods Seedling Growth

1. Sterilize Arabidopsis thaliana seeds (transgenic lines expressing GFP) by incubating them in 70% (v/v) ethanol for 15 min two times and 1 min in absolute ethanol. 2. After several rinse with sterile distilled water, sow seeds on agarsolidified nutrient medium (1/2 Murashige and Skoog salt). 3. Grow seedlings in vertically under 16 h light–8 h dark cycle at 22  C for 7 days before freezing.

3.2 Selection and Processing of Seedlings by HighPressure Freezing and Freeze Substitution

3.3 Sample Mounting, Sectioning, and Immunofluorescence Labeling

1. Remove the Petri dish cover and examine root tips with a stereo-microscope equipped for detecting GFP fluorescence. Mark seedlings displaying unambiguous fluorescence for high pressure freezing (HPF). Root tips positive for fluorescence were quickly dissected from seedlings and transferred to planchettes with sucrose solution. The root tip samples were frozen with an HPM100 HPF machine and subsequently freeze-substituted at 80  C for 24 h in anhydrous acetone containing 0.25% glutaraldehyde and 0.1% uranyl acetate. Then samples were washed with precooled anhydrous acetone, embedded in a graduated Lowicryl HM20 (33, 66 and 100%) resin, and polymerized under UV light for 18 h at 45  C. 1. After resin polymerization, cut out root tip samples and mount on plastic stubs with superglue glue (see Note 2). 2. Trim the block (trapezoidal block face) and prepare 250–300 nm thin sections with an ultramicrotome (see Note 3). 3. Collect section ribbons on slot grids and air dry. 4. Image the section by both fluorescence microscopy and TEM. Figure 1a shows an epifluorescence micrograph from Arabidopsis root tip cells expressing VHA1a-GFP. GFP-specific puncta are readily observed. Figure 1b is a TEM photo of the section in Fig. 1a. 5. For aligning micrographs from epifluorescence microscopy and TEM, we use the cell wall profiles visualized in the two microscopy modalities. For immunofluorescence labeling of cell walls, set up a humid chamber and float grids on drops of 2% nonfat milk in PBS for 30 min with the section side down (see Notes 4–6). 6. Transfer the grids to drops of primary antibody dilution (see Note 7) in 1% nonfat milk PBS and incubate for 2 h.

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Fig. 1 Low-magnification CLEM images of Arabidopsis root tip cells expressing the TGN marker VHAa1 fused to GFP (VHAa1-GFP). (a) Fluorescence micrograph of a root tip section from a high-pressure frozen, freeze-substituted, resinembedded VHAa1-GFP transgenic Arabidopsis seedling. (b) TEM micrograph of the same section shown in (a)

7. Wash three times with 0.5% nonfat milk PBS. 8. Incubate the grids in secondary antibody conjugated with Alexa Fluor 568 (1:60 dilution in 0.5% nonfat milk PBS) for 60 min. 9. Rinse the grids quickly and thoroughly. 3.4 Fluorescence Microscopy Imaging

We recommend capturing images from sections immediately after immunofluorescence labeling. 1. Dispense a drop of distilled water on the glass slide and place on top EM grids. Cover the grid with a coverslip carefully.

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Fig. 2 CLEM imaging of TGN in Arabidopsis root tip cells. (a) GFP fluorescence from a TEM section of an Arabidopsis seedling root expressing VHAa1-GFP; (b) Fluorescence micrograph of GFP (green) superimposed with immunofluorescence of xyloglucan cell wall polysaccharide visualized with CCRC-M1 primary antibodies

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2. Examine the grids under an epifluorescence microscope (see Note 8). We use a FITC filter and a rhodamine filter for GFP and Alexa Fluor 568 imaging, respectively. Figure 2 shows CLEM imaging of Arabidopsis root tip cells expressing VHAa1-GFP. Since GFP is more susceptible to photobleaching than Alexa dyes, take micrographs of GFP under the FITC filter first and then, change to the rhodamine channel. 3.5 Recovery, Washing, and Poststaining

1. After imaging sample by fluorescence microscopy, recover grids from glass slide carefully (see Note 9). 2. Rinse grid with distilled water and let air-dry completely. 3. Poststain sections with uranyl acetate and lead citrate as explained in Kang (2010) [17].

3.6 TEM Analysis and Cell Wall-Based Correlation

1. Locate cells with GFP fluorescence and record TEM micrographs. 2. Compare cell wall shapes from fluorescence micrographs with those in TEM images to find the area with TGN-specific puncta (see Note 10). 3. Images from epifluorescence microscopy and TEM can be electronically merged by overlaying cell wall profiles using Adobe Photoshop. Figure 2d–g shows two circled areas in TEM images superimposed with fluorescence micrographs. For accurate morphometric analysis (volumes, surface areas and lengths, etc.) of TGN cisternae, tilt series from the same section are collected for electron tomography reconstruction and 3D modeling (see Note 11).

4

Notes 1. Distilled water used for ultramicrotomy and subsequent steps should be neutral because fluorescence from GFP is sensitive to acidic environment. 2. We use superglue to mount sample blocks on stubs at room temperature. Do not place mounted stubs in 65  C to

ä Fig. 2 (continued) and Alexa Fluor 568–conjugated secondary antibodies (red). (c) TEM micrograph of the section shown in (a) and (b). (d–g) High-magnification TEM micrographs of the two areas marked with circles in (b). TGNs were identified in those areas. The TGN in the circle 1 (d and e) is associated with both VHAa1-GFP and CCRC-M1 specific fluorescence. Note that the TGN overlapping with VHAa1-GFP exhibits membrane profiles and luminal staining different from those of the TGN associated with CCRC-M1-specific fluorescence (d and e). The TGN in F and G is a free-TGN, associated with VHAa1-GFP but not with CCRC-M1. G Golgi, TGN trans-Golgi Network

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accelerate drying glue. High temperature could weaken or kill GFP fluorescence. 3. We have been able to detect GFP puncta reliably from sections thicker than 200 nm. 4. We use PBS instead of PBST because Tween 20 may be autofluorescent, enhancing nonspecific fluorescence. 5. For immunolabeling of plastic sections, some protocols recommend HCl treatment for glutaraldehyde removal [17]. Avoid this step as it may affect GFP fluorescence. 6. Other methods for aligning correlated micrographs can employed. For instance, TEM sections could be coated with quantum dots that are readily located under FM and TEM. 7. We usually use label xyloglucan for Arabidopsis cell walls with CCRC-M1 antibody (1:50 dilution). Another quick method is to visualizing the cell wall with Calcofluor White that stains cellulose [18]. 8. For FM imaging, capture micrographs quickly, because GFP are vulnerable to photo-bleaching in plastic resin. Also, do not use a confocal laser scanning microscope because laser excitation could kill GFP fluorescence almost immediately. 9. Be careful when you recover slot grids from glass slides or coverslips so formvar film and sections are not damaged. 10. We recommend slot grids instead of mesh or finder grids for serial section analysis, because areas of interest could be blocked from imaging by the metallic grid bars. 11. We have described methods for electron tomography analysis of plant Golgi stacks, TGN cisternae, and other organelles in detail in our earlier articles explaining our protocols [19–22].

Acknowledgments This work was supported by Hong Kong Research Grant Council (GRF14126116, AoE/M-05/12, C4002-17G), Rural Development Administration of Korea (Project No. 10953092019), and Chinese University of Hong Kong (Direct Grant 14101218). References 1. Rosquete MR, Drakakaki G (2018) Plant TGN in the stress response: a compartmentalized overview. Curr Opin in Plant Biol 46:122–129 2. Staehelin LA, Kang B-H (2008) Nanoscale architecture of endoplasmic reticulum export sites and of Golgi membranes as determined

by electron tomography. Plant Physiol 147:1454–1468 3. van de Meene AML, Doblin MS, Bacic A (2016) The plant secretory pathway seen through the lens of the cell wall. Protoplasma 254:1–20

Correlative Light and Electron Microscopy Imaging of the Plant trans... 4. LaMontagne ED, Heese A (2017) Trans -Golgi network/early endosome: a central sorting station for cargo proteins in plant immunity. Curr Opin in Plant Biol 40:114–121 5. Ruiz Rosquete M, Davis DJ, Drakakaki G (2017) The plant Trans-Golgi network. Not just a matter of distinction. Plant Physiol 176:01239.2017–01239.2198 6. Dettmer J, Hong-Hermesdorf A, Stierhof Y-D, Schumacher K (2006) Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell 18:715–730 7. Luschnig C, Vert G (2014) The dynamics of plant plasma membrane proteins: PINs and beyond. Development 141:2924–2938 8. Kang B-H, Nielsen E, Preuss ML, Mastronarde D, Staehelin LA (2011) Electron tomography of RabA4b- and PI-4Kβ1-labeled trans golgi network compartments in Arabidopsis. Traffic 12:313–329 9. Uemura T, Nakano RT, Takagi J, Wang Y, Kramer K, Finkemeier I, Nakagami H, Tsuda K, Ueda T, Schulze-Lefert P, Nakano A (2019) A Golgi-released subpopulation of the trans-Golgi network mediates protein secretion in Arabidopsis. Plant Physiol 179:519–532 10. Wang P, Chen X, Goldbeck C, Chung E, Kang B-H (2017) A distinct class of vesicles derived from the trans-Golgi mediates secretion of xylogalacturonan in the root border cell. Plant J 92:596–610 11. Wang P, Liang Z, Kang B-H (2019) Electron tomography of plant organelles and the outlook for correlative microscopic approaches. New Phytol 223:1756–1761 12. Seguı´-Simarro JM, Staehelin LA (2006) Cell cycle-dependent changes in Golgi stacks, vacuoles, clathrin-coated vesicles and multivesicular bodies in meristematic cells of Arabidopsis thaliana: a quantitative and spatial analysis. Planta 223:223–236 13. Kukulski W, Schorb M, Welsch S, Picco A, Kaksonen M, Briggs JAG (2011) Correlated fluorescence and 3D electron microscopy with

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high sensitivity and spatial precision. J Cell Biol 192:111–119 14. Bell K, Mitchell S, Paultre D, Posch M, Oparka K (2013) Correlative imaging of fluorescent proteins in resin-embedded plant material. Plant Physiol 161:1595–1603 15. Avinoam O, Schorb M, Beese CJ, Briggs JAG, Kaksonen M (2015) ENDOCYTOSIS. Endocytic sites mature by continuous bending and remodeling of the clathrin coat. Science 348:1369–1372 16. Zachari M, Gudmundsson SR, Li Z, Manifava M, Shah R, Smith M, Stronge J, Karanasios E, Piunti C, Kishi-Itakura C, Vihinen H, Jokitalo E, Guan J-L, Buss F, Smith AM, Walker SA, Eskelinen E-L, Ktistakis NT (2019) Selective autophagy of mitochondria on a ubiquitin- endoplasmic-reticulum platform. Dev Cell 50(5):627–643 17. Kang B (2010) Electron microscopy and highpressure freezing of Arabidopsis. Methods Cell Biol 96:259–283 18. Wang P, Kang B-H (2018) The trans-Golgi sorting and the exocytosis of xylogalacturonan from the root border/border-like cell are conserved among monocot and dicot plant species. Plant Signal Behav 13:e1469362–e1469363 19. Toyooka K, Kang B-H (2014) Reconstructing plant cells in 3D by serial section electron tomography. In: Zarsky V, Cvrckova F (eds) Plant cell morphogenesis. Humana Press, Totowa, NJ, pp 159–170 20. Kang B-H (2016) STEM tomography imaging of hypertrophied Golgi stacks in mucilagesecreting cells. Methods Mol Biol 1496:55–62 21. Mai KKK, Kang B-H (2017) Semiautomatic segmentation of plant Golgi stacks in electron tomograms using 3dmod. Methods Mol Biol 1662:97–104 22. Liang Z, Zhu N, Mai KK, Liu Z, Liu Z, Tzeng D, Tzeng DTW, Osteryoung KW, Zhong S, Staehelin LA, Staehelin A, Kang B-H (2018) Thylakoid-bound polysomes and a dynamin-related protein, FZL, mediate critical stages of the linear chloroplast biogenesis program in greening Arabidopsis cotyledons. Plant Cell 30:1476–1495

Chapter 7 Imaging Plant Cells by High-Pressure Freezing and Serial block-face scanning electron microscopy Kirk Czymmek, Abhilash Sawant, Kaija Goodman, Janice Pennington, Pal Pedersen, Mrinalini Hoon, and Marisa S. Otegui Abstract This chapter describes methods to enhanced contrast of plant material processed by high-pressure freezing and freeze substitution for improved visualization by serial block-face scanning electron microscopy (SBEM). The contrast enhancing steps are based on a protocol involving the sequential incubation of samples in heavy metals and sodium thiocarbohydrazide (OTO staining). We also describe the pipeline for imaging plant tissues in a commercial SBEM system (Gatan 3View®) and routines for the image analysis and three-dimensional reconstructions using open-source and commercial software packages. Key words High-pressure freezing, Serial block-face electron microscopy, Three-dimensional electron microscopy

1

Introduction Conventional transmission EM (TEM) of fixed and resin-embedded biological specimens have been essential for the understanding of basic cell organization, organelle structure and distribution. Compared to other popular modalities of microscopy used in cell biology, such as fluorescence-based confocal and super-resolution microscopy, TEM has the advantage of providing higher lateral resolution (~1–3 nm for resin-embedded sample) and an untargeted analysis of the cellular structure as it does not require fluorescence labeling of a preselected cellular component. However, whereas photons can be used to image microns to millimeters into the sample, the depth of electron beam penetration ranges from nanometers to a few microns, depending on the acceleration voltage. Thus, imaging a whole organ such as a plant root at the electron microscope level, typically would require slicing the sample into thousands of serial sections.

Marisa S. Otegui (ed.), Plant Endosomes: Methods and Protocols, Methods in Molecular Biology, vol. 2177, https://doi.org/10.1007/978-1-0716-0767-1_7, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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Three-dimensional (3D) reconstruction of whole cells and tissues can thus be achieved by imaging individual serial sections between 50 and 100 nm [1]. This approach requires manual sectioning of a resin-embedded sample using an ultramicrotome. The serial sections are maintained sequentially on slotted grids stained with heavy metal solutions such as lead citrate and uranyl acetate, imaged one grid at a time in a TEM, and subsequently all collected images are aligned. As an example of applications in plants, this approach was successfully used to reconstruct the distribution of organelles in columella cells of tobacco roots through the collection of 200 and 300 serial section sets [2]. However, serial thin sectionbased 3D reconstructions have the following disadvantages: (1) it is extremely difficult and time-consuming to manually obtain such a large collection of serial sections without losing/damaging slices during sectioning, grid handling, staining, or imaging; (2) although the lateral resolution of this approach is 1–3 nm, the axial resolution is rather poor since, as other serial section-based methods, it is limited to twice the section thickness [3] and therefore commonly restricted to ~100–200 nm); (3) sections can be wrinkled, warped, and deformed making registration of serial images problematic. To overcome the challenging task of collecting serial sections for TEM imaging, in 1981, Leighton [4] proposed the inclusion of a small ultramicrotome within the chamber of a scanning electron microscope (SEM) to image a resin-embedded squid fin. As the microtome shaved off sections (20–30 nm) from the sample, the block face was imaged using backscattered electrons. Nowadays, fully automated serial block-face SEM (SBEM) systems control the sectioning and imaging of the sample with minimal intervention by the user [5]. In addition, SBEM systems can enhance visualization of non-conductive samples by introducing small amount of water vapor [5] or nitrogen gas [6]. In a different modality of block-face methods, the sample surface is milled by a focused ion beam scanning electron microscope (FIB SEM), removing an ~5 nm layer of the surface producing datasets with isotropic voxels [7, 8]. Both SBEM and FIB SEM avoid section handling and distortions due to section warping all together and make registration of serial images straightforward. Both techniques can achieve a minimum lateral resolution of 3–5 nm with the axial resolution limited to the thickness of the material remove in each cycle. While both serial block-face approaches result in lower resolution than serial section TEM and destroy the sample while imaging; they offer the significant advantage of fast, automated, and reliable image acquisition of large sample volumes without the need of tedious manual sectioning and section handling/staining [9]. Since there is no post-section staining in SBEM, the samples have to be properly contrasted before resin embedding. Here, we provide a method to prepare plant samples for SBEM by highpressure freezing and freeze-substitution followed by osmiophilic

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Fig. 1 Key steps in serial block-face EM workflow for freeze-substituted plant samples

thiocarbohydrazide (TCH) incubation steps to enhance contrast [10, 11]. While conventional fixation of plant samples has been shown with SBFEM [12], high-pressure freezing followed by freeze substitution achieves much better preservation of plant and other tissues than chemical fixation [13–15]. To improve contrast after cryo-substitution with osmium tetroxide in acetone, samples are incubated in the mordant TCH serving as a bridge to amplify binding of osmium to samples. This is achieved by including a subsequent incubation step with osmium tetroxide in acetone (here the name “OTO” for this contrast enhancing approach) [10, 11, 16], an additional step of uranyl acetate and/or lead can further increase contrast of cellular components. We have adapted a protocol developed for Caenorhabditis elegans [17] for plant samples, such as roots and anthers. SBEM imaging of plant tissues processed with this approach allows for the analysis of plant organelles, including plant endosomes and the subcellular alteration of mutant plants affected in endosomal trafficking components. Here, we describe a pipeline for sample preparation, SBEM imaging using a 3View® system, and image analysis using open-source and commercial software packages (Figure 1).

2 2.1

Materials Plant material

1. Arabidopsis thaliana seedlings, 3–7 days after germination. 2. 0.5 Murashige and Skoog (MS) agar plates:0.8% agar plates containing 2.2 g of Murashige and Skoog (MS) basal medium powder per liter. 3. Arabidopsis plants in reproductive stage with floral buds Arabidopsis thaliana plants at reproductive stage.

2.2 High-Pressure Freezing and Freeze Substitution, and Resin Embedding

1. High-pressure freezer (Leica EM ICE or Wohlwend HPF Compact 02). 2. Cryoprotectant solution: 0.1 M sucrose in purified H2O. 3. Freezing planchettes planchettes).

(“hats”)

4. 2% OsO4 in anhydrous acetone.

(e.g.,

type

B

freezing

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5. Anhydrous acetone. 6. 0.3% sodium thiocarbohydrazide (TCH) in acetone (see Note 1). 7. 1% uranyl acetate (UA) in acetone (see Note 2). 8. Eponate resin (hard resin formulation). 9. Dry ice. 2.3 Serial Block-Face Imaging

1. Scanning electron microscope equipped with an ultramicrotome for automated sectioning (e.g., Gatan 3View with focal charge compensations from Zeiss or VolumeScope SEM from ThermoFisher). Here we used the Gatan 3View, Gatan Digital Micrograph v3.30 on a ZEISS GeminiSEM 300. 2. Gatan 3View™ specimen pin stub or Thermo Scientific VolumeScope™ pin stub with specimen flat. 3. Silver conductive epoxy. 4. Sputter coater.

2.4

Software

1. TrakEM2 module (inbuilt plugin) of Image J (freely accessible on the Fiji/Image J download at https://imagej.net/Fiji/ Downloads). 2. KNOSSOS software (freely downloadable online at https:// knossos.app/) 3. Amira software (Thermofisher).

3

Methods

3.1 High-Pressure Freezing and Sample Preparation

1. Excise root tips from seedlings grown on 0.5 MS with 1% sucrose plates for 4–7 days. For anthers, under a dissecting scope select developing flower buds of appropriate developmental stage from an Arabidopsis inflorescence. Excise anthers using a pair of sharp forceps and a needle in a drop of 0.1 M sucrose solution. 2. Load samples into type B freezing planchettes with cryoprotectant such that the planchette chamber is completely full without empty air spaces or bubbles to minimize collapse of tissue upon freezing. 3. Close chamber with the flat side of a second type B freezing planchettes and transfer to the cryoholder. 4. Freeze sample with a high-pressure freezer. 5. Open freezing planchettes under liquid nitrogen and transfer the hat containing your sample to a cryovial containing liquid nitrogen for storage or a cryovial with 2% OsO4 in anhydrous acetone.

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6. Perform freeze substitution by placing the cryovials with frozen samples in 2% OsO4 in anhydrous acetone in dry ice overnight, followed by warming to room temperature on a rocking shaker allowing slow agitation for 2–3 h. Keep cryovials at room temperature for approximately 1 h. 7. Discard the 2% OsO4 and wash between 4 and 6 times with anhydrous acetone, ensuring the sample has dislodged completely from the freezing planchettes. 8. Remove freezing planchettes (see Note 3). 9. Incubate samples in 0.3% TCH in acetone at room temperature for 60 min in the dark with slow agitation. 10. Discard TCH solution and wash samples with acetone 4–6 times. 11. Incubate samples in 2% OsO4 in acetone at room temperature for 60 min. 12. Discard 2% OsO4 solution and wash with acetone between 4 and 6 times. 13. Incubate samples in 1% UA in acetone for 60 min in the dark. 14. Discard 1% UA solution and wash with acetone between 4 and 6 times. 15. For resin infiltration, incubate samples in increasing concentration of hard formulation Eponate resin: 25% (v/v) resin in acetone for at least 1 h, 50% resin in acetone for at least 1 h, 75% resin in acetone for at least 1 h, 3 changes 100% 1–3 h each. 16. Embed samples in flat embedding molds and polymerize for 24 h at 60  C. 3.2 Serial Block-Face Imaging

1. Flat-embedded anthers were prescreened using a stereomicroscope in transmitted light mode and intact anthers with minimal/no evidence of mechanical damage were selected. 2. Excise a 1 mm  1 mm piece of targeted region of sample using a single-edged razor blade. 3. With a single-edged razor blade, gently scrape and remove excess resin from the sample side that would be mounted to the specimen pin stubs. The goal is to ensure the conductive osmicated sample tissue is exposed and in direct contact with the conductive silver epoxy in the subsequent step. 4. Apply a small drop of freshly mixed silver conductive epoxy to the end of the specimen pin stubs (standard 1.4 mm flat) and place the sample onto the conductive epoxy and place in 60  C oven overnight (Fig. 2a, b). 5. Trim the specimen block sides with a single edged razor blade as needed to remove excess resin and silver epoxy.

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Fig. 2 (a) Oblique perspective of epoxy embedded OTO sample (asterisk) mounted on Gatan 3View specimen pin stub and stage holder. (b) Top-down view of epoxy embedded OTO sample (asterisk) mounted with silver epoxy prior to final trimming and sputter coating. (c) Oblique perspective of 3View stage with sample (asterisk), Focal Charge Compensation needle (FCC) and retracted Diamond Knife arm (K). (d) Top-down enlarged view of sample with FCC needle in final and optimal position

6. If desired, mount the sample on the aluminum pin in an ultramicrotome chuck and face the block, collecting and staining sections as needed to confirm the presence of the targeted structures of interest and location on the block face. 7. Sputter-coat the sample with a generous layer (~50+nm) of Au or Au/PD to ensure a good conductive surface completely surrounds the specimen block. 8. Mount specimen pin in SBEM system, home the 3View stage in the z-direction, and approach the diamond knife with the coarse brass screw until the sample is nearly cutting. 9. While observing in the stereomicroscope attached to the 3view, do the final approach of the block face with diamond knife using the Digital Micrograph approach function. Set the slice thickness to 100 nm and 100 cuts. Observe the approach and stop the cutting when the sample surface has been faced off with the diamond knife. If the surface has not been reached and/or faced off after 100 cuts, repeat the approach. 10. Retract the knife and position the Focal Charge Compensation needle as closely as possible to the block face without touching it (Fig. 2c, d). Bring the knife back to cutting position and retract it again to ensure that the needle will stay at its intended position. 11. Pump down SEM chamber and turn on filament in normal gun mode with a voltage appropriate for your sample (range between 1.5 and 3 kV) with the 30 μm aperture. 12. Apply N2 gas via the Focal Charge Compensation (FCC) needle to 40% (pressure ¼ 3  103 mbar). 13. At low magnification, take an overview image of the block face in backscatter mode to use as a reference to target the area of interest for SBEM 3D Volume.

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14. Use the following parameters for anther image acquisition parameters: Image x-y pixel dimensions: 8000  8000 pixels Gun voltage: 2.2 kV Z-slice thickness: 70 nm Pixel size: 5 nm Pixel dwell-time: 0.8 μs 15. Optimize imaging conditions and number of slices for individual samples and experimental requirements (see Note 4). 3.3 Data Segmentation, Visualization, and Analyses

Two image segmentation and image analysis software packages are commonly used for segmenting and analyzing EM data sections generated by SBEM. These are listed below: 1. TrakEM2 module (inbuilt plugin) of Image J (freely accessible on the Fiji/Image J download at https://imagej.net/Fiji/ Downloads; the 2017 version of Fiji/Image J running on Java 6 platforms is recommended). 2. KNOSSOS software (freely downloadable online at https:// knossos.app/) These software packages can be coupled with the Amira software (ThermoFisher Scientific) for final rendering, visualization and coloring of the 3D EM segmented structures (see Note 5). Here we describe the work pipeline for analysis and rendition of data collected in a 3View system.

3.3.1 The Analyses Routine Through TrakEM2

1. Generate .tiff image stacks of the sequence of EM images and save all the .tiffs for one reconstruction within the same folder. The 3View system commonly saves each EM image of the stack as a .dm4 file format which needs to be converted into a .tiff format to be imported into TrakEM2. An automated script can be generated and saved as an ImageJ plugin for converting each image plane of the EM stack from the .dm4 file into a .tiff format together with applying a band-pass filter for enhancing the edges of the structures in the electron micrograph and sharpening the boundaries. The .tiff files that are generated are normally in an 8-bit format to enable import into the TrakEM2 module. This script can be run on a batch of EM images upon selecting the appropriate source and destination folder. 2. Generate an Excel sheet containing the list of names of the EM tiff files along with the corresponding X, Y, and Z coordinates of the image plane for correct placement of the imported file into TrakEM2. If only one tile has been generated in the 3View, then the X and Y coordinate values will remain at

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0 and only the value for the Z coordinate will increase correspondingly with each plane. If multiple tiles have been acquired, then the position of each image tile needs to reflect the correct location of the tile in the montage. For example, when arranging the position of an adjacent tile the column of the X coordinate needs to increase by the pixel resolution value of the preceding tile. Similarly, when arranging the position of the tile below another tile the Y coordinate will increase by the pixel resolution value of the tile above. Normally, the 3View captures a low-resolution “montage” image prior to high resolution scanning of individual tiles to enable assessment of the correct placement/positioning of each tile in the montage. Each tile will overlap with surrounding tiles such that similar structure can be identified and correct location of tiles in the montage ascertained. Once the Excel sheet containing the X, Y and Z coordinate information for each tiff has been generated, save the Excel sheet as a .txt file format in the same folder as the one containing all the tiff EM images. This text file sheet will be used as reference to correctly import the tiff EM files and accurately position them in the TrakEM2 module to reconstruct the 3D image montage that has been acquired by the serial block-face scanning electron microscope. 3. Open Fiji/Image J and select a new TrakEM2 file under the File tab. The user will be asked to select a storage folder for the TrakEM2 file and the folder containing the tiffs of the EM image planes should be selected. A new TrakEM2 window is thereafter accessible. To import the EM dataset into the TrakEM2 window right click to select import and thereafter the import from text file option. Then select the saved .txt file containing all the coordinate information of the tiff EM files. The layers tab of TrakEM2 begins to get populated as individual EM image planes get loaded into the display window of the TrakEM2 workspace. Once all the files have been imported, the images are loaded into appropriate planes and one can scroll through the entire dataset. Remember to save the file as an .xml file once the import of the dataset has been completed. Also remember to select the same folder as the one containing the original tiffs, for saving the .xml file. The .xml file is the one that will be used for subsequent opening, re-opening and analyses of the dataset. A detailed list of tools for editing and annotating data in TrakEM2 and the keyboard shortcuts for each command are readily available on the online TrakEM2 tutorials. Remember to set the Z distance between each image plane in terms of the pixel resolution of the acquired EM data. This will ensure correct visualization of the 3D objects detailed in steps 6 and 7.

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4. Once the stack is imported the user can choose to run an image plane alignment routine on the entire stack by right clicking on the TrakEM2 browser with the image stack loaded and selecting the align layers option. When different montages need to be correctly placed, one can right click on the TrakEM2 window and select the montage multiple layers option. 5. To volume reconstruct a structure of interest first generate an areatree and insert nodes at the center of the structure of interest on each plane. This creates a 3D “skeleton” of the structure of interest that needs to be reconstructed. One can thereafter paint from the center of each node until the periphery of the structure of interest to fill in the 3D volume of a particular structure of interest. Several areatrees, each corresponding to a specific structure of interest, can be created to 3D reconstruct multiple profiles within the dataset. These will populate in the project object tab of the TrakEM2 browser. 6. To paint-in or highlight specific structures that do not form a continuous skeleton across several planes, an area_list function can alternatively be used. Each reconstructed structure can be assigned as an areatree or area_list profile and added as a separate project object. Both areatree and area_list features can be visualized in the 3D viewer window of TrakEM2 after selecting a downsampling metric. Choose a project object to be visualized and right click to select the show in 3D option. If the 3D structures appear jagged the user can select a smoothing filter via a smooth control option on the 3D viewer browser. The smooth control option also allows the user to select the number of smoothing iterations to be applied. The 3D objects can be exported from the 3D viewer in the STL (ASCII) format to enable import and visualization in the Amira software (see example in Fig. 3). 7. After importing ASCII format files representing 3D reconstructed areatree and area_list profiles into Amira, a surface view along with a colormap needs to be assigned for each imported element. Amira offers better visualization of 3D reconstructed profiles and also extended flexibility with the color and transparency choices for the reconstructed structures. A varying degree of smoothing can be applied for the reconstructed structures before saving the profile in the ASCII format and also after import into Amira. The degree of profile smoothening needed will vary depending on the representation of the structure being reconstructed. If a separate coloring and transparency profile needs to be assigned to each reconstructed profile, remember to export each profile as a separate ASCII file. Also note that the individual nodes of each reconstructed structure will be lost during the ASCII file conversion and

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High-Pressure Freezing

Sample Preparation

Freezesubstitution with Thiocarbohydrazid e Resin Embedment

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Fig. 3 Example of three consecutive serial block-face EM images of a highpressure frozen/freeze-substituted Arabidopsis thaliana anther. For reconstruction, developing pollen grains were pseudocolored in magenta and surrounding (tapetal) cells, in green. Separate areatree profiles were made for these structures in TrakEM2. Right panel shows reconstruction of two developing pollen grains and two tapetal cells across 132 serial block-face EM planes. Scale bar ¼ 6 μm

subsequent import into Amira and only the painted-in areatree planes and area_lists profiles will appear in the 3D objects imported into Amira.

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1. Generate .tiff image stacks of the sequence of EM images (refer to Subheading 3.1 for additional details) and name each file in an ordered dictionary format (e.g., 1.tiff, 2.tiff, 3.tiff, and so on). Save all files in one source folder. A python-based application that converts images into KNOSSOS-readable format is readily available at https://github.com/knossos-project/ knossos_cuber. This knossos_cuber script takes in 3 arguments—source folder, image format (format of the source files), and target folder (sufficient storage space for KNOSSOS-compatible data) as parameters—and generates cubes of different magnifications in a format readable by KNOSSOS. This dataset is then accessible by a simultaneously generated KNOSSOS.conf configuration file for the converted dataset. Additionally, knossos_cuber script can also convert .png or .jpg files into a KNOSSOS-readable format. 2. Load the KNOSSOS app compatible with the operating system on your computer. Click on the choose dataset dialog, navigate to the KNOSSOS.conf file of the converted dataset and load the dataset. Optimal settings for “Field-of-View” pixel sizes are 256, 512, or 768. Once the dataset loads, KNOSSOS displays a two-dimensional representation of each side (XY view, YZ view, and ZY view) in 3 separate viewports and a 3D viewport for visualization of the 3D skeleton. Use the zoom window to navigate through different magnifications of these viewports. 3. One can parse through the dataset across Z-planes by using the scroll button on the computer mouse and in XY-planes by clicking and dragging the left-mouse button. Make sure the drag dataset and recenter options are enabled in the navigation window. Upon identifying a structure of interest, nodes can be placed by right-clicking in one of the 3 slice plane viewports. Consecutive nodes are automatically linked and included as part of the 3D skeleton. Information about coordinates of all nodes and skeleton is accessible through the annotation window from the Windows option in the toolbar. One can create new branches of the skeleton by pressing B on the keyboard, which converts an active node to a branch node. Progress of the 3D skeleton can be constantly monitored through dynamically changing views in each slice port and visualizing the skeleton in the 3D viewport. The user can refer to the XY, YZ, and ZY view simultaneously to confirm connectivity and continuity of the 3D skeleton. For details of shortcuts on other features, refer to the KNOSSOS documentation available online at https:// knossos.app/documentation/. 4. The generated skeleton data can be saved in an .xml annotation file through the save annotation option. A new skeleton tree can be initiated by pressing C on the keyboard, which enables the user to trace a new structure. Likewise, one can generate

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skeletons for multiple structures of interest within the same dataset. The skeleton data of multiple traced structures can be saved onto the same annotation file. 5. A helpful visualization feature of KNOSSOS is a dynamically changing inbuilt scalebar option, available for gauging widths or diameters of structures and 3D objects at any magnification. This can be accessed through the Preferences>Viewports>Show scalebar option. 6. Like TrakEM2, KNOSSOS allows rectification of incorrectly traced segments or randomly connected structures by merging, deleting, or separating the different segments of a 3D skeleton. This is achieved by selecting the pair of mismatched nodes in the annotation window and choosing the relevant option through the right-click menu. Additionally, for quickly capturing an important plane of the stack, KNOSSOS offers an inbuilt snapshot feature along with a scalebar imprinted onto the captured image.

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Notes 1. To dissolve TCH in acetone is recommended to start with a 10% solution of TCH adding 0.1 g TCH to 1 mL of purified water and vortex several times. Store 10% TCH solution in the dark. To prepare the 0.3% TCH solution add 300 μL of 10% stock solution to 9.7 mL of acetone. 2. UA has very limited solubility in acetone. We recommend preparing a stock solution of 10% UA in methanol and then add 500 μL of 10% UA solution to 4.5 mL of acetone. 3. Freezing planchettes can be sonicated in acetone and reused if not bent or damaged after freezing. 4. Sample preparation is key for successful 3D SBEM imaging, namely enhancing metallization of your samples as described in Subheading 3.1 above. Samples with soft resins and reduced conductivity (isolated cells, many air spaces, low metallization during sample prep) are more sensitive to electron beam dosage and interactions. In such cases, FCC and variable pressure modes can applied to reduce/eliminating charging effects, with FCC being the most effective while maintaining the highest resolution [6]. Additional parameter adjustments such as increasing z-section step size, binning pixels or increasing pixel size, reducing dwell time and/or reducing kV can be effective strategies to mitigate beam damage. 5. Workstations with a Xeon processor (~128GB RAM/solid state drives) are recommended for fast visualization and analyses of large stacks of the EM data. The workstations can be coupled with a Wacom tablet for extending the display of the

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3D EM profiles and for ease of coloring in the outlines of the 3D structures to be reconstructed. 6. OTO prepared epoxy resin blocks are highly amenable to X-ray microscopy allowing for a lower resolution 3D perspective (up to 500 nm isotropic voxels) of the entire sample block. This is especially beneficial when trying to efficiently target a specific structure for SBEM or identify mechanical damage that would limit the value of the 3D volume.

Acknowledgments This work was partially funded by NSF grant MCB1614965 to MSO and UW2020 WARF Discovery award to M.H. References 1. Rieder CL (1981) Thick and thin serial sectioning for the three-dimensional reconstruction of biological ultrastructure. In: Turner JN (ed) Methods in cell biology. Academic Press, New York, pp 215–249 2. Zheng HQ, Staehelin LA (2001) Nodal endoplasmic reticulum, a specialized form of endoplasmic reticulum found in gravity-sensing root tip columella cells. Plant Physiol 125:252–265 3. McEwen BF, Marko M (2001) The emergence of electron tomography as an important tool for investigating cellular ultrastructure. J Histochem Cytochem 49:553–564 4. Leighton SB (1981) SEM images of block faces, cut by a miniature microtome within the SEM - a technical note. Scan Electron Microsc 73–76. 5. Denk W, Horstmann H (2004) Serial blockface scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLOS Biol 2:e329 6. Deerinck TJ et al (2018) High-performance serial block-face SEM of nonconductive biological samples enabled by focal gas injection-based charge compensation. J Microsc 270:142–149 7. Peddie CJ, Collinson LM (2014) Exploring the third dimension: volume electron microscopy comes of age. Micron 61:9–19 8. Wei D et al (2012) High-resolution threedimensional reconstruction of a whole yeast cell using focused-ion beam scanning electron microscopy. BioTechniques 53:41–48 9. Smith D, Starborg T (2019) Serial block face scanning electron microscopy in cell biology: applications and technology. Tissue Cell 57:111–122

10. Seligman AM, Wasserkrug HL, Hanker JS (1966) A new staining method (OTO) for enhancing contrast of lipid—containing membranes and droplets in osmium tetroxide— fixed tissue with osmiophilic thiocarbohydrazide (TCH). J Cell Biol 30:424–432 11. Deerinck TJ et al (2010) Enhancing serial block-face scanning electron microscopy to enable high resolution 3-D nanohistology of cells and tissues. Microsc Microanal 16:1138–1139 12. Kittelmann M, Hawes C, Hughes L (2016) Serial block face scanning electron microscopy and the reconstruction of plant cell membrane systems. J Microsc 263:200–211 13. Otegui MS, Pennington JG (2019) Electron tomography in plant cell biology. Microscopy (Oxf) 68:69–79 14. Bourett TM, Czymmek KJ, Howard RJ (1999) Ultrastructure of chloroplast protuberances in rice leaves preserved by high-pressure freezing. Planta 208:472–479 15. Kiss JZ et al (1990) Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted root tips of Nicotiana and Arabidopsis. Protoplasma 157:64–74 16. Willingham MC, Rutherford AV (1984) The use of osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells. J Histochem Cytochem 32:455–460 17. Hall DH, Hartweig E, Nguyen KCQ. OTO fixation for SEM and blockface imaging. Wormatlas - anatomical methods. https://www. wormatlas.org/EMmethods/OTOFix.htm

Chapter 8 Functional Analysis of Plant FYVE Domain Proteins in Endosomal Trafficking Wenjin Shen, Juan Wei, and Caiji Gao Abstract The FYVE domain is a double zinc finger-like domain that predominantly binds phosphatidylinositol 3-phosphate. The FYVE domain is usually found in proteins primarily involved in regulating various aspects of endomembrane homeostasis, including endosome tethering, endocytic recycling, membrane protein sorting, and autophagosome maturation. Whereas FYVE domain proteins have been extensively studied in mammals and yeast, only a few FYVE domain proteins have been identified and characterized in plants. Here, by using as an example FREE1 (FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1), a protein previously identified by us as a critical factor for endosomal trafficking, we describe methods to determine its lipid binding properties and endosomal localization. In addition, we also demonstrate a method to quickly test whether an FYVE domain protein is involved in endosomal sorting in plant cells. Key words Endosomal sorting, FYVE domain, Lipid binding, Phosphatidylinositol 3-phosphate, Vacuole

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Introduction Phosphatidylinositol 3-phosphate (PI3P), one of the major phosphoinositides found in endosomes, controls diverse aspects of membrane dynamics in eukaryotes by recruiting effector proteins [1]. One class of effectors that are specifically recruited to endosomal membranes by PI3P are proteins containing an FYVE domain, named after the first letter of the four proteins in which this domain was originally discovered: Fab1, YOTB, Vac1, and EEA1. The basic FYVE domain contains eight Zn2+ coordinating cysteines, which are surrounded by three conserved regions: the N-terminal WxxD, the central R(R/K)HHCR, and the C-terminal R(V/I)C motifs (Fig. 1a). The basic motif in the (R/K)(R/K)HHCR region forms a β-strand, which creates a positively charged pocket that specifically binds to PI3P [2].

Marisa S. Otegui (ed.), Plant Endosomes: Methods and Protocols, Methods in Molecular Biology, vol. 2177, https://doi.org/10.1007/978-1-0716-0767-1_8, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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Fig. 1 Lipid binding assay and subcellular localization of plant FYVE domain-containing protein FREE1. (a) Sequence alignment of FYVE domains from FREE1, HsEEA1, HsHrs, and ScFab1p proteins. (b) His-SumoFREE1 protein was expressed in E. coli, purified using a His SpinTrap column, and stained with Coomassie blue after separation by SDS-PAGE. M stands for protein maker; asterisk indicates purified protein. (c) Purified His-Sumo-FREE1 protein was used in an in vitro lipid-binding assay, followed by immunodetection with an anti-His antibody. (d) Colocalization of GFP-FREE1 with the MVB marker mRFP-Rha1 in the transgenic Arabidopsis root cells. Wortmannin treatment caused the enlargement of MVB to form ring-like structures (arrows). Localization of GFP-FREE1 on the membrane of enlarged MVB induced by wortmannin treatment indicates that the PI3P lipid binding is dispensable for the MVB localization of GFP-FREE1. Bars are 10 μm

To data, over 100 FYVE domain proteins have been identified in yeast, plant, and mammals. Most FYVE-domain proteins are primarily associated with endomembrane trafficking. As examples, the best known mammalian FYVE-domain protein, EEA1, is an effector of the Rab5 small GTPase that mediates tethering of endosomes and concomitant fusion of early endosomes [3]; in mammal and yeast, HRS/Vps27p acts as an ESCRT (Endosomal sorting complex required for transport)-0 component to regulate the endosomal recruitment of other ESCRT modules and the sorting of ubiquitinated membrane proteins [4, 5]; mammalian FYCO1 is a Rab7 effector that mediate microtube-directed transport of vesicles and autophagosomes [6, 7]. Besides canonical endosomal trafficking, recent studies also demonstrate the critical role of several FYVE domain proteins in autophagosome formation and autophagic degradation (for review, please see [8, 9]). The Arabidopsis genome contains 15 genes encoding FYVEdomain-containing proteins [10]. Two are homologues of FAB1/ PIKfyve, which is a PtdIns 3,5-kinase and catalyzes the production of PI(3,5)P2 from PI3P [11]. The other 13 FYVE-domain proteins

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seem to be plant-specific as there are no homologous sequences in mammals and yeast. Up to now, only a few FYVE-domain proteins have been characterized in plants. The two Arabidopsis PtdIns 3,5-kinases, FAB1A and FAB1B, localize predominantly to sorting nexin1 (SNX1)-residing endosomes and regulate the endosomal recruitment of ARA7 and SNX1 [11]. Downregulation or depletion of FAB1A/B expression leads to disturbed endomembrane homeostasis, including delayed endocytosis, abnormal vacuole formation and acidification, as well as pollen abortion [12, 13]. FREE1 (also known as FYVE1) is a well-characterized plant FYVE-domain protein that specifically binds PI3P and functions as a plant-specific ESCRT component to regulate multivesicular body (MVB) biogenesis, vacuole formation, ubiquitinated membrane protein sorting, and autophagic degradation [14–16]. More recently, FREE1 was also found to shuttle to the nucleus to negatively regulate the transcription of abscisic acid-responsive genes [17]. Another characterized plant FYVE domain protein is CFS1 (also known as FYVE2); CFS1 binds PI3P through its FYVE domain and localizes to ARA7-labeled late endosomes [18]. Depletion of CFS1 leads to accumulation of autophagosomes and ubiquitinated proteins, indicating a role of CFS1 in both endosomal trafficking and autophagy [18]. Here, using FREE1 as an example, we describe methods to test protein binding to PI3P through in vitro lipid-binding assay and to determine whether an FYVE-domain protein is involved in endosomal trafficking by analyzing the transport of vacuolar storage proteins and the degradation of ubiquitinated membrane proteins in plants.

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Materials

2.1 Purification of Epitope-Tagged FYVEDomain Proteins from Escherichia coli

1. LB Broth: 1 L containing 10 g tryptone, 5 g yeast extract, 10 g NaCl, with or without 10 g agar, adjust pH to 7.0 with NaOH. The antibiotics, kanamycin (final concentration 50 mg/L) or ampicillin (final concentration 100 mg/L), are added to the LB medium just before use. 2. E. coli strain: BL21 Rosetta or BL21S transformed with a His-Sumo-FREE1 expression vector. 3. 0.8 M isopropyl β-D-thiogalactoside (IPTG) stock solution: 2 g of IPTG in 10 mL distilled H2O. Filter-sterilize with a 0.22 μm syringe filter. Store in 1 mL aliquots at 20  C. 4. Binding buffer: 20 mM Na2HPO4, 500 mM NaCl, 30–60 mM imidazole, pH 7.4. 5. Elution buffer: 20 mM Na2HPO4, 500 mM NaCl, 500–800 mM imidazole, pH 7.4 (see Note 1).

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6. His SpinTrap column. 7. Sonicator. 2.2 Lipid Binding Assay

1. Membrane lipid strips (e.g., from Echelon Biosciences). 2. 10 PBS stock buffer: 1 L stock solution containing 80 g NaCl, 2 g KCl, 2.3 g NaH2PO4lH2O, 13.9 g Na2HPO4l7H2O, adjust pH to 7.4. 3. Membrane washing buffer: dilute the 10 PBS stock buffer into 1 solution and add tween-20 to a final concentration of 0.1% (v/v). 4. Membrane blocking buffer: add 3 g BSA to 100 mL of the above membrane washing buffer to a final concentration of 3%. 5. Anti-His antibody (e.g., from Abcam). 6. Horseradish peroxidase (HRP)-conjugated secondary antibodies to detect anti-His antibodies (e.g., from Abcam). 7. Enhanced chemiluminescence (ECL) western blotting substrate (e.g., from Millipore).

2.3 Arabidopsis Transformation and Crossing

1. Arabidopsis MS medium: 2.2 g/L Murashige and Skoog Basal Salt Mixture, 10 g/L sucrose, 8 g/L agar, pH 5.7 (with KOH). Omit adding agar for liquid medium. The antibiotics, kanamycin (final concentration 50 mg/L) or hygromycin (final concentration 25 mg/L), are added to the MS medium just before use. 2. Agrobacterium strain: GV3101 transformed with a binary vector for the expression of GFP-FREE1 or other fluorescently tagged FYVE-domain protein. 3. YEP medium: 10 g/L yeast extract. 10 g/L Bacto peptone. 5 g/L NaCl. Adjust pH to 7.0 with NaOH. 4. Silwet L-77. 5. 50 g/L sucrose, prepared freshly. 6. 100% ethanol and 70% (v/v) ethanol containing 0.05% (v/v) Tween 20. 7. Fine forceps. 8. Transgenic plants expressing fluorescently tagged endosomal markers (e.g., mRFP-Rha1). 9. Dissection microscope.

2.4 Wortmannin Treatment and Confocal Microscopy

1. Wortmannin stock solution: 3.3 mM wortmannin in DMSO. Aliquot and store at 20  C (see Note 2). 2. Confocal microscope. 3. Microscope slides and coverslips.

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1. Wild type and free1 mutant Arabidopsis seeds. 2. 7-days-old wild type and free1 Arabidopsis seedlings. 3. Cell soluble (CS) and Cell membrane (CM) separation buffer: 40 mM HEPES-KOH at pH 7.5, 1 mM EDTA, 10 mM KCl, 0.4 M sucrose with 1 Complete Protease Inhibitor Cocktail. 4. Seed protein extraction buffer: 30 mM Tris–HCl at pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM KCl with 1 Complete Protease Inhibitor Cocktail. 5. Ground quartz or sand glass powder. 6. Electric grinder: a mini handheld and motor-driven tissue grinder used for homogenizing plant tissues (e.g., OSE-Y10; Tiangen Biotech Co., Ltd., Beijing, China). 7. Temperature-controlled ultracentrifuge.

benchtop

centrifuge

and

8. SDS–polyacrylamide gel electrophoresis (SDS-PAGE) system and protein transfer system for western blotting (e.g., Bio-Rad). 9. 5X SDS sample buffer: 250 mM Tris–HCl at pH 6.8, 100 g/L SDS, 5% (v/v) β-Mercaptoethanol, 50% (v/v) glycerol. 10. Coomassie blue staining solution: 2.5 g/L Coomassie brilliant blue R-250, 10% (v/v) acetic acid, 45% (v/v) methanol. 11. Nitrocellulose or PVDF membrane. 12. Anti-ubiquitin antibody (e.g., from Abcam). 13. Anti-12S globulin antibody (homemade by using 12S globulin protein extracted from Arabidopsis seed as antigen to immunize rabbit). 14. Horseradish antibodies.

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Methods

3.1 In Vitro Lipid Binding Assay Using Purified FYVE domain Proteins from E. coli

1. Inoculate a single colony of E. coli BL21 Rosetta strain cells transformed with a His-Sumo-FREE1 expression vector into 20 mL LB liquid medium with appropriate antibiotics in a flask and incubate the culture at 37  C under shaking at 250 rpm until OD600 reach 0.6–0.8. Add IPTG to the culture at a final concentration of 0.4 mM, and incubate the culture at 20  C overnight under shaking at 200 rpm to induce the expression of the His-tagged protein (see Note 3). As negative control, a construct with truncation of FYVE domain, for example His-Sumo-FREE1(ΔFYVE), is suggested to make for protein purification and lipid binding assay.

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2. Centrifuge to collect the cell pellet. Add 1 mL of binding buffer containing 1 mM PMSF to resuspend cell paste, and disrupt cells by sonication. Centrifuge the lysate in a benchtop centrifuge at 17,000  g speed for 10 min to remove insoluble material, and collect the supernatant. 3. Centrifuge the His SpinTrap columns to remove the storage liquid. Equilibrate the column by adding 600 μL of binding buffer, and then centrifuge for 30 s at 100  g to remove the binding buffer. 4. Add the supernatant of the E. coli cell lysate to the equilibrated column. Invert and shake the column for 10 s, and centrifuge for 30 s at 100  g. Wash the column with 600 μL of binding buffer. Centrifuge at 100  g for 30 s. Repeat this wash step twice. 5. Elute the His-Sumo-FREE1 protein twice with 200 μL of elution buffer. Centrifuge for 30 s at 100  g and collect the purified protein. The first 200 μL will contain most of the target protein (Fig. 2b). The purified protein can be kept at 4  C for up to 1 week before the lipid binding assay (see Note 4). 6. To perform the lipid binding assay, incubate the membrane strips spotted with various lipids in 10 mL of membrane blocking buffer for 1 h with gentle agitation at room temperature (RT) and in the dark. 7. Discard membrane blocking buffer and incubate membrane lipid strip with 2 μg/mL purified His-sumo-FREE1 protein in 10 mL membrane blocking buffer. Incubate for 1 h at RT with gentle agitation (see Note 5). 8. Discard the protein solution and wash the membrane lipid strips with PBS-T washing buffer for 5 min with gentle agitation. Repeat wash step three times. 9. Add anti-His primary antibody solution (1:1000 dilution in membrane blocking buffer), incubate for 1 h at RT with gentle agitation. 10. Discard the primary antibody solution and wash the membrane lipid strips as explained in step 8. 11. Incubate membrane lipid strips with HRP-conjugated secondary antibodies (1:10000 dilution in membrane blocking solution) for 1 h at RT with gentle agitation. 12. Discard the secondary antibody and wash the membrane as three times as explained in step 8. Use ECL western blotting substrate to detect signal Fig. 2c). 3.2 Arabidopsis Transformation and Crossing

The expression FREE1 or other FYVE-domain proteins with a fluorescent tag in Arabidopsis plants allows for their subcellular localization analysis. Once GFP-FREE1 transgenic plants are

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Fig. 2 Analysis seed storage protein processing and ubiquitinated membrane protein sorting in the free1 mutant. (a, b) Protein extracts from wild-type and free1 seeds either stained with Coomassie blue (a) or subjected to immunoblotting with anti-12S globulin antibodies show the abnormal accumulation of storage proteins precursors p12S) in free1 mutant seeds. (c) Cell soluble (CS) and cell membrane (CM) fractions of proteins extracted from the WT and free1 plants were subjected to immunoblot analysis with anti-ubiquitin antibody. Note that more ubiquitin conjugates accumulate in free1 mutant compared to WT, indicating a defect in endosomal sorting of ubquitinated proteins (especially membrane proteins) in free1 mutant

selected, they can be crossed with other lines explaining relevant endomembrane markers tagged with other fluorescent tags (e.g., RFP-ARA7 for late endosomes/MVBs). 1. Grow Arabidopsis plants under the long-day condition (16 h light/8 h dark, 22  C). Remove the first inflorescence to promote the growth of more inflorescences. When several flowers are open, proceed with the floral dip step (see Note 6). 2. Three days prior to floral dip, inoculate the transformed Agrobacterium into 5 mL liquid YEP medium with the appropriate antibiotics and incubate at 28  C with vigorous agitation for 2 days. Then, inoculate 100 μL of the Agrobacterium culture into 10 mL of YEP medium and incubate for 24 h at 28  C with vigorous agitation.

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3. Pellet down the Agrobacterium cells by centrifugation at 4000  g for 10 min, remove the supernatant and gently resuspend the pellet in 1 volume of freshly made 5% (w/v) sucrose solution. Before dipping, add Silwet L-77 to a final concentration of 0.05% (v/v) and mix gently (see Note 7). 4. Dip the whole inflorescence in the Agrobacterium suspension or use a disposable Pasteur pipette to apply the Agrobacterium culture to the inflorescence meristem. 5. Cover plants with a plastic cover or wrap them with plastic film to maintain high humidity overnight. Remove the cover the following day and allow plants to grow normally until seed collection (see Note 8). 6. For screening transgenic plants, sterilize seeds by vigorous washing in 70% (v/v) ethanol containing 0.05% (v/v) Tween 20 for 10 min, followed by another two washing steps in 100% ethanol for 5 min each. After letting ethanol to evaporate, plate the sterilized seeds on MS agar medium plate containing appropriate antibiotics for selection. 7. To cross GFP-FREE1 plants with other organelle marker lines, such as RFP-ARA7, select an inflorescence and remove all the flowers that already open as they are already self-pollinated. Choose those closed floral buds that are to undergo anthesis in the next 24–36 h. Carefully remove the sepals, petals, and anthers under the dissection microscope to expose the stigma. Pollen transfer is achieved by brushing the exposed stigma with a fully open flower from the other parental line. Make sure there is plenty of pollen on the recipient stigma (see Note 9). 8. Label the cross accordingly. After a day or 2, check the crossed flower. If the cross was successful, the young silique will be expanding. 3.3 Wortmannin Treatment and Confocal Imaging

Wortmannin is a covalent inhibitor of PI3 kinases and as such, can cause important alterations in PI3P abundance and endomembrane dynamics. As the FYVE domain binds PI3P, imaging of fluorescently tagged FYVE proteins and relevant endosomal markers (e.g., mRFP-Rha1), can provide useful information about their function and dynamics. 1. Dilute the 3.3 mM wortmannin stock solution in Arabidopsis MS liquid medium to reach a final concentration 16.5 μM (200 dilution of the stock solution). Add 5 days old transgenic seedlings expressing both GFP-FREE1 and mRFPRha1, mix them gently, and incubate at RT for 1 h. 2. Transfer a plant seedling to a microscope slide and place gently coverslip on top for immediate confocal imaging (see Note 10).

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3. Image fluorescent proteins in Arabidopsis root cells using a 63 water-immersion lens (see Note 11). As shown in Fig. 1d, GFP-FREE1 colocalize with the MVB marker mRFP-Rha1. However, wortmannin treatment induces the homotypic fusion and enlargement of MVBs [19]. Under wortmannin treatment, GFP-FREE1 localizes to abnormally large endosomes also labeled by mRFP-Rha1, confirming the MVB localization of GFP-FREE1 in plant cells (see Note 12). 3.4 Analysis of Seed Storage Proteins and Ubiquitinated by Western Blotting

3.4.1 Analysis of 12S Globulin Processing

Endosomal and vacuolar sorting defects can be tested by analyzing the processing of seed storage proteins and the abundance of ubiquitinated proteins. Storage proteins, such as the 12S albumins are synthesized as precursors and undergo proteolytic cleavage in late endosomes and vacuole. Mutants defective in endosomal/vacuolar trafficking usually accumulate abnormal amounts of 12S globulin precursors in their seeds. In addition, if ESCRTdependent sorting in endosomes is abnormal, cells tend to accumulate higher levels of ubiquitinated proteins. Using mutants lacking important FYVE-domain proteins, such as the free1 mutant, it is possible to analyze abnormal seed storage protein processing and accumulation of ubiquitinated protein by western blotting. 1. Place 50 mature seeds into 100 μL seed protein extraction buffer. Add a ground quartz or sand glass powder to the buffer and grind the seeds thoroughly with an electric grinding rod. 2. Centrifuge the lysate at 20,000  g for 10 min at 4  C and transfer the supernatant to a new tube. Add SDS sample buffer to the extracted protein in supernatant and boil it at 95  C for 5 min. Load 20 μL or 5 μL of boiled protein samples for SDS-PAGE separation followed by Coomassie blue staining or immunoblotting with anti-12S globulin antibody, respectively. As shown in Fig. 2a, b, depletion of FREE1 leads to abnormal accumulation of 12S globulin precursors, indicating the potential function of FREE1 in vacuolar protein sorting in plants.

3.4.2 Abundance of Ubiquitinated Proteins in Cell Soluble (CS) and Cell Membrane (CM) Fractions

1. Add 0.5–1 mL of ice-cold cell soluble (CS) and cell membrane (CM) fraction isolation buffer to 0.2–0.4 g of 7-day-old Arabidopsis seedlings and grind the seedlings thoroughly using mortar and pestle on ice. 2. Centrifuge the lysate at 600  g for 3 min to remove large cellular debris. Remove supernatant and place it into a new tube for centrifugation at 100,000  g for 30 min at 4  C. The supernatant and pellet are the CS and CM fractions, respectively (see Note 13). 3. Dissolve proteins in CS and CM fractions in 1 SDS sample buffer by boiling at 95  C for 10 min (see Note 14). Separate

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proteins by SDS-PAGE and detect ubiquitinated proteins by immunoblotting using an anti-ubiquitin antibody. As shown in Fig. 2c, depletion of FREE1 leads to accumulation of ubiquitinated proteins, especially in the CM fraction, indicating the critical function of the FYVE domain-containing protein FREE1 in the regulation of endosomal sorting and degradation of membrane proteins [15].

4

Notes 1. The concentration of imidazole in the binding and elution buffers can vary and it should be optimized for each protein. We routinely use binding buffer with 50 mM imidazole and elution buffer with 500 mM imidazole. 2. The drug wortmannin should be stored in small aliquots at 20  C. For each experiment, pick a new aliquot to avoid repeated thawing/freezing. 3. The E. coli strain as well as the incubation conditions (e.g., temperature, time) after adding IPTG to the culture should be optimized for each protein. We routinely use E. coli BL21 Rosetta or BL21 Soluble strains and incubate the culture at 20  C or 28  C from 6 h to overnight to induce the expression of the target protein. 4. We recommend performing the lipid binding assay immediately after purifying the protein under study. 5. The optimal concentration of purified FYVE-domain protein for the lipid binding assay varies depending on the properties of each protein. We recommend using proteins at a final concentration of 0.5–2 μg/mL. 6. Three- to four-week-old plants are usually ready for floral dip; 3–5 plants are enough for each transformation. Healthy plants are critical for successful transformation. 7. Since Silwet L-77 is harmful to Agrobacterium, this chemical should be added just before the dipping step. 8. For higher transformation rates, especially when few plants are used, it is better to dip inflorescence three times in a 7-day interval. 9. Extra care should be taken to avoid self-pollination in Arabidopsis flowers to be crossed. Be sure to remove the anthers before anther dehiscence. Cleaning forceps between different crosses is essential. Be very careful to avoid damaging the stigma while removing anthers. 10. To avoid the movement of seedlings during imaging, use 2% (w/v) low melting agar to mount seedlings on the microscope slide.

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11. Line sequential scanning mode is recommended to avoid possible crosstalk between different fluorophores. 12. Since wortmannin is an inhibitor of PI3K and inhibits the production of PI3P, wortmannin treatment usually leads to the dissociation and release of FYVE domain-containing proteins from PI3P-enriched endosomes. However, some FYVEdomain containing protein might be recruited to endosomes independently of binding to PI3P, through binding to other endosome-localized interaction partners. In the latter case, wortmannin treatment may not result in the dissociation of FYVE-domain protein from endosomes. In our example, wortmannin treatment does not cause dissociation of FREE1 from MVB, but rather results in the localization of FREE1 on enlarged MVBs. 13. Centrifugation at 100,000  g is required for clear separation of soluble and membrane fractions. In addition, it is recommended to wash the membrane pellet using the CS/CM isolation buffer twice to avoid contamination of the membrane fraction with the soluble fraction. 14. We routinely dissolve the cell membrane pellet using 1 SDS sample buffer at a one tenth of the liquid volume of the cell soluble fraction. For example, if the volume of the cell soluble fraction is 500 μL, the volume of 1 SDS sample buffer used to dissolve the cell membrane pellet will be 50 μL.

Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (31671467 and 31870171), the China 1000-Talents Plan for young researchers (C83025) to C. G., and the National Natural Science Foundation of China (31701246) to W.S. References 1. Schink KO, Tan KW, Stenmark H (2016) Phosphoinositides in control of membrane dynamics. Annu Rev Cell Dev Biol 32:143–171 2. Hayakawa A, Hayes SJ, Lawe DC et al (2004) Structural basis for endosomal targeting by FYVE domains. J Biol Chem 279:5958–5966 3. Mills IG, Urbe´ S, Clague MJ (2001) Relationships between EEA1 binding partners and their role in endosome fusion. J Cell Sci 114:1959–1965 4. Bilodeau PS, Winistorfer SC, Kearney WR et al (2003) Vps27-Hse1 and ESCRT-I complexes

cooperate to increase efficiency of sorting ubiquitinated proteins at the endosome. J Cell Biol 163(2):237–243 5. Katzmann DJ, Stefan CJ, Babst M et al (2003) Vps27 recruits ESCRT machinery to endosomes during MVB sorting. J Cell Biol 162 (3):413–423 6. Olsvik HL, Lamark T, Takagi K et al (2015) FYCO1 contains a C-terminally extended, LC3A/B-preferring LC3-interacting region (LIR) motif required for efficient maturation of autophagosomes during basal autophagy. J Biol Chem 290(49):29361–29374

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7. Pankiv S, Alemu EA, Brech A et al (2010) FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end– directed vesicle transport. J Cell Biol 188 (2):253–269 8. Lystad AH, Simonsen A (2016) Phosphoinositide-binding proteins in autophagy. FEBS Lett 590:2454–2468 9. Hayakawa A, Hayes S, Leonard D et al (2007) Evolutionarily conserved structural and functional roles of the FYVE domain. Biochem Soc Symp 74:95–105 10. Wywial E, Singh SM (2010) Identification and structural characterization of FYVE domaincontaining proteins of Arabidopsis thaliana. BMC Plant Biol 10:157 11. Hirano T, Munnik T, Sato MH (2015) Phosphatidylinositol 3-phosphate 5-kinase, FAB1/ PIKfyve kinase mediates endosome maturation to establish endosome-cortical microtubule interaction in Arabidopsis. Plant Physiol 169:1961–1974 12. Whitley P, Hinz S, Doughty J (2009) Arabidopsis FAB1/PIKfyve proteins are essential for development of viable pollen. Plant Physiol 151(4):1812–1822 13. Hirano T, Matsuzawa T, Takegawa K et al (2011) Loss-of-function and gain-of-function mutations in FAB1A/B impair endomembrane

homeostasis, conferring pleiotropic developmental abnormalities in Arabidopsis. Plant Physiol 155:797–807 14. Gao C, Zhuang X, Cui Y et al (2015) Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation. Proc Natl Acad Sci U S A 112:1886–1891 15. Gao C, Luo M, Zhao Q et al (2014) A unique plant ESCRT component, FREE1, regulates multivesicular body protein sorting and plant growth. Curr Biol 24:2556–2563 16. Kolb C, Nagel MK, Kalinowska K et al (2015) FYVE1 is essential for vacuole biogenesis and intracellular trafficking in Arabidopsis. Plant Physiol 167:1361–1373 17. Li H, Li Y, Zhao Q et al (2019) The plant ESCRT component FREE1 shuttles to the nucleus to attenuate abscisic acid signalling. Nat Plants 5(5):512 18. Sutipatanasomboon A, Herberth S, Alwood EG et al (2017) Disruption of the plant-specific CFS 1 gene impairs autophagosome turnover and triggers EDS1-dependent cell death. Sci Rep 7(1):8677 19. Wang J, Cai Y, Miao Y et al (2009) Wortmannin induces homotypic fusion of plant prevacuolar compartments. J Exp Bot 60 (11):3075–3083

Chapter 9 Assessing Extrinsic Membrane Protein Dependency to PI4P Using a Plasma Membrane to Endosome Relocalization Transient Assay in Nicotiana benthamiana Mehdi Doumane and Marie-Ce´cile Caillaud Abstract Phosphoinositides are key players from which the various membranes of the cells acquire their identity. The relative accumulation of these low-abundant anionic phospholipids in the cytosolic leaflet of the plasma membrane and of various organelles generates a landmark code, responsible for the selective recruitment of extrinsic proteins at given membranes. One of the key players in the protein/lipid interaction at the plasma membrane in plant cells, is phosphatidylinositol 4-phosphate (PI4P), which patterns the recruitment of effector proteins from the plasma membrane to organelles along the endocytic pathway. Here we describe a fast assay to assess the requirement of PI4P for membrane localization of extrinsic membrane proteins in vivo. This system relies on perturbing PI4P distribution in plant cells via the action of a PI4P phosphatase that depletes the pool of PI4P at a given membrane. This system efficiently decreases PI4P levels, and can therefore be easily used to assess requirement of PI4P (and electrostatics) for the targeting of extrinsic membrane proteins to the plasma membrane or endosomes. Ultimately, this system could also be extended to test the phosphatase activity in planta of enzymes putatively involved in PI4P metabolism. Key words Anionic lipids, Phosphoinositides, Fluorescent-tagged protein, Membrane proteins, Phosphatase assay, Membrane trafficking, Plasma membrane, Electrostatics, Agrobacterium transformation

1

Introduction Biological membranes act as selectively permeable barriers between two compartments. For instance, the plasma membrane (PM) limits cytoplasm and extracellular space. That way, the PM allows physicochemical parameters to be different inside the cell compared to the outside medium, facilitating or even allowing metabolic reaction to occur [1]. In some Prokaryotes and in Eukaryotes, additional biological membranes are present inside the cells. These endomembrane structures define intracellular compartments in which additional conditions, different from cytoplasm, can exist: the lumen of the

Marisa S. Otegui (ed.), Plant Endosomes: Methods and Protocols, Methods in Molecular Biology, vol. 2177, https://doi.org/10.1007/978-1-0716-0767-1_9, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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endoplasmic reticulum is an oxidizing environment whereas the cytoplasm is reducing; the lumen of the vacuole is acid whereas the cytosol is neutral, and so on. Biological membranes are made of lipids and proteins and their biophysical attributes derive from the individual properties of their components as well as the emergent properties generated by their assembly at the supramolecular scale. Membrane lipids are amphipathic molecules that self-organize in a bilayer of two leaflets. Each leaflet exposes polar groups toward water solvent, interacting with either water molecules or other lipid polar groups. On the other hand, proteins can be associated with membranes via several means. Intrinsic proteins possess hydrophobic domains, such as hydrophobic alpha helixes, embedded inside membranes. Extrinsic proteins are associated with membranes, either by a covalent bound, or by more labile non-covalent bounds with membrane lipids or proteins [2]. Interestingly, extrinsic proteins that associate to the cytosolic leaflet of a membrane appear to be targeted mainly or exclusively to a specific membrane. For example, the SidM protein from Legionella pneumophila localizes only to the membrane surrounding this intracellular bacteria in infected mammalian cells [3–5], whereas Arabidopsis MEMBRANE ASSOCIATED REPRESSOR KINASE 2 (MARK2) localizes to the plasma membrane [6]. How is this selective subcellular association with membrane achieved? SidM directly interacts with phosphatidylinositol-4-phosphate (PI4P) and localize to PI4P-enriched membranes and MAKR2 is targeted to the highly electronegative electrostatic field beneath the plasma membrane, partially driven by PI4P [6, 7]. Phosphoinositides, including PI4P, are indeed key players that confer their identity to the various cellular membranes. These low abundant membrane lipids derive from phosphatidic acid (PA, the simplest phospholipid) bearing in addition an inositol head that can be virtually phosphorylated at three different positions. So far, only phophatidylinositol (PI), three phosphatidylinositol-phosphate (PI3P, PI4P, and PI5P) and two phosphatidylinositol-bisphosphate [PI(3,5)P2 and PI(4,5)P2] species have been reported in plants. The differential accumulation of theses lipids in the cytosolic leaflet of the plasma membrane and of organelles generates a landmark code, responsible for the selective recruitments of extrinsic proteins [8, 9]. Altogether, protein recruitments at the molecular level can occur by direct binding to a cognate lipid like SidM does, and/or via the recognition of lipid induced biophysical properties such as membrane curvature, local electrostatic field or packing defects [2, 8]. We generated a system that allows the targeting at the plasma membrane—or at both plasma membrane and endosomes—of yeast SUPPRESSOR OF ACTIN 1 (Sac1) PI4P-4phosphtase domain (Sac12-518; Fig. 1a) [6, 7, 10, 11]. In this system, a

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Fig. 1 When targeted at the plasma membrane, the yeast SUPPRESSOR OF ACTIN 1 (Sac1) PI4P-4phosphtase domain efficiently depletes PI4P in plant cells. (a) Genetic constructs used to dephosphorylate PI4P at the plasma membrane, and possibly to a lesser extent at endosomes. (b) MAP-FP-Sac1C392S2-518 is targeted at the plasma membrane when expressed in N. benthamiana. (c) PI4P sensors are composed of a lipid-binding domain fused to a fluorescent protein. PI4P sensors (e.g., mCIT-P4MSidM) specifically binds PI4P but not PI. (d) In resting conditions, PI4P sensors only (mCIT-P4MSidM) or mainly (mCIT-PHFAPP1-E50A) label the plasma membrane (PM), which contains the major pool of PI4P in plant cells. (e) Upon PI4P depletion at the PM, PI4P biosensors are expected to relocalize to endosomes. (f) PI4P depletion may also solubilize PI4P sensors into the cytoplasm. (g) mCIT-P4MSidM localizes at the PM when infiltrated alone (see diagram d). (h) In cells expressing MAP-mCH-Sac12-518, mCIT-P4MSidM relocalizes to endosomes (white arrowhead) and is seen in cytoplasmic threads and around large organelles (orange arrowhead), showing it is soluble in the cytosol (see diagrams e and f). (i) mCIT-P4MSidM localization is not affected by the expression of MAP-mCH-Sac1C392S22518. Scale bars: 40 μm

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catalytically dead version of Sac1 phosphatase domain (Sac1C392S2518 in which a catalytic cysteine was mutated into a serine) serves as negative control (Fig. 1a). The N-terminal myristoylation and palmitoylation sequence (MAP) was added to the construct to target the catalytic domain of the phosphatase to the plasma membrane. The addition of a fluorescent protein (FP) to this reporter allowed for the detection MAP-FP-Sac12-518 in transiently transformed cells of N. benthamiana leaves and confirmed that MAP-FPSac12-518 localizes at the plasma membrane. Depending on the level of expression, MAP-FP-Sac12-518 can localize to both plasma membrane and endosomes, which allowed us to test the role of both pools of PI4P on the localization of our protein of interest (Fig. 1b). We confirmed the efficiency and specificity of this PI4P reporter by coexpressing MAP-FP-Sac12-518 together with anionic lipids biosensors. PI4P biosensors, composed of an FP fused to a PI4P lipid-binding domain, can dynamically track PI4P (Fig. 1c) [6, 12, 13]. For instance, mCITRINE-P4MSidM binds to PI4P and highlights the most abundant pool of PI4P at the plasma membrane of plant cells (Fig. 1d, g). When coexpressed with MAP-FP-Sac12-518, mCITRINE-P4MSidM; however, it relocalizes to endosomes where a minor pool of PI4P is found [13]. Depending on the level of expression of MAP-FP-Sac12-518, mCITRINEP4MSidM can solubilize into the cytoplasm when both pools of PI4P, at the plasma membrane and in the endosomes, are depleted (Fig. 1e, f, h). Consistently, MAP-FP-SacC392S2-518 does not cause mCITIRNE-P4MSidM delocalization (Fig. 1i). This system efficiently decreases PI4P levels, and can therefore be used to assess requirement of PI4P for the targeting of extrinsic membrane proteins to the plasma membrane or endosomes. Thus, it is possible to transiently express in Nicotiana benthamiana a fluorescent protein of interest together with MAP-FP-Sac12-518 to deplete PI4P and PI4P biosensors as internal controls. To extend the colors available on our toolbox, we generated mTURQUOISE2 (mTU2, blue) and mCHERRY (mCH, red) fluorescent MAP-FP-Sac12-518 and MAP-FP-SacC392S2-518 as well as mTU2, mCITRINE (mCIT, yellow) and red fluorescent PI4P sensors (Table 1) [6, 13]. Ultimately, this system could also be extended to test the phosphatase activity in planta of enzymes supposedly important for the metabolism of PI4P.

As proof of concept, we successfully coexpressed MAP-mTU2Sac12-518, mCIT-P4MSidM, and 2xmCH-KA1MARK1 (Fig. 2) [7]. The KA1 domain from human MARK1 protein corresponds to a cationic stretch targeting the protein to the plasma membrane via electrostatic interactions [14]. Since PI4P is the main anionic lipid responsible for electronegativity of the PM in plants, the plasma membrane localization of KA1MARK1 in plant cells depends

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Table 1 Possible combinations for double or triple transformation of N. benthamiana

2x35Sprom::MAP-mTU2-Sac11-511

Red (2xmCH-KA1MARK1)

2x35Sprom::MAP-mTU2-Sac1C392S1-511 2x35Sprom::MAP-mCH-Sac11-511 Yellow/Green

Blue

Yellow/Green

Protein tog be tested

Phosphatase assay

Tag for the Anionic lipid biosensors Yellow (mCIT-P4MSidM) Blue Red (mCIT-PHFAPP1-E50A) Lact (mCIT-C2 )

2x35Sprom::MAP-mCH-Sac1C392S1-511 For coinfiltration, anionic lipid biosensor (controls) and the protein of interest to be tested need to be transformed in different leaves, together with the 2x35Sprom::MAP-FP-SAC12-518 and the corresponding catalytic dead version. Transformed cells will express two constructions. For triple transformation of N. benthamiana, anionic lipid biosensor (controls) and the protein of interest need to be transformed in the same leave (transformed cells will coexpress three constructions) together with the 2x35Sprom::MAP-FP-SAC12-518 and the corresponding catalytic dead version

on PI4P [6]. 2xmCH-KA1MARK1 localization was unchanged when coexpressed with MAP-mTU2-Sac1C392S (Fig. 2a–d). On the other hand, 2xmCH-KA1MARK1 delocalized from the plasma membrane upon MAP-mTU2-Sac12-518 induced PI4P-depletion, and colocalized with also delocalized mCIT-P4MSidM (Fig. 2e–g) [6].

2

Materials

2.1 Plants and Agrobacteria

1. Three- to five-week-old Nicotiana benthamiana plants, before vegetative to reproductive transition—no flower buds or flowers formed yet. 2. GV3101 Agrobacterium tumefaciens Strain: Genotype C58 (rifampicin genomic resistance) Ti pMP90 (pTiC58DT-DNA; gentamycin resistant).

2.2

Cell Culture

1. Lysogeny broth (LB) liquid medium: 10 g/L peptone, 5 g/L Yeast Extract, 5 g/L (86 μM) NaCl. 2. LB plates: LB liquid medium supplemented with 14 g/L (~1.4%) agar. 3. Yeast extract broth (YEB) plates: 5 g/L beef extract, 1 g/L yeast extract, 5 g/L peptone, 5 g/L (41 μM) sucrose, 14 g/L (~1.4%) agar, pH 7.2, supplemented with 2 mM MgSO4 (or MgCl2) after autoclaving. 4. Liquid half Murashige and Skoog basal medium (½ MS). 5. 1 M MgCl2 stock solution.

Fig. 2 Assessment of PI4P requirement for subcellular localization of a protein of interest. Here we used 2xmCH-KA1MARK1 as example of protein under study. (a) Description of the schematic representation of the molecular actors drawn in b and e. (b–d) When expressing MAP-FP- Sac1C392S2-518, both PI4P sensor and a protein relying on PI4P for its localization are properly localized. (e–g) Upon expression of MAP-FP-Sac11-511, both mCIT-P4MSidM and 2xmCH-KA1MARK1 change their subcellular localization. They are enriched in intracellular compartments (white arrowheads) and/or solubilized in the cytosol as indicated by cytoplasmic threads and surroundings of large organelles (orange arrowheads). (h and i) Example of quantification. Scale bars: 40 μm

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Fig. 3 First 2 weeks of culture of Nicotiana benthamiana. Seeds are sowed by simply dropping them onto soil. Seedlings sprout under a cut bottle to prevent dehydration (left). The week after, the bottle is removed (right) and seedlings can be pricked out into individual pots

6. 1 M Tris–HCl stock solution, pH 7.5. 7. 10 mM MgCl2 10 mM Tris–HCl pH 7.5. 8. Rifampicin stock solution: 50 mg/mL (60.8 μM) rifampicin (0.5 g rifampicin dissolved in 1.5 mL 0.5 N HCl and brought to 10 mL with water). 9. Gentamycin sulfate stock solution: 20 mg/mL gentamycin sulfate in water. 10. Spectinomycin stock solution: 100 mg/mL (301 μM) in water. 2.3 Supplies and Equipment

1. Half liter plastic bottle cut in half (Fig. 3). 2. 1000 μL pipette tips. 3. Needleless syringes. 4. Spectrophotometer measuring optic density at 600 nm. 5. Microscope slides. 6. Coverslips. 7. Urgopore tape. 8. Scalpel or/and leaf tissue puncher. 9. Confocal microscope equipped with 488 or 515 nm laser lines to excite mCIT; a 561 nm laser line to excite mCH and a 445 nm laser line to excite mTU2.

2.4

Plasmids

Between 50 and 1000 μg/mL of the following vectors compatible for agrobacteria-mediated transient expression in Nicotiana benthamiana leaf epidermis (Table 1):

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1. PI4P biosensor genetic construct in Gateway destination vector UBQ10prom::mCIT-P4MSidM [6]. 2. PI4P biosensor genetic construct in Gateway destination vector UBQ10prom::mCIT-PHFAPP1-E50A [6]. 3. Phosphatidylserine (PS) biosensor genetic construct in Gateway destination vector UBQ10prom::mCIT-C2Lact [7]. 4. Your protein of interest to be tested for delocalization upon PI4P depletion fused to a fluorescent protein cloned in a plant expression vector; for instance, pH7m34GW. As an example in this protocol, we used UBQ10prom::2xmCH-KA1MARK1/ pH7m34GW [7]. 5. PI4P-depletion system in Gateway destination vector: 2x35Sprom::MAP-mTU2-SAC12-518 (or 2x35Sprom::MAPmCH-SAC12-518) and the corresponding mutated version to be used as negative control: 2x35Sprom::MAP-mTU2SAC1C392S2-518 or 2x35Sprom::MAP-mCH-SAC1C392S2-518 (see Note 1).

3

Methods

3.1 Growth of Nicotiana benthamiana

1. Sow N. benthamiana seeds by simply dropping them on a wet soil. Keep the seedlings under plastic bottle cut in half for a week to prevent dehydration (Fig. 3). Keep seeds in a dark and well-ventilated location at 15–20  C. 2. Between 1 and 2 weeks after sowing, prick out seedlings in individual pots (see Note 2) and place plants in a greenhouse or growth chamber.

3.2 Transient Transformation of Nicotiana benthamiana

Steps 1–3 should be conducted in sterile conditions (see Note 3). 1. Transform agrobacteria with each of the vectors of interest (Table 1 and see Subheading 2.4 and [10] for a detailed procedure). 2. Grow agrobacteria strains in the dark on LB agar plates supplemented with the appropriate antibiotics for 48 h at 29  C. Antibiotics should be used at the following concentrations for agrobacteria culture: rifampicin 50 μg/mL; gentamycin 20 μg/mL; spectinomycin 250 μg/mL (five times the concentration used for Escherichia coli). Alternatively, scoop few agrobacteria from a LB plate kept at 4  C for few days or weeks, and spread it on a new one. 3. The day before agroinfiltration, use a pipette tip to scoop few colonies. Gently resuspend them in 300 μL of liquid LB by pipetting in and out several times.

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4. Plate the re-suspended agrobacteria on a YEB agar plate supplemented with the appropriate antibiotics. Spread using sterile glass beads or a cell spreader, and let dry for 30 min. Close the petri dish and incubate in the dark for 16–24 h at 29  C. 5. Using a 1000 μL pipette tip, scoop the bacteria and gently re-suspend them in 1.5 mL of MgCl2 10 mM Tris–HCl 10 mM pH 7.5. Measure the optic density at 600 nm (OD600) (see Note 4). 6. Prepare premixes of agrobacteria containing the different vectors to be coexpressed to reach a total OD600 ¼ 1. 7. For a coinfiltration of two types of agrobacteria, mix them so each has a final OD600 ¼ 0.5 (see Note 5). Double infiltration mixes could, for instance, correspond to: – 2x35Sprom::MAP-mCH-SAC12-518 and UBQ10prom:: mCIT-P4MSidM (technical positive control). – 2x35Sprom::MAP-mCH-SAC1C392S2-518 and UBQ10prom:: mCIT-P4MSidM (technical negative control). – 2x35Sprom::MAP-mCH-SAC12-518 and a construct encoding your protein of interest tagged with green, yellow, or blue fluorescent protein (here UBQ10prom::2xmCHKA1MARK1). – 2x35Sprom::MAP-mCH-SAC1C392S2-518 and a construct encoding your protein of interest tagged with green, yellow, or blue fluorescent protein (negative control). 8. For a coinfiltration of three types of agrobacteria, mix them so that each has an OD600 ¼ 0.33. – 2x35Sprom::MAP-mTU2-SAC12-518 and UBQ10prom:: mCIT-P4MSidM together with your protein of interest (here UBQ10prom::2xmCH-KA1MARK1). – 2x35Sprom::MAP-mTU2-SAC12-518 and UBQ10prom:: mCIT-PHFAPP1-E50A together with your protein of interest (here UBQ10prom::2xmCH-KA1MARK1). – 2x35Sprom::MAP-mTU2-SAC12-518 and UBQ10prom:: mCIT-C2Lact together with your protein of interest (here UBQ10prom::2xmCH-KA1MARK1). – 2x35Sprom::MAP-mTU2-SAC1C392S2-518 and UBQ10prom::mCIT-P4MSidM together with your protein of interest (here UBQ10prom::2xmCH-KA1MARK1). – 2x35Sprom::MAP-mTU2-SAC1C392S2-518 and UBQ10prom::mCIT-PHFAPP1-E50A together with your protein of interest (here UBQ10prom::2xmCH-KA1MARK1).

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– 2x35Sprom::MAP-mTU2-SAC1 C392S2-518 and UBQ10prom:: mCIT-C2Lact together with your protein of interest (here UBQ10prom::2xmCH-KA1MARK1) (see Notes 6 and 7). 9. Use needleless syringes to infiltrate the abaxial side of Nicotiana benthamiana leaves (see Note 8). 10. Place back N. benthamiana plants to the greenhouse or growth chamber (see Note 9). 11. Perform imaging 36 h after agroinfiltration (see Notes 10 and 11). 3.3 Imaging Using Confocal Microscopy

1. Add a droplet of water or ½ MS on a microscope glass slide. 2. Cut a piece of agroinfiltrated leaves using a scalpel or a leaf tissue puncher. 3. Place the leaf piece on the glass slide with the abaxial side facing up. 4. Add another droplet of water or ½ MS on top of the piece of leaf, and cover it with a coverslip. 5. Gently tether the coverslip to the slide with Urgopore tape. 6. Image using a confocal fluorescence microscope. 7. Start by visualizing MAP-mTU2-Sac1C492S2-518 using 445 nm laser excitation, the PI4P biosensor mCIT-P4MSidM, mCITPHFAPP1-E50A or the negative control mCIT-C2Lact using a 488 or 515 nm laser excitation, and your protein of interest fused to mCherry using 561 nm laser excitation. All these proteins should be localized at the plasma membrane of pavement cells (Fig. 2) (see Note 12). 8. Identify and image cells in which MAP-mTU2-Sac12-518 is at the plasma membrane of leaf pavement cell. In cells expressing MAP-mTU2-Sac12-518, the PI4P biosensor mCIT-P4MSidM or mCIT-1xPHFAPP1-E50A should be relocalized to endosomes due to the depletion of PI4P plasma membrane pool (Fig. 2) (see Note 13). 9. In cells expressing MAP-mTU2-Sac12-518, the PS biosensor mCIT-C2Lact should remain at the plasma membrane (see Notes 14–16). 10. Assess the effect of the depletion of PI4P on the localization of your protein of interest by imaging the corresponding fluorescence signal. For example, when coinfiltrated together with MAP-mTU2-Sac12-518, 2xmCH-KA1MARK1 relocalized to the cytoplasm (Fig. 2e–g) (see Note 17).

3.4

Quantification

1. For quantification, only consider cells that have been successfully transformed with all agroinfiltrated constructs. 2. Count the proportion of cells displaying regular localization of the protein of interest when coexpressed with MAP-FP-Sac12-

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and MAP-FP-Sac1C392S2-518. Make sure to sample at least 15 cells per condition. The experiment should be independently replicated three times. 518

3. In case of triple transformations, you can restrict the sampling to cells where the PI4P sensor is efficiently delocalized (Fig. 2h). In case of double transformations, make sure to also quantify the effect of MAP-FP-Sac11-518 and MAP-FPSac1C392S2-518 on PI4P sensor subcellular localization (Fig. 2i) (see Note 18). 4. Perform a statistical analysis on the data obtained using an Fisher’s exact test (suited for small sample size) or a Chi-squared test. In both cases, assess the dependency of the number of cells displaying normal or relocalized distribution of the protein of interest when coexpressed together with MAP-FP-Sac11-518 and MAP-FP-Sac1C392S2-518. For instance, if (a) 22 out of 25 cells coexpressing MP-mCH-Sac1C392S2-518 and the protein of interest, display a normal subcellular localization of the protein of interest at the PM and/or endosomes, and (b) 23 out of 31 cells coexpressing MP-mCH-Sac12-518 and the protein of interest, relocalized the protein of interest; then the corresponding R-commands would be fisher.test (matrix(c(22,3,8,23),2,2, byrow¼TRUE)) and chisq.test (matrix(c(22,3,8,23),2,2, byrow¼TRUE), correct¼FALSE).

4

Notes 1. MAP-mTU2 and MAP-mCH were amplified to add the 12 N-terminal residues (myristoylation and palmitoylation sites) of AtGPA1 (At2g26300) to mTURQUOISE2 using mTURQUOISE2/pEGFP-C1 as template (gift from Joachim Goedhart) or to mCHERRY using mCHERRY/pDONR207 as template. The resulting PCR products were cloned into pDONR221 by BP recombination to generate the MAPmTU2noSTOP/pDONR221 and MAP-mCHnoSTOP/ pDONR221 vectors. The catalytic domains of the Saccharomyces cerevisiae Sac1p (AA 2 to 518 of Sac1p) and Sac1pC392S (carrying a C392S mutation) were amplified using the PSEUDOJANIN (PJ) and PJ-INPP5E plasmids (gift of Gerald Hammond, University of Pittsburg, USA, Addgene #37999) and recombined into pDONR-P2R-P3 by BP recombination to generate SAC1/pDONR-P2R-P3 and SAC1C392S/pDONRP2RP3. Final destination vectors for Agrobacterium-mediated transformation were obtained using LR recombination with pK7m34GW (spectinomycin resistance for bacteria, kanamycin resistance in plants) destination vector for mTU2 constructs,

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and pH7m34GW for mCH constructs (spectinomycin resistance for bacteria, hygromycin resistance in plants) [6]. 2. Four-week-old plants are generally fine for agroinfiltration. Do not use plants that have started to flower. 3. The agroinfiltration protocol described does not require acetosyringone. 4. Be extra careful when resuspending MgCl2Tris solution and avoid vortexing.

agrobacteria

in

5. OD600 generally changes linearly with particle (bacteria) concentration between 0.2 and 1. Therefore, it is necessary to dilute the resuspended agrobacteria prior to assess OD600. In our conditions, when resuspending the content of a petri plate in 2 mL of MgCl2, a 1:40 v/v dilution roughly corresponds to OD600 between 0.2 and 1. 6. Larger volumes of the culture are needed with the construct coding for protein of interest (here UBQ10prom::2xmCHKA1MARK1) than for the other constructs. 7. 2x35Sprom::MAP-mCH-SAC12-518 and 2x35Sprom::MAPmCH-SAC1C392S2-518 can be used instead of the mTU2 constructs. In this case, either use a blue-tagged protein of interest and perform a triple transformation. Alternatively, use a green/ yellow tagged protein of interest and perform double transformations. 8. Carefully chose the leaves to agroinfiltrate. Flat heart-like shaped are the best, rather than roundish that are too old, or than small wavy leaves that are too young. Also, we recommend agroinfiltrating two leaves with the same premix and to image both to reduce variability. 9. Agrobacteria are light sensitive. Therefore, infiltrated Nicotiana benthamiana plants can be kept for few hours in a shadowed location, not taken right away to a growth chamber or greenhouse. 10. When coinfiltrating two constructs with a final OD600 ¼ 1 for the mix, we generally achieved 60–90% cotransformed epidermal cells. 11. Do not exceed a 48 h-window after agroinfiltration to image the leaves. When using strong or mild promoters, proteins start to adopt artefactual localizations, possibly due to aggregation. 12. If using MAP-mCH-Sac1C392S2-518, use 561 nm laser excitation. 13. Upon depletion of PI4P from the plasma membrane, only some PI4P remains in endomembrane compartments. These PI4P-containing organelles move fast in N. benthamiana epidermal cells, and therefore, a spinning disc microscope may be

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better more appropriate for imaging than a scanning confocal microscope. 14. mCIT-P4MSidM sometimes also, or instead, relocalizes to the cytoplasm as fluorescence is seen in cytoplasmic threads and surrounding intracellular compartments (Fig. 1e, f, h). The expression level may be the cause of this effect: the stronger the expression, the more cytosolic the localization (data not shown). 15. Use similar confocal settings when comparing fluorescence intensity or for quantification. 16. For quantitative imaging, images of epidermal root cells were taken with detector settings optimized for low background and no pixel saturation. 17. Mosaic expression is common. Therefore, we recommend coimaging MAP-mTU2-Sac11-511 or MAP-mCH-Sac11-511 together with the protein of interest for subsequent quantification. 18. As blue fluorescence corresponding to MAP-mTU2-Sac12-518 and MAP-mTU2-Sac1C392S2-512 proteins might prove hard to detect, a general quantification can also be performed. However, it requires the transformation efficiency to be high enough (>75%) (Fig. 2i).

Acknowledgments We thank Yvon Jaillais (RDP laboratory, ENS LYON, France) for comments on the manuscript, as well as Romain Boisseau (OBEE department, University of Montana, USA) and Antoine Grissot (Department of Vertebrate Ecology and Zoology, University of Gdansk, Poland) for their advice concerning the statistical analysis. This work was supported by Seed Fund ENS LYON-2016 and Junior Investigator grant ANR-16-CE13-0021. M.D. is funded by a fellowship from the French Ministry of Higher Education. References 1. Monnard PA, Walde P (2015) Current ideas about Prebiological compartmentalization. Life (Basel) 5:1239–1263 2. Platre MP, Jaillais Y (2017) Anionic lipids and the maintenance of membrane electrostatics in eukaryotes. Plant Signal Behav 12: e1282022 3. Zhu Y, Hu L, Zhou Y, Yao Q, Liu L, Shao F (2010) Structural mechanism of host Rab1 activation by the bifunctional legionella type IV effector SidM/DrrA. Proc Natl Acad Sci U S A 107:4699–4704

4. Brombacher E, Urwyler S, Ragaz C, Weber SS, Kami K, Overduin M, Hilbi H (2009) Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of legionella pneumophila. J Biol Chem 284:4846–4856 5. Del Campo CM, Mishra AK, Wang YH, Roy CR, Janmey PA, Lambright DG (2014) Structural basis for PI(4)P-specific membrane recruitment of the legionella pneumophila effector DrrA/SidM. Structure 22:397–408

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6. Simon ML, Platre MP, Marques-Bueno MM, Armengot L, Stanislas T, Bayle V, Caillaud MC, Jaillais Y (2016) A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants. Nat Plants 2:16089 7. Platre MP, Noack LC, Doumane M, Bayle V, Simon MLA, Maneta-Peyret L, Fouillen L, Stanislas T, Armengot L, Pejchar P, Caillaud MC, Potocky M, Copic A, Moreau P, Jaillais Y (2018) A combinatorial lipid code shapes the electrostatic landscape of plant endomembranes. Dev Cell 45(465–80):e11 8. Lemmon MA (2008) Membrane recognition by phospholipid-binding domains. Nat Rev Mol Cell Biol 9:99–111 9. Noack LC, Jaillais Y (2017) Precision targeting by phosphoinositides: how PIs direct endomembrane trafficking in plants. Curr Opin Plant Biol 40:22–33 10. Noack LC, Pejchar P, Sekeres J, Jaillais Y, Potocky M (2019) Transient gene expression as a tool to monitor and manipulate the levels

of acidic phospholipids in plant cells. Methods Mol Biol 1992:189–199 11. Guo S, Stolz LE, Lemrow SM, York JD (1999) SAC1-like domains of yeast SAC1, INP52, and INP53 and of human synaptojanin encode polyphosphoinositide phosphatases. J Biol Chem 274:12990–12995 12. Hammond GR, Machner MP, Balla T (2014) A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi. J Cell Biol 205:113–126 13. Simon ML, Platre MP, Assil S, van Wijk R, Chen WY, Chory J, Dreux M, Munnik T, Jaillais Y (2014) A multi-colour/multi-affinity marker set to visualize phosphoinositide dynamics in Arabidopsis. Plant J 77:322–337 14. Moravcevic K, Mendrola JM, Schmitz KR, Wang YH, Slochower D, Janmey PA, Lemmon MA (2010) Kinase associated-1 domains drive MARK/PAR1 kinases to membrane targets by binding acidic phospholipids. Cell 143:966–977

Chapter 10 Subcellular Localization of PI3P in Arabidopsis Han Nim Lee, Hyera Jung, and Taijoon Chung Abstract Phosphatidylinositol-3-phosphate (PI3P) is a signaling phospholipid enriched in the membranes of late endosomes (LE) and vacuoles. PI3P mediates vacuolar and endosomal trafficking through recruiting PI3Pbinding effector proteins to the LE. PI3P is produced from phosphatidylinositol by the PI 3-kinase complex containing VACUOLAR PROTEIN SORTING 34 (VPS34). The role of PI3P has been elucidated by using genetically encoded PI3P biosensors. We previously showed that Arabidopsis VPS38, a component of the VPS34 complex, localized at the LE and that VPS38 is essential for proper PI3P distribution in endosomal and vacuolar trafficking routes. In this chapter, we describe methods for microscopic imaging of PI3P using the PI3P biosensor citrine-2  FYVE and the PI 3-kinase inhibitors. Key words Phosphoinositide, Late endosomes, Multivesicular body, Wortmannin, 3-methyladenine, Arabidopsis thaliana

1

Introduction Plant vacuoles are the destination of various cargo proteins. First, biosynthetic cargoes like storage proteins and vacuolar hydrolases are synthesized at the endoplasmic reticulum (ER), typically move through the Golgi apparatus, trans-Golgi network (TGN), and multivesicular body (MVB) to reach the vacuole [1]. The cargo proteins are often recognized by VACUOLAR SORTING RECEPTORs (VSRs), whose retrograde transport requires the retromer complex [2]. Secondly, a portion of endocytosed plasma membrane proteins, such as PINFORMED 2 (PIN2) and BRASSINOSTEROID-INSENSITIVE 1 (BRI1), is targeted to the vacuoles for degradation. This endosomal trafficking route comprises the early endosome (EE) and the late endosome (LE), which in plant cells are equivalent to the TGN and the MVB, respectively [1, 3, 4]. The ENDOSOMAL SORTING COMPLEXES REQUIRED FOR TRANSPORT (ESCRTs) are required for the internalization of ubiquitinated membrane proteins into intraluminal vesicles of the LE/MVB. A third route to the vacuole

Marisa S. Otegui (ed.), Plant Endosomes: Methods and Protocols, Methods in Molecular Biology, vol. 2177, https://doi.org/10.1007/978-1-0716-0767-1_10, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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is autophagy, by which cytoplasmic constituents are sequestered into double membrane-bound organelles, autophagosomes, and delivered to the vacuole for degradation [5, 6]. Phosphoinositides, or the phosphorylated derivatives of phosphatidylinositol, represent less than 1% of the membrane phospholipids [7, 8]. Their inositol head groups can be phosphorylated and dephosphorylated at the D-3, D-4, or D-5 position by the combined action of kinases and phosphatases. Through the different locations of each phosphorylated species in the endomembrane system, phosphoinositides confer identity to membrane compartments and are involved in dynamic membrane trafficking [9, 10]. In plant cells, D4-phosphorylated phosphoinositides are enriched in the plasma membrane, whereas D3-phosphorylated phosphoinositides are associated with endosomal and vacuolar membrane [9, 11]. Phosphatidylinositol-3-phosphate (PI3P) is a D3-phosphorylated phosphoinositide important for endosomal, vacuolar, and autophagic trafficking in yeast. PI3P is mostly enriched at the LE/MVB membrane and produced from phosphatidylinositol by the phosphatidylinositol 3-kinase (PI 3-kinase) VACUOLAR PROTEIN SORTING 34 (VPS34). In yeast and mammals, Vps34 homologs are found in at least two distinct PI 3-kinase complexes. The Vps34 complex I, consisting of Vps34, Vps15, Autophagy-related 6 (Atg6)/Vps30, and Atg14, regulates an early step of autophagy at the site of autophagosome formation, whereas the Vps34 complex II containing Vps34, Vps15, Atg6/ Vps30, and Vps38 (or UVRAG in mammals) is involved in vacuolar protein sorting and localized to the endosome [12, 13]. Genetically encoded biosensors are valuable tools for determining the subcellular distribution of PI3P and other phosphoinositides [11]. The 2  FYVE biosensor employs tandem repeats of the FYVE (Fab1, YOTB/ZK632.12, Vac1, and EEA1) domain, which specifically binds to PI3P. In Arabidopsis, the 2xFYVE biosensor mostly decorates the LE/MVBs and sometimes the tonoplast [11, 14]. Both chemical and genetic inhibition of PI3P metabolism has been used to understand the essential functions of PI3P in plant development and protein trafficking. Wortmannin (Wm), an inhibitor of PI 3-kinases, interferes with vacuolar protein sorting [15, 16] and induces the enlargement of LE/MVBs [17, 18]. Wm also appears to affect endosomal and autophagic trafficking, because vacuoles of Wm-treated Arabidopsis seedlings do not accumulate endocytosed PIN2 [19] and autophagic marker ATG8 [20]. Nevertheless, nonspecific PI-3-kinase inhibitors like Wm, LY294002, and 3-methyladenine (3-MA) also inhibit phosphoinositide 4-kinases and other related kinases in mammals [21, 22], necessitating independent validation by genetic intervention of PI3P production and action. Genetic ablation of PI 3-kinase

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activity in Arabidopsis has provided limited information because pollen development is abnormal in knockout mutants for shared components of two VPS34 complexes (VPS34, VPS15, and ATG6/VPS30) [23–25]. Alternative approaches may include an inducible knockdown strategy and mutations for specific PI3P effectors. PI3P effectors involved in plant endosomal trafficking and their specific functions have been characterized. For example, the plantspecific ESCRT component FREE1/FYVE1 binds PI3P [26, 27]. The retromer subcomplex SORTING NEXINs (SNXs) are targeted to both the TGN/EE and LE/MVBs by binding PI3P [28, 29]. Complete knockout mutation in FYVE1/FREE1 causes lethality [30], whereas snx1 snx2a snx2b triple mutants are viable and show minor developmental phenotypes [29]. More investigation is needed to determine which SNX mutant combinations are useful to study the role of PI3P in endosomal sorting [2]. Moreover, FREE1/FYVE1 and other potential PI3P effectors may play multiple roles in both endosomal and autophagic trafficking [10]. Recent work from our laboratory [31] and others’ [32] has identified an Arabidopsis homolog of yeast Vps38 that is specific to the Vps34 complex II. Arabidopsis vps38 mutants are defective in endosomal sorting of PIN2 and showed abnormal distribution of VSR2;1 [31]. Although the mutants contain enlarged endosomes and show an altered distribution of PI3P biosensors, PI3P content in the vps38 mutants does not differ from that in wild type. Moreover, vps38 mutations mimic the effects of Wm treatment on endosomal and retromer-mediated pathways but not on the autophagic pathway [31]. These data indicate that VPS38 is specifically responsible for the local production of PI3P in endosomes. Here, we describe procedures for generating Arabidopsis vps38 mutants (or any mutants of choice) expressing the PI3P biosensor citrine2  FYVE and detecting subcellular distribution of PI3P biosensors. We also describe our protocol for treatment of Arabidopsis seedlings with Wm and 3-MA.

2

Materials

2.1 Plant Media and Growth

1. Sterile hood. 2. 50% (v/v) bleach. 3. Sterile water. 4. Sterile 1 mL pipette tips. 5. Arabidopsis seeds expressing citrine-2  FYVE (see Notes 1 and 2) [11]. 6. 1 Murashige and Skoog (MS) medium containing 1% (w/v) sucrose and 0.6% (w/v) Phytagel, pH 5.7.

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7. Petri dishes. 8. Micropore tape. 2.2 Crossing Arabidopsis Mutants

1. Fine forceps. 2. Scissors with fine tips. 3. Micropore tape. 4. Ethanol. 5. Magnifying glasses. 6. 5 mg/mL glufosinate ammonium (Basta) in sterile water. 7. Solid medium containing 1 MS, 1% (w/v) sucrose, 0.6% (w/v) Phytagel, pH 5.7, supplemented with 5 μg/mL Basta.

2.3 Inhibitor Treatment and Confocal Microscopy

1. 1 MS liquid medium with 1% (w/v) sucrose, pH 5.7 adjusted with 1 M KOH. 2. 24-well plates. 3. Dimethyl sulfoxide (DMSO). 4. 30 mM Wm in DMSO. 5. 1 MS liquid medium containing 5 mM 3-MA (see Notes 3 and 4). 6. Confocal laser scanning microscope. 7. Glass slides. 8. Coverslips.

2.4 Image Analysis Software

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1. FIJI [33].

Methods

3.1 Preparation of Arabidopsis Plants for Crossing

1. Place Arabidopsis seeds in a 1.5 mL tube and add 50% bleach. 2. Gently vortex the tube and briefly spin down for 5 s. Incubate for 15 min. 3. In a sterile hood, discard bleach solution using sterile pipette tips. Rinse the seeds three times with sterile water. 4. Sow the surface-sterilized seeds onto MS solid medium in a petri dish. 5. Seal the petri dish with micropore tape. 6. Stratify the seeds by placing the petri dish at 4  C for 3 days. 7. Incubate the petri dish at 22  C under a long-day photoperiod (16-h light/8-h dark). 8. Transfer 2-week-old plants to pots. Allow the plants to grow further and flower.

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1. Clean tips of forceps and scissors with ethanol and Kimwipes. Briefly air-dry them. 2. Use magnifying subsequent steps.

glasses

or

dissecting

microscope

for

3. For the female parent (a mutant of your choice; see Note 5), choose one or two young flowers in which petals are not open yet and anthers do not reach the stigma. Using forceps and scissors, remove all the flower organs except the carpel. 4. Cut other flowers on the inflorescence except those selected for crossing. 5. For the male parent (see Note 5), select an open flower with fresh pollen. Use forceps to take one or two stamens from the flower. 6. Pollinate by gently rubbing anthers of the dissected stamen to the stigmatic surface of the carpels on the female part. 7. Label the crosses with a small piece of micropore tape around the peduncle (flower stem) of the female parent. 8. For another round of crossing, repeat steps 1–8. 9. After 2–3 weeks, collect siliques containing F1 seeds when they start to turn yellow. 10. Air dry the siliques in a microcentrifuge tube until the silique dehisces (opens). 3.3 Selection of F1 and F2 Plants Expressing Citrine2  FYVE Reporter

1. Plate all F1 seeds on a solid growth medium containing 5 μL/mL Basta (see Note 5), as described in steps 1–7 of Subheading 3.1. 2. Select Basta-resistant seedlings about 1 week later (see Note 5). 3. Transfer selected F1 plants to soil. Allow the plants to flower and self-pollinate. 4. Collect siliques containing F2 seeds. 5. Germinate ~40 F2 seeds, as described in Subheading 3.1. Use solid growth medium containing appropriate antibiotics/herbicide for selection. 6. Perform genotyping test to select homozygous mutants expressing the Citrine-2  FYVE transgene (see Note 6).

3.4 Inhibitor Treatment and Confocal Microscopy

1. Surface-sterilize wild-type and mutant seeds expressing Citrine-2  FYVE, as described in steps 1–3 in Subheading 3.1. 2. Prepare 24-well plates containing 1 mL liquid medium per well. Place 10 to 15 seeds on each well. 3. Seal the plate with micropore tape and stratify the seeds at 4  C in darkness for 3 days.

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Fig. 1 vps38 affects the intracellular distribution of PI3P. Confocal microscopic images of wild-type (a and c) and vps38 (b) roots expressing Citrine-2  FYVE. Wild type and vps38 seedlings were treated with either 30 μM Wm (c) or DMSO (a and b) for 1 h before confocal imaging. Note that Citrine-2  FYVE-labeled compartments are larger and ring-shaped in vps38 and Wm-treated wild type than DMSO-treated wild type. Scale bars: 5 μm

4. Place the plates on a rotary shaker (~100 rpm). Incubate the plates at 22  C under a long-day photoperiod (16-h light/8h dark). 5. In a sterile hood, remove liquid medium and transfer 5-day-old seedlings into a new 1.5 mL tube. Add either 1 μL of 30 mM Wm stock solution or DMSO to each tube and mix by pipetting. Transfer media containing Wm or DMSO back to the well (see Note 7). Alternatively, fresh medium containing 3-MA can be used. 6. Incubate the seedlings for 1 h at 22  C on a rotary shaker before imaging. 7. Place the seedling on a slide glass and remove shoot parts. 8. Add a drop of liquid medium in which the seedlings were incubated and cover with a coverslip. 9. Observe the meristematic/transition zone of root epidermal cells using confocal microscopy. To detect citrine-2  FYVE fluorescence, use a standard setting for GFP. A typical confocal image obtained is shown in Fig. 1. 10. Use Fiji (https://fiji.sc/) to quantify the number and the size of intracellular compartments labeled by citrine-2  FYVE. Convert confocal microscope images to 8-bit and use Analyze Particles function followed by adjusting threshold.

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Notes 1. The Citrine-2  FYVE seed is available from the Arabidopsis Biological Resource Center (ABRC, Columbus, OH) with Germplasm ID CS2105611. More PIPline biosensors with

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Fig. 2 Late endosomes (LE) in Arabidopsis roots are enlarged by treatment with 3-MA, a nonspecific inhibitor of PI 3-kinases. Seven-day-old seedlings expressing YFP-RabF2b/ARA7 (ABRC stock CS781647) were incubated in nitrogendeficient liquid medium for 24 h, then incubated with new nitrogen-deficient medium containing 5 mM 3-MA (b) or not (a) for 24 h. Confocal images were obtained from root tips. Scale bars: 5 μm

different fluorescent tags and affinities to other phosphoinositides are also available from the ABRC. 2. Overexpression of lipid-binding biosensors can interfere with the function of endogenous phosphoinositides. To avoid strong overexpression, all PIPline transgenes are expressed under the control of Arabidopsis UBIQUITIN 10 gene [11]. No apparent morphological abnormality was observed in the Citrine-2  FYVE transgenic line. 3. The medium containing 5 mM 3-MA should be prepared freshly prior to use. Add 3.7 mg of 3-MA to 5 mL of liquid medium, heat the medium to ~40  C for dissolving 3-MA, and filter through a 0.45-μm syringe filter. 4. Figure 2 shows the effect of 3-MA on an endosomal marker. Although 3-MA has been used as an autophagy inhibitor in plant cells, 3-MA treatment also leads to enlarged endosomes. Thus, 3-MA is not a specific inhibitor of autophagy. 5. If Citrine-2  FYVE plants are used as a pollen donor and if the carpel donor (or female parent) is sensitive to Basta, a successful crossing will result in F1 plants with Basta resistance, and seeds from unwanted self-pollination events will be Basta-sensitive. If antibiotics/herbicide selection is not possible (e.g., the female parent is also Basta-resistant), inspect with a fluorescence microscope and select the seedlings with fluorescence. In any case, it is recommended to confirm F1 candidates by genotyping. 6. About 75% of F2 individuals will express Citrine-2  FYVE transgene and show Basta resistance. Two-thirds of the Bastaresistant F2 individuals will be heterozygous for Citrine-

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2  FYVE transgene, whereas one-third will be homozygous. Basta selection and microscopic observation can be used to select F3 progeny self-pollinated from the homozygote, as the homozygous F3 population will uniformly express a Bastaresistance gene and Citrine-2  FYVE transgene. 7. The working concentration (~30 μM) of Wm inhibits not only PI 3-kinases but also PI 4-kinases and affects the morphology of both the MVB and TGN [34, 35]. References 1. Viotti C, Bubeck J, Stierhof YD, Krebs M, Langhans M, van den Berg W, van Dongen W, Richter S, Geldner N, Takano J, Jurgens G, de Vries SC, Robinson DG, Schumacher K (2010) Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle. Plant Cell 22 (4):1344–1357. https://doi.org/10.1105/ tpc.109.072637 2. Heucken N, Ivanov R (2018) The retromer, sorting nexins and the plant endomembrane protein trafficking. J Cell Sci 131(2). https:// doi.org/10.1242/jcs.203695 3. Paez Valencia J, Goodman K, Otegui MS (2016) Endocytosis and endosomal trafficking in plants. Annu Rev Plant Biol 67:309–335. https://doi.org/10.1146/annurev-arplant043015-112242 4. Scheuring D, Viotti C, Kruger F, Kunzl F, Sturm S, Bubeck J, Hillmer S, Frigerio L, Robinson DG, Pimpl P, Schumacher K (2011) Multivesicular bodies mature from the trans-Golgi network/early endosome in Arabidopsis. Plant Cell 23(9):3463–3481. https:// doi.org/10.1105/tpc.111.086918 5. Kim SH, Kwon C, Lee JH, Chung T (2012) Genes for plant autophagy: functions and interactions. Mol Cells 34(5):413–423. https:// doi.org/10.1007/s10059-012-0098-y 6. Ding X, Zhang X, Otegui MS (2018) Plant autophagy: new flavors on the menu. Curr Opin Plant Biol 46:113–121. https://doi. org/10.1016/j.pbi.2018.09.004 7. Heilmann I (2016) Phosphoinositide signaling in plant development. Development 143 (12):2044–2055. https://doi.org/10.1242/ dev.136432 8. Munnik T, Nielsen E (2011) Green light for polyphosphoinositide signals in plants. Curr Opin Plant Biol 14(5):489–497. https://doi. org/10.1016/j.pbi.2011.06.007 9. Noack LC, Jaillais Y (2017) Precision targeting by phosphoinositides: how PIs direct

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Subcellular Localization of PI3P Odorizzi G, Otegui MS (2007) The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. Plant Cell 19(4):1295–1312. https://doi.org/10.1105/tpc.106.049346 18. Wang J, Cai Y, Miao Y, Lam SK, Jiang L (2009) Wortmannin induces homotypic fusion of plant prevacuolar compartments. J Exp Bot 60 (11):3075–3083. https://doi.org/10.1093/ jxb/erp136 19. Kleine-Vehn J, Leitner J, Zwiewka M, Sauer M, Abas L, Luschnig C, Friml J (2008) Differential degradation of PIN2 auxin efflux carrier by retromer-dependent vacuolar targeting. Proc Natl Acad Sci U S A 105(46):17812–17817. https://doi.org/10.1073/pnas.0808073105 20. Zhuang X, Wang H, Lam SK, Gao C, Wang X, Cai Y, Jiang L (2013) A BAR-domain protein SH3P2, which binds to phosphatidylinositol 3-phosphate and ATG8, regulates autophagosome formation in Arabidopsis. Plant Cell 25 (11):4596–4615. https://doi.org/10.1105/ tpc.113.118307 21. Ronan B, Flamand O, Vescovi L, Dureuil C, Durand L, Fassy F, Bachelot MF, Lamberton A, Mathieu M, Bertrand T, Marquette JP, El-Ahmad Y, Filoche-Romme B, Schio L, Garcia-Echeverria C, Goulaouic H, Pasquier B (2014) A highly potent and selective Vps34 inhibitor alters vesicle trafficking and autophagy. Nat Chem Biol 10 (12):1013–1019. https://doi.org/10.1038/ nchembio.1681 22. Bago R, Malik N, Munson MJ, Prescott AR, Davies P, Sommer E, Shpiro N, Ward R, Cross D, Ganley IG, Alessi DR (2014) Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase. Biochem J 463(3):413–427. https://doi.org/10.1042/BJ20140889 23. Xu N, Gao XQ, Zhao XY, Zhu DZ, Zhou LZ, Zhang XS (2011) Arabidopsis AtVPS15 is essential for pollen development and germination through modulating phosphatidylinositol 3-phosphate formation. Plant Mol Biol 77 (3):251–260. https://doi.org/10.1007/ s11103-011-9806-9 24. Lee Y, Kim ES, Choi Y, Hwang I, Staiger CJ, Chung YY, Lee Y (2008) The Arabidopsis phosphatidylinositol 3-kinase is important for pollen development. Plant Physiol 147 (4):1886–1897. https://doi.org/10.1104/ pp.108.121590

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25. Fujiki Y, Yoshimoto K, Ohsumi Y (2007) An Arabidopsis homolog of yeast ATG6/VPS30 is essential for pollen germination. Plant Physiol 143(3):1132–1139. https://doi.org/10. 1104/pp.106.093864 26. Gao C, Luo M, Zhao Q, Yang R, Cui Y, Zeng Y, Xia J, Jiang L (2014) A unique plant ESCRT component, FREE1, regulates multivesicular body protein sorting and plant growth. Curr Biol 24(21):2556–2563. https://doi.org/10.1016/j.cub.2014.09.014 27. Kolb C, Nagel MK, Kalinowska K, Hagmann J, Ichikawa M, Anzenberger F, Alkofer A, Sato MH, Braun P, Isono E (2015) FYVE1 is essential for vacuole biogenesis and intracellular trafficking in Arabidopsis. Plant Physiol 167 (4):1361–1373. https://doi.org/10.1104/ pp.114.253377 28. Phan NQ, Kim SJ, Bassham DC (2008) Overexpression of Arabidopsis sorting nexin AtSNX2b inhibits endocytic trafficking to the vacuole. Mol Plant 1(6):961–976. https://doi. org/10.1093/mp/ssn057 29. Pourcher M, Santambrogio M, Thazar N, Thierry AM, Fobis-Loisy I, Miege C, Jaillais Y, Gaude T (2010) Analyses of sorting nexins reveal distinct retromer-subcomplex functions in development and protein sorting in Arabidopsis thaliana. Plant Cell 22 (12):3980–3991. https://doi.org/10.1105/ tpc.110.078451 30. Barberon M, Dubeaux G, Kolb C, Isono E, Zelazny E, Vert G (2014) Polarization of IRON-REGULATED TRANSPORTER 1 (IRT1) to the plant-soil interface plays crucial role in metal homeostasis. Proc Natl Acad Sci U S A 111(22):8293–8298. https://doi.org/ 10.1073/pnas.1402262111 31. Lee HN, Zarza X, Kim JH, Yoon MJ, Kim SH, Lee JH, Paris N, Munnik T, Otegui MS, Chung T (2018) Vacuolar trafficking protein VPS38 is dispensable for autophagy. Plant Physiol 176(2):1559–1572. https://doi.org/ 10.1104/pp.17.01297 32. Liu F, Hu W, Vierstra RD (2018) The vacuolar protein sorting-38 subunit of the Arabidopsis phosphatidylinositol-3-kinase complex plays critical roles in autophagy, endosome sorting, and gravitropism. Front Plant Sci 9:781. https://doi.org/10.3389/fpls.2018.00781 33. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an opensource platform for biological-image analysis.

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35. Takac T, Pechan T, Samajova O, Samaj J (2013) Vesicular trafficking and stress response coupled to PI3K inhibition by LY294002 as revealed by proteomic and cell biological analysis. J Proteome Res 12(10):4435–4448. https://doi.org/10.1021/pr400466x

Chapter 11 Immunopurification of Intact Endosomal Compartments for Lipid Analyses in Arabidopsis Yoko Ito, Magali Grison, Nicolas Esnay, Laetitia Fouillen, and Yohann Boutte´ Abstract Endosomes play a major role in various cellular processes including cell–cell signaling, development and cellular responses to environment. Endosomes are dynamically organized into a complex set of endomembrane compartments themselves subcompartmentalized in distinct pools or subpopulations. It is increasingly evident that endosome dynamics and maturation is driven by local modification of lipid composition. The diversity of membrane lipids is impressive and their homeostasis often involves crosstalk between distinct lipid classes. Hence, biochemical characterization of endosomal membrane lipidome would clarify the maturation steps of endocytic routes. Immunopurification of intact endomembrane compartments has been employed in recent years to isolate early and late endosomal compartments and can even be used to separate subpopulations of early endosomes. In this section, we will describe the immunoprecipitation protocol to isolate endosomes with the aim to analyze the lipid content. We will detail a procedure to identify the total fatty acid and sterol content of isolated endosomes as a first line of lipid identification. Advantages and limitations of the method will be discussed as well as potential pitfalls and critical steps. Key words Organelles, Immunopurification, Golgi, Endosomes, Lipids, Fatty acids, Mass spectrometry, Plants

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Introduction The endosomal network is a highly coordinated set of endomembrane compartments which host materials coming from early endocytic events at the plasma membrane through early endosomes (EEs), which are then dispatched over recycling pathways back to the plasma membrane or degradation pathways through late endosomes (LEs). However, plants have no EEs as described in animals, and endocytic vesicles converge directly to the trans-Golgi Network (TGN) where the endocytic tracer FM4-64 was found to accumulate relatively fast (couple of minutes) during endocytosis

Yoko Ito and Magali Grison contributed equally to this work. Marisa S. Otegui (ed.), Plant Endosomes: Methods and Protocols, Methods in Molecular Biology, vol. 2177, https://doi.org/10.1007/978-1-0716-0767-1_11, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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before reaching LEs [1–3]. TGN is a tubulovesicular membrane network that originates from the trans-most cisternae of the Golgi apparatus, acting as a main central station in endosomal trafficking. In plants, the TGN can undergo homotypic fusion and can associate transiently with Golgi apparatus, similarly to what is found for EEs and TGN in mammalian cells [2, 4]. Moreover, plant TGN can detach from the Golgi apparatus to form a highly dynamic Golgiindependent structure (Golgi-Independent TGN; GI-TGN) [2, 4]. From electron microscopy (EM) ultrastructural studies, at least three populations of TGN-associated vesicles could be identified, the secretory vesicles (TGN-SVs) that do not bear any apparent coat and which size vary between 50 and 100 nm in diameter, the clathrin vesicles (TGN-CCVs) that possess a clathrin coat and measure 35 nm, and the COPIb-type of vesicles that carry a COPI coat and measure 45 nm in diameter [5–7]. From confocal colocalization studies we know that TGN-SVs are marked with the V-ATPase VHA-a1, the syntaxin proteins SYP61 and SYP43 and the protein ECHIDNA while TGN-CCVs are marked with the RAB-GTPase RAB-A2a [1, 6, 8–14]. It is not completely clear if TGN-SVs and TGN-CCVs can both contain endocytic materials; however, EM and live-cell observations suggest that endocytosis tracers are found at GI-TGN within a few minutes after endocytosis, and both the CCV marker clathrin and the SV marker VHA-a1 localize on GI-TGN more than on Golgi-associated TGN [2, 3, 6]. Hence, to completely understand the time frame and maturation steps of endocytic routes, there is a stumbling block to overcome. Cell biology approaches indicate that TGN and endosomes are made of subpopulations that might not have the same lipid and protein content, reflecting the maturation and dynamics of these endomembrane compartments. This view should now be scaled up with multidisciplinary approaches to understand the chemistry and biochemistry of membranes including proteins and lipids. In recent years, the development of methods allowing for the purification of organelles using different specific baits opened the door to the biochemical characterization of distinct endocytic compartments. Isolation of intracellular organelles was done previously by classical density gradient centrifugation but these methods have several limitations due to similar densities of distinct intracellular compartments and the diversity of small compartments found in one fraction. Localization of organelle proteins by isotope tagging (LOPIT) has been used on iodixanol gradients to group proteins using principal component analysis and allowed to clearly separate Golgi proteins from endoplasmic reticulum (ER) proteins [15]. The Golgi apparatus was also isolated by combination of density gradient and the surface charge separation technique called free-flow electrophoresis (FFE) [16]. Very recently, a subtle combination of LOPIT and FFE was used to determine the proteins and glycan content of Golgi cisternae from the ER to the trans-most

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side of the Golgi apparatus [17]. However, these methods do not allow for separation of subpopulations of endosomes, and compartments such as TGN-SVs and TGN-CCVs would be hard to isolate independently. Nonetheless, EM studies have identified the presence of proteins to specific subpopulations of TGN or endosomes [6, 9, 13]. Hence, these hallmarks can be used as tags to isolate compartments in a target specific way with high specificity using immunoaffinity approaches. The first study used SYP61 fused to GFP as a bait for TGN using anti-GFP antibodies coupled to agarose-beads as affinity ligand [18]. This method was also adapted to magnetic beads using VHA-a1 fused to YFP as TGN bait and coupled to LOPIT technique [19]. Immunoisolation was further used on a range of markers belonging to the Wave collection [20] labeling the Golgi, TGN, EEs, or LEs [21]. Immunoisolation procedures have proved to be very useful to determine the proteome of intact and full Golgi cisternae, TGN, EEs, or LEs and get a clearer view of the identity of these compartments. It has also become increasingly evident that lipids are key determinants of membrane identity, sorting mechanisms and maturation processes. A well-known process accompanying endocytosis and maturation of endosomes in mammalian cells is the conversion of phosphoinositides (PIPs), which are negatively charged phosphorylated forms of phospholipids [22]. This change in lipid composition drives the recruitment of distinct RAB GTPases and defines a new identity to the compartment. Another example of lipid involvement in membrane maturation is the accumulation of phosphatidic acid (PA), a lipid which favor vesicle fission, at the neck of constricted COPI vesicles through the action of phospholipase D (PLD) that converts phosphatidylcholine (PC) into PA [23]. In plants, immunoisolation of distinct subdomains of TGN using magnetic beads coupled to anti-GFP antibodies has successfully reached the separation of TGN-associated SVs (using SYP61-CFP as a bait) and TGN-associated CCVs (using RAB-A2a-YFP as a bait) to analyze lipid composition [14]. Strikingly, an enrichment of α-hydroxylated very-long-chain fatty acids (hVLCFAs), a specific signature of sphingolipids (SLs), was observed together with an enrichment of sterols at SVs as compared to CCVs [14]. Characterizing the complete fatty acids (FAs) and sterols profile of immunoisolated compartments by gas chromatography–mass spectrometry (GC-MS) is the first step to the in-depth characterization of lipid composition. Certainly, lipidomic profiling using liquid chromatography mass spectrometry (LC-MS) would definitely be the most powerful approach to identify several classes and species of lipids in one sample. However, the diversity of lipids is estimated to include more than forty thousand molecular species as referenced in LIPID MAPS online database (https://www. lipidmaps.org/). A general definition of lipids would be a hydrophobic or amphiphilic molecular assembly soluble in organic

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solvents. This definition includes a huge structural diversity ranging from but not limited to (1) glycerolipids composed from a glycerol backbone on which one, two or three FAs are esterified, (2) glycerophospholipids composed from a glycerol backbone on which one or two FAs, a phosphate group and amino alcohols are esterified, (3) sphingolipids composed of a ceramide backbone (made of a sphingoid base amidified with a FA) which can be further modified by glycosylation, phosphorylation, or addition of an inositol phosphate residue which can be glycosylated, (4) sterols constituted from a steroid backbone which can be modified by glycosylation and/or addition of a FA chain in the case of the sterylesters, glycosylesters, or acetylglycosylesters. The acyl chains of lipids can be hydroxylated and unsaturated to different degree and position resulting in even more diverse structures. The polar head grafted to the hydrophobic chains confers diverse polarity index to the molecular assembly. Hence, depending on the polarity of the mix of solvents used to extract lipids, some classes or species of lipids might be more efficiently extracted as compared to others, introducing a bias in their quantification. Some lipids such as the phosphorylated lipids, are more unstable than others, also skewing lipid quantification. Hence, using only one extraction method to quantify all lipids at once is not feasible. In the future, improved extraction and LC-MS methodologies will need to address these pitfalls to identify and compare the different lipid classes found in immunopurified compartments. LC-MS clearly has the potential to identify major and minor lipid species without potential biases from the separation and desorption of lipids from high-performance thin layer chromatography (HPTLC) plates. For this purpose, performing a complete FA and sterol profile is a useful first-line of identification and characterization. Here, we describe the immunoisolation procedure used for lipid analyses. We also explain the procedure to perform a complete FA and sterol profiling of immunoisolated compartments, both at the preparative and analytical level.

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Materials Because this protocol is for immunoprecipitation of intact membrane compartments, the GFP tag has to be exposed to the cytosolic side of the membrane so that the anti-GFP antibody can bind to it. We recommend using transmembrane proteins (e.g., SNAREs [18] or V-ATPases [19]) as baits. However, some membrane associated proteins (e.g., RABs) work as well [14, 21]. However, as RAB proteins can potentially dissociate from the membranes during purification and therefore not yield enough purified organelles, this protocol also describes and illustrates how to evaluate the efficiency of IP and validation of the extraction. The amount of

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the plant material is one of the most crucial points of this experiment. At least 10 g (fresh weight) of seedling materials should be obtained for one IP. Seedlings can be collected from MS plates (12  12 cm) or from liquid culture. However, to reach 10 g of fresh materials 200 μL of seeds (120–160 mg of dry seeds) need to be sowed. In this protocol we will describe a liquid culture method allowing for easy production of fresh material in a relatively small amount of time. 2.1 Plant Material and Growth

1. Arabidopsis thaliana seeds expressing a GFP-tagged organelle marker that has been chosen as a bait. 2. Sterilization solution: 1% (w/v) sodium dichloroisocyanurate in sterile deionized water. 3. MS medium: 4.4 g/L Murashige and Skoog medium including vitamins, 10 g/L sucrose, and 0.5 g/L 2-(N-morpholino) ethanesulfonic acid (MES). Adjust the pH to 5.8 with KOH and autoclave at 110  C for 30 min. 4. 70% (v/v) ethanol in H2O. 5. 500 mL baffled flasks.

2.2 Membrane Isolation and Protein Quantification

1. 50 mM HEPES in H2O. Adjust the pH to 7.5 with KOH and keep at 4  C. 2. Vesicle isolation buffer: 0.45 M sucrose, 5 mM MgCl2, 1 mM dithiothreitol (DTT), and 0.5% Polyvinylpyrrolidone (PVP, see Note 1) dissolved in 50 mM HEPES pH 7.5. Store at 20  C and keep at 4  C for use. Just before use, add phenylmethylsulfonyl fluoride (PMSF) at the final concentration of 1 mM. 3. Wash buffer: 0.25 M sucrose, 1.5 mM MgCl2, 0.2 mM EDTA (pH 8), and 150 mM NaCl dissolved in 50 mM HEPES pH 7.5. Store at 20  C and keep at 4  C for use. 4. Resuspension buffer: 1 mM PMSF and 1% (v/v) protease inhibitor cocktail in wash buffer. Prepare immediately before use and keep at 4  C. 5. Sucrose solutions: 38%, 33%, and 8% sucrose (w/v) in 50 mM HEPES, pH 7.5. 6. Bicinchoninic Acid Protein Assay Kit. 7. Mortar (with the diameter about 20 cm) and pestle. 8. Funnel. 9. Gauze or Miracloth (Millipore).

2.3 Immunoprecipitation

1. Anti-GFP, rabbit IgG, polyclonal (e.g., Thermo Fisher Scientific, A-11122). 2. PBS-T: 150 mM NaCl, 2.7 mM KCl, 11.5 mM Na2HPO4, 1.76 mM KH2PO4, 0.02% (v/v) Tween 20. Keep at 4  C.

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3. Dynabeads Protein A for immunoprecipitation and magnetic tube holder. 4. Bis[sulfosuccinimidyl]suberate (BS3). 5. BS3 conjugation buffer: 20 mM Na2HPO4, 0.15 M NaCl. 6. BS3 working solution: 5 mM in BS3 conjugation buffer. Prepare before use. 7. BS3 quenching buffer: 1 M Tris–HCl pH 7.5. 2.4 SDS-PAGE and Western Blotting

1. Primary antibodies: mouse anti-CFP/GFP/YFP for IP (e.g., Roche 118144600001 at 1/1000); rabbit anti-SYP61 at 1/1000 [9] or, rabbit anti-ECHIDNA at 1/1000 [11] for TGN detection; antibodies to detect contamination from other compartments (e.g., rabbit anti-MEMB11 at 1/1000 for Golgi [24], rabbit anti-V-ATPase-E [Agrisera, AS07 213] at 1/2000 for vacuole, rabbit anti-PMA2 at 1/1000 for plasma membrane [25], rabbit anti-PM-ATPase at 1/1000 for plasma membrane [Agrisera, AS07 260]). 2. Secondary antibodies: goat anti-mouse IgG-HRP conjugate, goat anti-rabbit IgG-HRP conjugate. 3. 5 loading buffer: 200 mM Tris–HCl pH 6.8, 50% (v/v) glycerol, 5% (w/v) sodium dodecyl sulfate (SDS), 250 mM DTT, and 0.05% (w/v) bromophenol blue. 4. 10% TGX Stain-Free FastCast Acrylamide Kit. 5. N,N,N0 -tetramethylethylenediamine (TEMED). 6. 10% (w/v) ammonium peroxodisulfate (APS). 7. Running buffer: 25 mM Tris–HCl, 192 mM glycine, 0.1% SDS. 8. SDS-PAGE molecular weight standards. 9. Transfer buffer: 25 mM Tris–HCl, 192 mM glycine, 20% (v/v) ethanol. 10. TBS-T: 20 mM Tris–HCl, 140 mM NaCl, 2.5 mM KCl, 0.1% (v/v) Tween 20. 11. Blocking buffer: 5% (w/v) nonfat dried milk in TBS-T. 12. Enhanced chemiluminescence (ECL) reagents. 13. Polyvinylidene difluoride (PVDF) membrane. 14. Blotting paper.

2.5 Fatty Acid Profiling (Fatty Acid Methyl Esters: FAMEs)

1. Hydrolysis solution: 5% H2SO4 in methanol. 2. Hexane 99%. 3. 2.5% NaCl in H2O. 4. 100 mM Tris–HCl, 0.09% NaCl, pH 8. 5. Distillated H2O.

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6. 100 mM Tris–HCl. 7. BSTFA-TMCS N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane. 2.6

Sterol Profiling

1. Ethanol. 2. 11 N KOH. 3. Hexane 99%. 4. Distillated H2O. 5. BSTFA-TMCS N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane.

2.7

Lipid Standards

1. Heptadecanoic acid (17:0). 2. 2-hydroxytetradecanoic acid (h14:0). 3. Cholestan-2-ol (also known as α-cholestanol). Optional: 4. 1-Pentadecanol (OH-15:0). 5. 15-hydroxypentadecanoic acid (ωOH-15:0).

2.8

Consumables

1. Ice. 2. 50 mL Falcon tubes. 3. 10 mL and 25 mL disposable pipettes. 4. 1.5 mL microcentrifuge tubes. 5. 38 mL thin wall open-top ultracentrifugation tubes. 6. Glass Pasteur pipettes. 7. Screw glass tubes of 8 mL. 8. Screw caps with Teflon seal.

2.9

Equipment

1. Shaker for flasks installed in plant incubator/growing facility. 2. 1.5 mL tube rotator. 3. Refrigerated centrifuge for centrifugation of 50 mL tubes in a swinging-bucket rotor. 4. Ultraspeed refrigerated centrifuge for centrifugation of 38 mL tubes in a swinging – bucket rotor at 150,000  g (r max). 5. Centrifuge for centrifugation of 8 mL glass tubes in a fixedangle bucket rotor. 6. Water bath. 7. Dry bath. 8. Spectrophotometer and cuvettes. 9. Protein electrophoresis device. 10. Power supply.

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11. Stain-Free enabled imager (e.g., Bio-Rad ChemiDoc MP imaging system). 12. Protein transfer device. 13. Air flow evaporator system for lipids. 14. GC-MS simple Quadrupole system equipped with an autosampler, a mass detector, and a data processing station. An HP-5MS capillary column (5% phenyl-methyl-siloxane, 30 m, 250 mm, and 0.25 mm film thickness; Agilent) is used in our laboratory. 2.10

Software

1. MassHunter from Agilent. 2. NIST database. 3. Image J (https://imagej.net/Fiji).

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Methods

3.1 Growing Arabidopsis Seedlings in Liquid Culture

1. Prepare one 1.5 mL tube for each culture flask (usually 4 tubes for 4 flasks for one IP), and place approximately 50 μL of seeds (30–40 mg of seeds) per tube. 2. Sterilize the seed surface with 70% ethanol for 1 min. 3. Remove the ethanol, add 1 mL of sterilization solution, and incubate for 20–30 min with continuous mixing in a tube rotator. 4. Wash 3–4 times with sterile distilled H2O (see Note 2). 5. Keep seeds in the dark, at 4  C for 2 days in sterile distilled H2O. 6. Pour 250 mL of MS liquid medium into each 500 mL baffled flask and sterilize at 110  C for 30 min. 7. After the medium has cooled down at room temperature, place the seeds into each flask. Incubate for 8–9 days under 120 rpm and long-day condition (16 h light/8 h darkness) at 22  C (see Note 3).

3.2 Membrane Extraction and Fractionation

To obtain intact membrane compartments with high purity, we disrupt the seedlings in a detergent-free buffer, remove the debris, and perform a two-step process including a sucrose cushion and a sucrose step-gradient fractionation by ultracentrifugation. The samples should be kept at 4  C or on ice during all the manipulations; precool buffers and tubes. 1. Precool the vesicle isolation buffer, 50 mL Falcon tubes (for collecting the homogenate), mortar, and pestle in ice. Set the swing-rotor centrifuge at 4  C. 2. Collect and weigh the seedlings (see Note 4).

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3. Transfer the seedlings into the mortar on ice, and grind them in 2–3 volumes (e.g., 20 g of seedlings in 60 mL) vesicle isolation buffer (see Note 5). 4. Set the funnel and gauze/Miracloth on the precooled 50 mL Falcon tube on ice. Harvest the homogenate with a disposable pipette and filter it through the gauze/Miracloth. Squeeze the waste to get as much material as possible. 5. Centrifuge the filtered homogenate at 1600  g for 20 min at 4  C. Meanwhile, precool new 50 mL Falcon tubes in ice. 6. Transfer the supernatant to the precooled 50 mL Falcon tubes by decantation, and centrifuge at 1600  g for 15 min at 4  C. In total, step 5 should be performed 3–4 times. 7. Precool the 38% sucrose solution and 38 mL centrifuge tubes in ice. 8. Transfer 8 mL 38% sucrose solution to 38 mL centrifuge tubes, and load the last supernatant of step 6 on it (see Note 6, Fig. 1). Ultracentrifuge at 150,000  g for 3 h at 4  C with a swingingbucket rotor. Precool the 33% and 8% sucrose solutions in ice. 9. The membrane fraction appears at the interface between the sucrose cushion and the supernatant as a yellow layer (Fig. 1). Remove the supernatant using a 10 mL pipette (see Note 7). On top of the membrane fraction, carefully load 15 mL of 33% sucrose solution, followed by 10 mL of 8% sucrose solution (see Note 8, Fig. 1). 10. Ultracentrifuge at 150,000  g overnight (16 h) at 4  C. 11. Precool 50 mL Falcon tubes and new 38 mL centrifuge tubes in ice. Harvest separately the membrane fractions at the 38%/ 33% and 33%/8% interfaces using a Pasteur pipette in precooled 50 mL Falcon tubes (see Note 9). 12. Dilute the collected membrane fractions into 1/3 (v/v) by 50 mM HEPES, pH 7.5. Mix gently by inverting the tubes. Transfer it into the precooled 38 mL centrifuge tubes and ultracentrifuge at 150,000  g for 3 h at 4  C. Meanwhile, prepare resuspension buffer and keep it on ice. 13. Discard the supernatant by decantation. Wipe carefully the inside of the centrifuge tubes with a paper towel without touching the pellet. Resuspend the pellet (microsomal fraction) with 0.5–1 mL resuspension buffer depending on the size of the pellet. 3.3 Protein Quantification in the Total Membrane Fraction

If there are multiple microsomal fractions (e.g., when comparing the membranes of drug-treated and nontreated plants or mutants versus wild-type), the concentration of membrane material should be equilibrated among samples. The membrane concentration is assumed to be proportional to the protein concentration. We use

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a 8% Supernatant (discard)

Homogenate

33%

Ultracentrifugation

38%

38%

Total Membrane Fraction

38%

b

BS3 GFP

anti-GFP Dynabeads

Fig. 1 Immunoprecipitation of intact endomembrane compartments. (a) Two-step membrane fractionation. Left: load the homogenate of Arabidopsis seedlings on a 38% sucrose cushion and perform ultracentrifugation. Middle: After ultracentrifugation, the membrane fraction appears at the interface as a yellow layer. Discard the supernatant above the membrane fraction using a glass pipette. Right: By stepwise loading of 33% and 8% sucrose solutions, the membrane fraction is divided into two layers at the 8%/33% and 33%/ 38% interfaces. After an additional ultracentrifugation step, harvest those layers separately or together (see Note 9). (b) Schematic representation of membrane immunoprecipitation principle. Compartments which harbor the protein bait fused to GFP are recognized by the anti-GFP antibody. The antibody is conjugated to magnetic beads (Dynabeads) and cross-linked by BS3. GFP-labeled compartments will be pulled down together with beads using magnetism

the bicinchoninic acid (BCA) protein assay kit to reliably quantify protein concentration and adjust samples accordingly. 1. Dilute 5 μL of the microsomal fraction with 100 μL of resuspension buffer (1/20 dilution). Prepare 2 tubes for each sample. 2. Measure the protein concentration of the diluted microsomal fractions using a BCA protein assay kit. Average the results of

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the 2 tubes for each sample and calculate the original concentration (see Note 10). 3. Equilibrate the protein concentration of the microsomal fraction with the resuspension buffer to the one with the lowest concentration. 3.4 Immunoprecipitation

Conjugate the GFP antibody to Dynabeads by BS3, and immunoprecipitate the GFP-tagged compartments from the microsomal fraction. All the steps other than BS3 conjugation should be performed at 4  C or on ice. 1. Prepare 150 μL of Dynabeads in a 1.5 mL tube for each IP (see Note 11). 2. Wash the beads with cold 1 mL PBS-T by pipetting. Set the tubes on the magnetic tube holder, wait until the beads are pulled by the magnet, and remove the PBS-T. 3. Add 500 μL PBS-T and cool the tubes on ice. 4. Add 15 μL of anti-GFP and incubate for at least 1 h at 4  C under continuous rotation by the tube rotator (see Note 12). 5. Remove the buffer containing the antibodies and wash the beads with 500 mL of BS3 conjugation buffer (see Note 13). 6. Dissolve BS3 into BS3 conjugation buffer (BS3 working solution, see Note 14). 7. Remove the buffer from the beads, and resuspend the beads with the BS3 working solution. Incubate for 30 min at room temperature with rotation. 8. Quench the reaction by adding 25 μL BS3 quenching buffer. Incubate for 15 min at room temperature with rotation. 9. Transfer the tubes to ice. Wash the beads with 500 μL of cold PBS-T three times. 10. Resuspend the beads with 500–1000 μL of cold resuspension buffer, and cool them in ice for at least 10 min. 11. Remove the buffer from the beads, and add 900 μL of the equilibrated microsomal fraction from Subheading 3.3, step 3. Incubate for 1 h at 4  C with continuous rotation (see Note 15). 12. Remove the supernatant. Wash the beads with 1 mL of cold wash buffer four times, and finally resuspend in 70 μL of wash buffer; this is the IP output fraction (see Note 16). Store at 80  C if you do not use the samples for further analysis in a short term.

3.5

Controls for IPs

To check the level of nonspecific background in immunoprecipitated samples, we advise to perform an IP from the microsomal fraction using beads without GFP antibody as negative control (skipping the steps Subheading 3.4, steps 4–10 for the preparation

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of beads). An alternative negative control can be obtained by doing an IP with GFP antibody-conjugated beads from a microsomal fraction extracted from wild-type plants that do not express any GFP-labeled protein bait. 3.6 Enrichment and Purity Assessment of the IP Fraction

The IP fraction should be checked by Western blotting to determine the enrichment level (the IP output fraction is compared with the microsomal IP input fraction) of the GFP-tagged compartment and contamination from other membranes. Primary antibodies appropriate for detection of SYP61 and other organelles are listed at Subheading 2.4, item 1 (see Note 17). We used the TGX Stain-Free system from Bio-Rad, which enables rapid visualization of proteins by the fluorescence of tryptophan excited by UV light, to quantify the total amount of proteins. If the system is not available, Coomassie brilliant blue (CBB) staining is the standard alternative. 1. Add 5 μL of 5 loading buffer to 10 μL of the IP output fraction or 5 μL of the microsomal IP input fraction, adjust with 1 loading buffer to get a final volume of 25 μL in every tube (see Note 18). 2. Perform electrophoresis polyacrylamide gel.

using

TGX

Stain-Free

3. Visualize the total protein profile using Stain-Free system (Fig. 2). Using the ImageJ software, define equally sized

a

b

Stain-Free IP IP input output

(kDa)

(kDa)

250

250

150

150

100

100

75

75

50

50

37

37

25 20

25

mouse anti-GFP IP IP input output

Fig. 2 Analysis of IP output fraction. (a) Stain-Free fluorescence image of a SDS-PAGE gel loaded with equal protein amounts of the IP input microsomal fraction (IP input) and IP output fraction (after beads incubation) using GFP-SYP61 as the IP bait. (b) Western blotting image displaying enrichment of TGN-SV in the IP output fraction as revealed by GFP-SYP61 detection by mouse anti-GFP antibody

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boxes around each lane, analyze the signal intensity, and calculate the ratio of the total protein amount between samples. Dilute samples with 1 loading buffer to equalize the protein concentration to that of the lowest one. 4. Perform a new SDS-PAGE of the equilibrated samples and transfer the proteins to PVDF membranes using standard protocols. 5. Perform immunoblotting using appropriate antibodies by standard protocols (Fig. 2). Use mouse anti-GFP antibodies to know whether the bait has been pulled-down (Fig. 2). We advise to use several other antibodies recognizing endogenous proteins of the targeted compartments (like ECHIDNA for SYP61/SVs-TGN) and antibodies against other organelle marker proteins to check for potential contaminations from other membranes. A list of appropriate antibodies is given in Subheading 2.4, item 1. 3.7

Lipid Extraction

3.7.1 Total Fatty Acid Extraction (Fatty Acid Methyl Esters: FAMEs)

In each experiment, a negative control should be prepared by following the protocols described below replacing the IP output fraction by IP wash buffer in step 1. In the negative control, only the standards should be detected by GC-MS. In addition, a vial containing 100 μL of hexane should be prepared as negative control for hexane contaminants. Use the same hexane as used in Subheading 3.7, steps 1 and 2. No components should be detected. In the case of contamination by plastics, alkanes will be detected, in that case, use new hexane and proceed with new lipid extraction. 1. Transfer 25–50 μL of the IP output fraction in a screw glass tube, named tube A (see Note 19). 2. Add 1 mL of the hydrolysis solution containing 5 μg/mL of internal standard 17:0 and h14:0 (see Note 20). 3. Incubate overnight at 85  C in a dry bath, be sure that the contents of tubes are not evaporating by retightening the screw caps every 5–10 min during the first 30 min. This step aims at releasing the FAs of different lipid classes from their respective backbone (Fig. 3, see Note 21). 4. Remove the tubes from the dry bath and let them cool down. 5. Add 1 mL of 2.5% NaCl and then 1 mL of hexane. 6. Mix vigorously (see Note 22). 7. Centrifuge for 5 min at 800  g at room temperature to separate the phases. 8. Prepare new screw glass tubes containing 1 mL of 100 mM Tris–HCl, 0.09% NaCl, pH 8, named tube B.

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Fig. 3 Schematic representation of the hydrolysis of fatty acids (FAs) from the lipid backbone. (a) FAs esterified on the glycerol backbone in glycerolipids and glycerophospholipids or amidified on the long chain base of sphingolipids are cleaved through acidic hydrolysis (Methanol + sulfuric acid, see Subheading 3.7.1, step 2). (b) Basic hydrolysis (Ethanol + KOH, see Subheading 3.7.2, step 2) cleaves esterified FAs chains grafted on a steroid backbone in the case of sterylesters, glycosylsterylesters, or acylated glycosylesters. Yellow circles are the radical groups esterified on glycerol backbone of glycerolipids and glycerophospholipids or esterified on the steroid backbone of sterols or amidified on the sphingoid backbone of sphingolipids; red scissors represent the cleavage of the ester or amid bonds; blue lines encircled the lipid part recovered after extraction and analyzed by GC-MS.

9. Collect the hexane upper phase (see Note 23) and transfer it to the tube B prepared in previous step. 10. Add 1 mL of hexane to the initial tube A. 11. Mix vigorously. 12. Centrifuge for 5 min at 800  g at room temperature to separate the phases. 13. Collect the hexane upper phase and add it to tube B. 14. Mix vigorously the content of tube B. 15. Centrifuge for 5 min at 800  g at room temperature to separate the phases. 16. Recover gently the hexane upper phase taking care not to contaminate the sample with the lower aqueous phase (see Note 24) and transfer to a new glass tube. 17. Evaporate the hexane through air flow evaporator.

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18. Add 200 mL of BSTFA. 19. Incubate for 15 min at 110  C in a dry bath. 20. Evaporate the BSTFA through air flux evaporator (see Note 25). 21. Once BSTFA is completely evaporated add 100 μL of hexane and close the lid immediately to avoid hexane evaporation. 22. Vortex. 23. Transfer into GC vials containing a conical insert. 24. Run the sample in a GC-MS instrument using the FAMES methods. 3.7.2 Sterol Extraction

1. Transfer 25–50 μL of the IP output fraction in a screw glass tube. 2. Add 1 mL of ethanol containing 5 μg/mL of cholestan-2-ol and then 100 μL of 11 N KOH; this is a saponification step which eliminates FAs. Please note that this method will not distinguish between sterols, sterylesters, sterylglycosides or acetylated sterylglycosides in which a FA chain and/or a glucose is grafted on a steroid backbone. 3. Incubate for 1 h at 80  C in a dry bath. 4. Let cool the samples. 5. Add 1 mL of hexane. 6. Vortex. 7. Add 2 mL of distillated H2O. 8. Mix vigorously. 9. Centrifuge for 5 min at 800  g at room temperature to separate the phases. 10. Recover gently the hexane upper phase taking care not to contaminate the sample with the lower aqueous phase (see Note 24). 11. Evaporate the hexane through air flow evaporator. 12. Add 200 mL of BSTFA. 13. Incubate for 15 min at 110  C in a dry bath. 14. Evaporate the BSTFA through air flux evaporator. 15. Once BSTFA is completely evaporated add 100 μL of hexane (see Note 26). 16. Vortex. 17. Transfer into GC vials containing a conical insert. 18. Run the sample in GC-MS instrument using the sterol methods.

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GC-MS Analysis

3.8.1 GC-MS Method

1. Set the helium carrier gas at 2 mL/min. 2. Use the splitless mode for injection. 3. Set the temperatures of injector to 250  C and the auxiliary detector to 352  C. 4. Set the oven temperature to be held at 50  C for 1 min. 5. Program a 25  C/min ramp to 150  C (2-min hold) and a 10  C/min ramp to 320  C (6-min hold). 6. MS Analyzer is set in scan only with mass range 40–700m/z in positive mode with an electron emission set to 70 eV. 7. The temperature of MS Source is set to 230  C and the MS Quad to 50  C.

3.8.2 Identification of Peaks

Quantification of FAMES and sterols was based upon peak areas that were derived from the total ion current. Each sample chromatogram is analyzed using the MassHunter software equipped with a NIST database. An automatic identification of the lipid species using algorithms developed by Agilent is recommended. However, manual integration could also be added to automatic integration results in order to consider lipids that are below the automatic threshold detection or lipids for which peaks would be too close to be separated by automatic detection (example in Fig. 4). An example of chromatogram resulting from a SYP61GFP IP output fraction is given in Fig. 4, see also Table 1. The retention times given in Fig. 4 are only indicative as these values change depending on the column and age of the column. The major FAs 16:0, 18:2, and 18:3 are typical for glycerolipids (DAG, TAG) and glycerophospholipids (phospholipids) while the α-hydroxylated FAs (hFAs) are a specific feature of sphingolipids with h24:1 and h24:0 being enriched in SYP61 IP as described before [14]. Nonhydroxylated 24:0 FA is strongly present in the sphingolipid pool but could also be detected in the glycerophospholipid pool [14]. The fatty alcohols OH-18:0 and OH-20:0 as well as the dicarboxylic acid 18:2-DCA are typical from extracellular lipids such as cutin and suberin, which precursors are probably transported through the SYP61 compartment to reach the plasma membrane and be secreted in the apoplast.

3.8.3 Calculation

The relative quantification is obtained by normalizing the peak area of one lipid species to the appropriate internal standard area. Nonhydroxylated FAs, including dicarboxylic acids (DCAs), are normalized to 17:0; hFAs are normalized to h14:0. If other types of FAs, such as fatty alcohols or ω-hydroxyFAs (ω-OH-FAs), are found, quantify them relatively to OH-15:0 (1-pentadecanol) or ω-OH-15:0 (15-hydroxypentadecanoic acid) standards, respectively. These standards will need to be added at Subheading 3.7.1, step 2 (see Note 20).

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x10 9

x10

8

7

73.100

7

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74.100

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4 3 3 2 2 1

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0 180

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18:3

7

h24:1 h24:0

h22:0

24:0

0.2

24:1

23:0

0.4

22:1 22:0

0.6

280

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3.5 3 2.5 2 1.5

0

12.4 12.6 12.8 13.0 13.2 13.4 13.6 13.8 14.0 14.2 14.4 14.6 14.8 15.0 15.2

260

4

18:2-DCA

1

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4.5

20:0 OH-20:0

1.2

0.8

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x10 6 5

20:2 20:1

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(standard)

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Fragmentation spectrum count vs Mass-to-charge (m/z)

18:0

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Fragmentation spectrum count vs Mass-to-charge (m/z)

1

β-sitosterol

160

campesterol stigmasterol

140

cholesterol

120

cholestan-2-ol

100

(standard)

80

26:0

60

25:0

40

0.5 0

15.4 15.6 15.8 16.0 16.2 16.4 16.6 16.8 17.0 17.2 17.4 17.6 17.8 18.0 18.2 18.4 18.6 18.8 19.0 19.2 19.4 19.6 19.8 20.0 20.2 20.4 20.6 20.8 21.0 21.2 21.4 21.6 21.8

22.2 22.4 22.6 22.8 23.0 23.2 23.4 23.6 23.8 24.0 24.2 24.4

h16:0

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18:0

20:1

Retention Time (min)

Fig. 4 Example of a typical FAMES and sterols chromatogram from a SYP61-GFP IP. Fragmentation spectrum of the α-hydroxylated FA standard h14:0 is shown in green. The TMS group at m/z 73.1 and the m/z 44 differences between the two major peaks are specific features of the α-hydroxylated FAs. Fragmentation spectrum of the nonhydroxylated FA standard 17:0 is shown in red. The TMS group is at m/z 74.1, which is typical of nonhydroxylated FA. All FAs are shown in blue. The dotted circles show two peaks close to one another for which manual integration could be helpful. All other peaks were automatically integrated

After normalization to the standard, the value obtained is expressed in μg and then ng. This value could be further transformed in nmol using the specific molecular weight of the specific FA. Different IP could yield different amount of material and thus different lipids quantity (in ng or nmol). We advise to always perform IPs with the same protein concentration of microsomal IP input fraction, and in the same volume. However, the yield of the IPs could still be different. Hence, we do not advise to compare directly the lipid quantity values (in ng or nmol) obtained from different IPs. Instead, these values should be normalized. A classical way is to normalize the lipid quantity values against the fresh or dry weight of starting seedling material. This is, however, not possible in this protocol as multiple steps of membrane fractionation are performed prior to the IP, potentially leading to different amounts of IP input material. Protein quantification of IP output fractions is still possible but not reliable as antibodies used for IPs and possibly the magnetic beads would interfere with most protein quantification assays (see Note 18). Moreover, the protein–lipid ratio in membranes is clearly variable from one endomembrane

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Table 1 List of FAs typically found in SYP61 IP fraction FA short name

FA nomenclature name

16:0

Hexadecanoic acid

h14:0

2-hydroxytetradecanoate

17:0

Heptadecanoic acid

18:2

Octadecadienoic acid

18:3

Octadecatrienoic acid

18:0

Octadecanoic acid

h16:0

2-hydroxyhexadecanoate

OH-18:0

1-octadecanol

20:2

Eicosadienoic acid

20:1

Eicosenoic acid

20:0

Eicosanoic acid

OH-20:0

1-eicosanol

18:2-DCA

Octadecadiynoic acid

22:1

Docosenoic acid

22:0

Docosanoic acid

26:1

Hexacosenoic acid

23:0

Tricosanoic acid

24:1

Tetracosenoic acid

h22:0

2-hydroxydocosanoate

24:0

Tetracosanoic acid

25:0

Pentacosanoic acid

h24:1

2-hydroxytetracosenoate

h24:0

2-hydroxytetracosanoate

26:0

Hexacosanoic acid

h26:0

2-hydroxyhexacosanoate

compartment to another or from one experimental condition to another. Hence, using the protein–lipid ratio as a way of normalizing lipid quantities is clearly not the best approach. Instead, we suggest that, for IP samples, the quantity of one specific FA species should be normalized to the total amount of FAs found in the profile. The value will then be expressed as ng% or nmol%.

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Notes 1. There are multiple products with different molecular weight for PVP. Use the one with the average molecular weight of 10,000. 2. Because we use large amounts of seeds, the culture can easily be contaminated with bacteria or fungi. Hence, seed sterilization should be performed carefully. 3. For IPs of intact membrane compartments, it is always preferable to work with fresh material. We advise not to keep plant material at 80  C before starting the membrane extraction, fractionation, and IP. 4. Be careful not to let the seedlings dry during manipulation. Fresh weight should be at least 10 g, ideally 15–20 g. 5. It is easier to start grinding with a small volume of buffer (e.g., 25 mL), and add the rest during grinding. Because of the toxicity of PMSF, the manipulation should be carried out under a chemical fume hood. 6. Load the supernatant slowly and gently in order not to disrupt the interface between the sucrose solution and the supernatant. It is easier to load the first several milliliters with a P1000 pipetman. The thin wall open-top centrifuge tubes must be filled up to 2–3 mm from the tube top to avoid deformation of the tubes during ultracentrifugation. If the amount of the supernatant is not enough to fill the tubes, add vesicle isolation buffer. 7. When removing the supernatant, keep a thin layer of supernatant of approximately the same thickness of the yellow layer to avoid pipetting membranes. 8. By loading 33% sucrose solution, the membrane fraction is separated into two layers beneath and above the 33% sucrose solution. The upper fraction can accidentally get into the pipette during this process. To avoid it, keep the sucrose solution continuously flowing out of the pipette while loading. The loading of the two sucrose solutions should be performed carefully; similar to the previous step, use a P1000 pipetman to load the first several milliliters. Do not disrupt the interfaces. 9. Most of the small early endosomal compartments, TGN, and Golgi will be located at the 33%/8% interface, while an important part of the late endosomal compartments will be located to both 38%/33% and 33%/8% interfaces. Hence, it is advisable to check beforehand by Western blot in which interface the targeted compartment carrying the protein-GFP bait is most enriched. In any case, it is alternatively possible to combine the two fractions together.

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10. The protein concentration of the original microsomal fraction should be at least 1 mg/mL, ideally around 2 mg/mL. If the concentration is lower than 1 mg/mL, we advise to start a new membrane extraction. 11. Before taking Dynabeads from the vial, resuspend the beads by vortex; beads sink to the bottom of the vial. 12. To thoroughly mix beads and antibodies, invert the tubes several times before setting the tubes on the rotator. The incubation can be overnight. 13. Collect the beads sticking on the lid of the tubes. 14. Prepare this solution freshly. 15. The incubation should neither be longer nor shorter than 1 h. 16. The IP output fraction contains the immunopurified membranes bound to the magnetic beads. As the beads sediment in the tube, homogenize by pipetting before using the IP output fraction for subsequent steps. 17. Because the IP is performed by using rabbit anti-GFP IgG, rabbit primary antibodies should not be used for the detection of GFP or proteins of similar molecular weight to IgG. 18. These amounts are suggested as a reference. Alternatively, protein quantification using a BCA protein assay could be performed. However, this method might be less reliable than visualizing the total protein profile on the TGX Stain-Free system due to (1) the presence of antibodies used for the IPs which create a bias in the protein quantification while they could easily be spotted on an SDS-PAGE, (2) the beads could interfere on their own with the protein quantification assay. 19. The beads are resistant to the hydrolysis solution and do not generate any molecules detectable by GC-MS. 20. The 17:0 standard is used to quantify the most common FAs coming from the glycerolipid and glycerophospholipid pool. The h14:0 standard is used to quantify the α-hydroxylated (2-hydroxy)-FAs coming exclusively from the sphingolipid pool. Dicarboxylic acids (DCA) are FAs typical of the suberin and cutin lipids, which could be found in the FAs profile; quantification of DCA compounds is made with the 17:0 standard. Other types of FAs, such as fatty alcohols or ω-hydroxyFAs, need to be quantified relative to OH-15:0 (1-pentadecanol) or ω-OH-15:0 (15-hydroxypentadecanoic acid) standards, respectively; add 5 μg of each in the hydrolysis solution. 21. Depending on the type of chemical bounds between FAs and the backbone, the hydrolysis will occur at different efficiency (Fig. 3). FAs of glycerolipids and glycerophospholipids are

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esterified on the glycerol backbone while FAs of sphingolipids are amidified on the sphingoid backbone, which is more difficult to hydrolyze than an ester bound. Hence, the overnight incubation is necessary to correctly visualize the FAs coming from the sphingolipid pool. Please note that FAs esterified on a steroid backbone, such as in the case of sterylesters, glycosylesters, or acetylglycosylesters, will also be hydrolyzed and thus be part of the FAs pool detected in the profiling. 22. Shake tube for homogeneous mixing. Vortex only may not result in efficient mixing. 23. Accidental collection of small amounts of the lower aqueous phase does not matter at this stage, which is aimed at adjusting the pH of the hexane phase. 24. Contrary to Subheading 3.7.1, step 9, it is important not to collect any of the lower aqueous phase as this will interfere with Subheading 3.7.1, step 18 resulting in misleading chromatogram results and may destroy the GC column. If some of the aqueous phase was accidentally collected, a white residue coming from the hydrolysis of trimethylsilyl (TMS) molecules that release silicon atoms, will appear in Subheading 3.7.1, step 20. 25. If a white residue appears in the tube, add 1 mL of hexane and then 1 mL of 100 mM Tris–HCl, 0.09% NaCl, pH 8 and resume at Subheading 3.7.1, step 14. 26. If a white residue appears in the tube, add 1 mL of hexane and then 1 mL of distillated water and resume at Subheading 3.7.2, step 8.

Acknowledgments This work is supported by the French National Research agency (ANR) grant “caLIPSO” to Y.B (ANR-18-CE13-0025) and Overseas Research Fellowship granted from Japan Society for Promotion of Science (JSPP) to Y.I. We gratefully acknowledge Pierre Van Delft for helpful discussions and support for lipidomic analyses (Plateforme MetaboHUB-Bordeaux) (ANR-11-INBS-0010). The authors would like to warmly acknowledge Natasha Raikhel (Distinguished Professor of Plant Cell Biology, Institute for Integrative Genome Biology, University of California Riverside, USA) for making available the SYP61-CFP Arabidopsis line and the support she originally provided to analyze lipid content in immunopurified TGN fraction. We thank Patrick Moreau and Sebastien Mongrand for helpful comments and critical reading of the manuscript.

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References 1. Dettmer J, Hong-Hermesdorf A, Stierhof YD, Schumacher K (2006) Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell 18 (3):715–730 2. Viotti C, Bubeck J, Stierhof YD, Krebs M, Langhans M, van den Berg W et al (2010) Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle. Plant Cell 22(4):1344–1357 3. Uemura T, Nakano RT, Takagi J, Wang Y, Kramer K, Finkemeier I et al (2019) A Golgireleased subpopulation of the trans-Golgi network mediates protein secretion in Arabidopsis. Plant Physiol 179(2):519–532 4. Uemura T, Suda Y, Ueda T, Nakano A (2014) Dynamic behavior of the trans-golgi network in root tissues of Arabidopsis revealed by superresolution live imaging. Plant Cell Physiol 55 (4):694–703 5. Donohoe BS, Kang BH, Staehelin LA (2007) Identification and characterization of COPIaand COPIb-type vesicle classes associated with plant and algal Golgi. Proc Natl Acad Sci U S A 104(1):163–168 6. Kang BH, Nielsen E, Preuss ML, Mastronarde D, Staehelin LA (2011) Electron tomography of RabA4b- and PI-4Kβ1-labeled trans Golgi network compartments in Arabidopsis. Traffic 12(3):313–329 7. Ito E, Fujimoto M, Ebine K, Uemura T, Ueda T, Nakano A (2012) Dynamic behavior of clathrin in Arabidopsis thaliana unveiled by live imaging. Plant J 69(2):204–216 8. Bru¨x A, Liu TY, Krebs M, Stierhof YD, Lohmann JU, Miersch O et al (2008) Reduced V-ATPase activity in the trans-Golgi network causes oxylipin-dependent hypocotyl growth inhibition in Arabidopsis. Plant Cell 20 (4):1088–1100 9. Sanderfoot AA, Kovaleva V, Bassham DC, Raikhel NV (2001) Interactions between syntaxins identify at least five SNARE complexes within the Golgi/prevacuolar system of the Arabidopsis cell. Mol Biol Cell 12(12):3733–3743 10. Uemura T, Ueda T, Ohniwa RL, Nakano A, Takeyasu K, Sato MH (2004) Systematic analysis of SNARE molecules in Arabidopsis: dissection of the post-Golgi network in plant cells. Cell Struct Funct 29(2):49–65 11. Gendre D, Oh J, Boutte´ Y, Best JG et al (2011) Conserved Arabidopsis ECHIDNA protein

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Chapter 12 Cell-Free Protein Translation System for Expression of Lipid-Binding Proteins Tagged with Small epitopes and Their Use in Protein–Lipid Overlay Assays Julio Paez-Valencia and Marisa S. Otegui Abstract We adapted an efficient cell-free protein synthesis–based protocol for the production of lipid-binding proteins. The experimental procedures are based on the following steps: (1) cell-free synthesis of soluble, lipid-binding proteins fused to small tags; (2) analysis by dot blot of the accessibility of antibodies to the small tags. (3) protein lipid overlay assay with, immunodetection of bound protein by either chemiluminescence or fluorescence. We also provide a fast and inexpensive protocol for homemade lipid nitrocellulose strips spotted with acidic lipids (mostly phosphoinositides) extracted from plant tissues. These homemade lipid strips can be used for preliminary screen and characterization of putative phosphoinositide-binding proteins. Key words Cell free protein synthesis system, Phosphoinositides, Protein–lipid overlay assay, Small tags

1

Introduction Membrane lipids interact with proteins and define membrane identities. For instance, protein–lipid interactions contribute to the formation of functional membrane platforms that regulate endosome biogenesis and function [1]. Protein–lipid interactions can be assayed by biochemical and biophysical techniques, including liposome-based assay, isothermal titration calorimetry, surface plasmon resonance, microscale thermophoresis, and native protein mass spectrometry. However, the elevated cost of the instrumentation required for these assays limits their use [2]. Protein–lipid overlay assay (PLOA), is powerful and relatively inexpensive method to identify lipid ligands [3]. PLOA facilitated the analysis of lipid binding specificity of many protein domains, such as the PX domain of the tSNARE Vam7 and p40phox toward phosphatidylinositol [3–5] phosphate PI(3)P. PLOA can be done with nitrocellulose strips using either serial dilution of one or several

Marisa S. Otegui (ed.), Plant Endosomes: Methods and Protocols, Methods in Molecular Biology, vol. 2177, https://doi.org/10.1007/978-1-0716-0767-1_12, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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A

B

IPTG

0.01mM MW

100 75 50 37 25 20

SF

IB

0.5mM SF

IB

T7P

1mM SF

IB

C

100 75

RBS

TB-C

HIS6

T7T

T7P: T7 promoter RBS: Ribosomal biding site TB-C: TUBBY-C terminal His6:Hexahistidine tag T7T: T7 terminator

50 37

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Fig. 1 (a) Purification of inclusion bodies of TUBBY-C-His6 expressed in BL21 Escherichia coli cells induced with different concentration of Isopropyl β- d-1-thiogalactopyranoside (IPTG) at 22  C for 4 h. Molecular weight markers (MW), soluble fraction (SF), inclusion bodies (IB). (b) Expression of TUBBY-C-His6 construct using in vitro protein synthesis kit. Schematic representation of the DNA template design applied in this study. (c) Qualitative analysis of in vitro synthesis of TUBBY-C-His6 by Western blotting developed with monoclonal anti-His6 antibodies coupled to HRP

immobilized lipid species of interest. The membrane is incubated with the protein of interest typically fused to a tag, washed, and probed for protein binding detection by antibodies against the epitope tag [3]. Although additional methods to prove protein– lipid interactions are always recommended, PLOA is often used as a first step to explore lipid binding properties/specificity. A standard PLOA requires 1–10 μg of a tagged protein, which is usually synthesizes and purified by standard methods. One of the most commonly used methods for protein production is expression and purification from bacteria. However, this approach is limited by the propensity of certain proteins to form inclusion bodies and by the fact that refolding of proteins in inclusion bodies is timeconsuming and often inefficient (Fig. 1a). In addition, the fusion of large tags commonly used for protein purification, such as GST, increases the likelihood of altering the native conformation and lipid binding capabilities of proteins under study [6]. In this context, a cell-free protein synthesis system is a valuable alternative method to synthetize protein that are difficult to produce in vivo [7]. For this method, it is generally recommended to add small tags (e.g., a poly-His tag) at the C-terminus for verifying full-length synthesis of the target protein by immunoblot detection. We described here an efficient cell-free protein synthesis–based protocol for the production of lipid-binding proteins that when expressed in bacteria, are predominantly sequestered into inclusion bodies. For standardization purpose, we use the C-terminal domain of the murine TUBBY protein (TUBBY-C), which binds PI(4,5) P2, PI(3,4)P2, and PI(3, 4, 5)P3 in vitro [8].

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1. DNA template for protein of interested (PCR product or plasmid). 2. Commercial kit for standard in vitro protein synthesis based on PURE system. 3. Incubator.

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1. Nitrocellulose membrane. 2. Narrow-pipette tips. 3. Tris-buffered saline (pH 8),150 mM NaCl.

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4. Washing buffer: 10 mM Tris–HCl (pH 8), 150 mM NaCl, 0.1% Tween. 5. Blocking buffer: 3% fatty acid-free bovine serum albumin (BSA) in washing buffer. 6. Monoclonal anti-His6 antibodies coupled to horseradish peroxidase (HRP) or to a fluorescent molecule (e.g., fluorescein isothiocyanate or FITC). 7. Chemiluminescent substrate with different sensitivities (e.g., Thermo Fisher SuperSignal West Pico, Thermo Fisher SuperSignal West Femto, standard ECL western blotting substrate). 8. Fluorescence/chemiluminescence imaging system. 9. Commercial or homemade lipid strips. 10. Cell-free protein synthesis reaction kit. 2.3 Extraction of Phosphoinositides from Plant Tissues

1. Arabidopsis thaliana seeds. 2. Grinding buffer: 50 mM HEPES–KOH (pH 8.0), 250 mM sorbitol, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF). 3. Filtration cloth (e.g., Miracloth). 4. Solution I: ice-cold chloroform–methanol–concentrated hydrochloric acid (100:100:0.7, v/v/v). 5. Solution II: chloroform–methanol–0.6 N hydrochloric acid (3:48:47, v/v/v). 6. Solution III: chloroform–methanol–water (65:35:8 v/v/v). 7. Hydrochloric acid 0.6 N. 8. Benchtop refrigerated centrifuge. 9. Microcentrifuge tubes.

2.4 Homemade Lipid Nitrocellulose Strips

1. Nitrocellulose membrane. 2. Tweezers.

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3. Scissor. 4. Soft pencil. 5. Phospholipids extracted from plants. 6. Aluminum foil.

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The protocol presented here can be performed with commercially available kits based on the PURE system technology originally developed by Dr. Takuya Ueda at the University of Tokyo [1]. The in vitro translation protocol is divided into three main steps: (1) Template preparation. (2) Protein synthesis reaction. (3) Analysis of the translated product by Western blotting. The DNA templates can either be cloned into a plasmid or provided as a PCR product. Standard expression vectors like pET or PIVEX derivates can be used. In addition to a coding sequence, the template must contain a T7 promoter upstream of the coding sequence, ribosome binding site, and a T7 terminator downstream of the stop codon (Fig. 1b). Linear PCR templates containing all required T7-promoter sequences can be generated by in Gibson assembly method. It is also recommended to include in the template small purification/detection tags such as Myc, FLAG, poly (His)n-tag, Strep or T7 at the N- or C-terminus of the protein to be expressed. A general protocol performed with commercially available kits involves the following steps: 1. Amplify and purify plasmid or PCR product of interest (see Note 1). 2. Add the PCR product or plasmid to of a PURE-based system kit following the manufacturer’s instructions. 3. Incubate at 37  C for 2 h. 4. Stop the reaction by placing the tube(s) on ice. 5. To determine the translation efficiency, remove 10% of your reaction (e.g., if the final reaction volume was 20 μL take 2 μL for this analysis) and analyze it by SDS-PAGE followed by western and dot blot (see below) using antibodies against the epitope tag fused to the protein of interest. 6. The reaction mixture can be used directly in the lipid overlay since its components do not interfere with the assay (see Note 2).

3.2 Protein Detection by Dot Blot

In a PLOA, proteins are directly applied to the nitrocellulose strips without a denaturalization step. As a result, small epitope tags can be hidden within the native structure of the protein under study

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and not fully accessible to antibodies. Dot blots provide a close approximation to whether epitope tags will be detectable by antibodies after a PLOA. If the antibody does not recognize the epitope tag fused to the protein in a dot blot, we recommend not to proceed with the PLOA and try a new tag instead. 1. Take 2 μL of the in vitro protein synthesis reaction and add 8 μL of TBS. Split the content in two aliquots of 5 μL each. Keep on in ice one of the aliquots (native), and incubate the other at 95  C for 5 min (denatured). 2. Using a narrow-pipette tip, apply a 1 μL drop of each of the two aliquots onto a nitrocellulose membrane. The distance between the two drops should be at least 1 cm. 3. Let dry for 10 min. 4. Block the membrane with blocking solution. Incubate at room temperature for 1 h with constant agitation. 5. Discard the blocking solution and add the primary antibody dissolved in blocking buffer (1:1000 to 1:10000 dilution). 6. Wash the membrane three times with washing buffer for 5 min each time, at room temperature and with gentle agitation. 7. Discard the washing solution and transfer the membrane strips into a clean tray. Do not let the membrane strips dry. 8. Detect the bound protein according to the antibody used. For HRP-conjugated antibodies, prepare the working solution of the chemiluminescent substrate. We recommend trying substrates with different sensitivity (e.g., Regular ECL Western blotting substrate, Super Signal West Pico or Femto) and different exposure times. For fluorescently labeled antibodies, expose the membrane using a fluorescence imaging system. 3.3 Analysis of Lipid Binding Capacity of Target Protein(s) Using PLOA

1. Using methanol-cleansed tweezer, transfer the lipid membrane strips individually into incubation trays keeping the lipid spots facing down (see Note 3). 2. Add 5 mL blocking buffer and incubate for 1 h at room temperature using a platform shaker. Cover the trays with aluminum foil throughout the experiment. 3. Discard the blocking buffer. Dilute the purified protein or in vitro translation reaction in 5 mL of blocking buffer or enough volume to cover the membrane. Incubate for 1 h at room temperature and with gentle agitation (see Note 4). 4. Discard the protein solution and wash the membrane with washing buffer three times for 10 min each at room temperature and with gentle agitation (see Note 5). 5. Prepare the antibody against the tagged protein. We recommend using antibodies directly conjugated to horseradish

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Fig. 2 Dot blot and protein lipid overlay assay of TUBBY-C-His6 detected with anti-His6 antibodies coupled to FITC. (a) Dot blot immunodetection of TUBBY-C-His6 under both native and denaturing conditions. (b) Protein–lipid overlay assay using the in vitro synthesis reaction containing TUBBY-C-His6. TUBBY-C-His6 binds to PI(4,5)P2 and PI(3,4,5)P3. PA, phosphatidic acid; PC, phosphatidylcholine; PE phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; PI(4)P, phosphatidylinositol 4-phoshate; PI(4,5)P2 phosphatidylinositol 4,5-bisphoshate; PI(3,4,5)P3 phosphatidylinositol 3,4,5trisphoshate

peroxidase (HRP) or a fluorescent molecule (Fig. 2) to avoid the incubation step with a secondary antibody; this is particularly important of the protein under study is prone to degradation. Fluorescent antibodies are light sensitive and should be cover with aluminum foil during the incubation. 6. Wash membrane three times with washing buffer for 10 min each, at room temperature and with gentle agitation. 7. Detect bound proteins as explain in Subheading 3.2, steps 7 and 8 (Fig. 3). 3.4 Extraction of Acidic Lipids

1. Grow Arabidopsis thaliana seeds under short day cycles (10-h light–14-h dark cycle at 23  C). Grow plants for 4 weeks and harvest leaf material prior to bolting. 2. Use 1 g of rosettes leaves (2 medium rosettes) per 2 mL of grinding buffer. Grind leaves on mortar in ice-cold grinding buffer. Keep all media, centrifuge tubes, and mortar and pestle on ice. 3. Filter the homogenate through Miracloth (prewet the cloth with grinding buffer). 4. Collect crude pellet by centrifugation at 10,000  g for 15 min at 4  C, discard the supernatant. Immediately resuspend the pellet with solution I (around 1 mL of solution I per 50 mg of

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Fig. 3 Dot blot and protein lipid overlay assay of TUBBY-C-His6 detected with anti-His6 antibodies coupled to HRP. (a) Dot blot immunodetection of TUBBY-C-His6. Monoclonal anti-His6 antibodies coupled to HRP recognize the epitope tag under both native and denaturing condition. (b) Protein–lipid overlay assay using the in vitro synthesis reaction containing TUBBY-C-His6. TUBBY-C-His6 binds to PI(4,5)P2 and PI(3,4,5)P3. Protein detection was performed with monoclonal anti-His6 antibodies coupled to HRP, followed by chemiluminescent detection with different substrates. The chemiluminescent substrate can affect the level of background signal from the membrane. The best result was obtained with standard ECL Western blotting substrate. DAG, 1,2-Diacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PE phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; PI(4)P, phosphatidylinositol 4-phoshate; PI(4,5)P2 phosphatidylinositol 4,5-bisphoshate; PI(3,4,5)P3 phosphatidylinositol 3,4,5trisphoshate

pellet; see Note 6). After homogenization a one-phase system should form (see Note 7). 5. Add 200 μL of 0.6 N HCl to generate two-phase: an acidified aqueous upper phase and an organic lower phase containing nonpolar compounds, such as phospholipids. 6. Remove and discard the top phase without disturbing the organic phase. 7. Wash the remaining bottom phase three times with 400 mL of Solution II, each time removing and discarding the upper phase. 8. Transfer the bottom phase and any interface material left to a tube and centrifuge at 5000  g for 5 min. Recover the bottom phase and transfer into a new tube. Dry the samples in a vacuum dryer (see Note 8). 9. Resuspend the lipids in a small volume (e.g., 20–50 μL) of solution III. For best results, lipid extracts should be spotted onto nitrocellulose membranes right after isolation (see Note 9).

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Fig. 4 Phosphoinositide binding assay using homemade lipid membrane strips. Crude organelle and endomembrane system were isolated by centrifugation and acidic lipids were extracted by a basic method. Serial dilutions of extracted lipids were spotted onto nitrocellulose membrane strips and incubated with 5 μL of the in vitro protein synthesis reaction mixture containing TUBBY-C-His6. The protein was detected with monoclonal anti-His6 antibodies coupled to HRP, followed by chemiluminescent detection. As a control, undiluted sample (2 μL) extracted from membrane pellet was spotted in the bottom

3.5 Blotting Lipids on Nitrocellulose Membranes

1. Cut strips of nitrocellulose membrane to fit at least four individual lipid spots (4 cm  2 cm). Cut a corner of the membrane for orientation purposes. 2. Using a pencil, mark the membrane lightly with a grid to guide the application of the lipid dots. There should be a minimum distance of 1 cm between dots. 3. After resuspending extracted plant acidic lipids in solution III (see Subheading 3.4) prepare three serial dilutions (e.g., 1, 1:10, 1:100). Keep dilutions in a cooled benchtop rack. 4. Pipet 1 μL of each lipid dilution onto the prelabeled nitrocellulose membrane with the most highly concentrated lipid sample at one end of the strip. 5. Dry the membranes for 1 h at room temperature. Protect them from light by wrapping them in aluminum foil. Store at 4  C. 6. For lipid–protein interaction assay follow the same steps as in PLOA (Fig. 4).

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Notes 1. The template should be free of salts and residual RNA. Plasmid DNA prepared from commercial kits may contain inhibitory amounts of RNase A. If RNase was used during plasmid preparation, phenol–chloroform deproteinization should be performed. In our hands 250 ng of DNA in 25 μL of final reaction works well. However, depending of the amplicon the optimal amount of DNA will fall in the range of 25–1000 ng. 2. If protein purification is required, we recommend using polyHis tagging and immobilized metal affinity chromatography. 3. Both commercial and custom-made lipid membrane strips can fit in a 5  7 cm incubation tray. 4. For commercial lipid membrane strips, it is recommended to start with 0.5 μg/mL or a lower concentration to avoid binding saturation. If the in vitro reaction is used, between 5 and 20 μL of reaction mixture should be used. 5. During the washing steps, do not use tweezer to hold the membrane strips. Instead, carefully tip the incubation tray into a waste container to discard the washing buffer; the membrane will attach to the bottom of the tray. Do not let the membrane strips ¼ dry out. 6. The volume of solution I depends on the amount of material to be extracted and its water content. For larger scale isolation (e.g., fresh tissue from whole rosettes), approximately 10 mL of solution should be added per 500 mg of fresh tissue. Tissue with lower water content (e.g., dry seeds) needs less solution I for pellet resuspension. 7. If water content is too high, a two-phase system will form. If this happens, a small amount of cold methanol must be added to obtain a single phase. 8. Ideally, solvents should be evaporated under a stream of O2-free N2 gas. 9. To avoid quick evaporation of the solvents in which lipids are dissolved, we recommend keeping the extracted lipids in a cooled rack.

Acknowledgements This work was supported by NSF grant MCB1614965.

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References 1. Paez Valencia J, Goodman K, Otegui MS (2016) Endocytosis and endosomal trafficking in plants. Annu Rev Plant Biol 67:309–335 2. Saliba AE, Vonkova I, Gavin AC (2015) The systematic analysis of protein-lipid interactions comes of age. Nat Rev Mol Cell Biol 16:753–761 3. Dowler S, Kular G, Alessi DR (2002) Protein lipid overlay assay. Sci STKE 2002:6 4. Cheever ML, Sato TK, de Beer T, Kutateladze TG, Emr SD, Overduin M (2001) Phox domain interaction with PtdIns(3)P targets the Vam7 t-SNARE to vacuole membranes. Nat Cell Biol 3:613–618 5. Zhan Y, Virbasius JV, Song X, Pomerleau DP, Zhou GW (2002) The p40phox and p47phoxPX domains of NADPH oxidase target

cell membranes via direct and indirect recruitment by phosphoinositides. J Biol Chem 277:4512–4518 6. Rathner P, Stadlbauer M, Romanin C, Fahrner M, Derler I, Mu¨ller N (2018) Rapid NMR-scale purification of (15)N,(13)C isotope-labeled recombinant human STIM1 coiled coil fragments. Protein Expr Purif 146:45–50 7. Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T (2001) Cell-free translation reconstituted with purified components. Nat Biotechnol 19:751–755 8. Santagata S, Boggon TJ, Baird CL, Gomez CA, Zhao J, Shan WS, Myszka DG, Shapiro L (2001) G-protein signaling through tubby proteins. Science 15:2041–2050

Chapter 13 Isolation and Glycomic Analysis of Trans-Golgi Network Vesicles in Plants Guangxi Ren, Michel Ruiz Rosquete, Angelo G. Peralta, Sivakumar Pattathil, Michael G. Hahn, Thomas Wilkop, and Georgia Drakakaki Abstract The dynamic endomembrane system facilitates sorting and transport of diverse cargo. Therefore, it is crucial for plant growth and development. Vesicle proteomic studies have made substantial progress in recent years. In contrast, much less is known about the identity of vesicle compartments that mediate the transport of polysaccharides to and from the plasma membrane and the types of sugars they selectively transport. In this chapter, we provide a detailed description of the protocol used for the elucidation of the SYP61 vesicle population glycome. Our methodology can be easily adapted to perform glycomic studies of a broad variety of plant cell vesicle populations defined via subcellular markers or different treatments. Key words Glycomics, Endomembrane trafficking, Immunoisolation, Vesicle isolation, SYP61, Trans-Golgi Network, Polysaccharides

1

Introduction The plant endomembrane system is a complex and dynamic network of membranous compartments playing crucial roles in plant growth, development, and adaptation to the environment. It facilitates the transport of proteins and other cargoes and is pivotal for cell wall biosynthesis and assembly [1–5]. The cell wall is a complex structure made of polysaccharides, structural proteins and other molecules that surrounds and protects plant cells and is essential for their development. While many enzymes responsible for polysaccharide biosynthesis have been identified, our understanding of how polysaccharides are transported and assembled is still limited. Polysaccharides originate at distinct cellular locations; cellulose and callose are synthesized at the plasma membrane, whereas the synthesis of hemicellulose and pectin and the glycosylation of proteins take place in the Golgi

Marisa S. Otegui (ed.), Plant Endosomes: Methods and Protocols, Methods in Molecular Biology, vol. 2177, https://doi.org/10.1007/978-1-0716-0767-1_13, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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apparatus and the trans-Golgi Network [6, 7]. Cell wall polysaccharides not synthesized in muro, associated enzymes and glycoproteins are carried to their specific cell wall deposition sites by vesicle transport pathways that remain elusive [1, 8–11]. The highly dynamic nature of the endomembrane system makes it challenging to assign unequivocal roles to specific vesicle populations in the transport of cell wall material and assembly of the cell wall. The plant trans-Golgi Network/Early Endosome (TGN/EE) is a distinct compartment on the Golgi trans-side comprising diverse vesicle populations and serving as a hub of secretory and endocytic traffic [12–16]. The plant TGN/EE is unique in that it orchestrates the trafficking of cell wall polysaccharides from the Golgi, the point of their synthesis, to the plasma membrane, for cell wall deposition, assembly, and modification (Fig. 1a) [1, 11, 17]. Because polysaccharide composition determines the biological function of plant cell walls, the dynamics of polysaccharide traffic is a decisive factor in mechanisms that control cell wall deposition and assembly. Thus, the development of approaches to illuminate the glycome of plant vesicles is of great significance. Protocols for the isolation of specific plant vesicle populations have recently become available [18–20]. They are allowing us to identify the protein cargo of vesicles and to shed light into their biochemical properties, such as membrane lipid composition [13], for a better understanding of vesicle heterogeneity and vesicle functional compartmentalization. Recent technical advances have made it possible to start piecing polysaccharides biosynthesis, assembly, and modification together. These include the application of chemical methods for compositional analysis of the plant cell wall [21] and the use of oligosaccharide mass profiling (OLIMP) to retrieve compositional data from preparations of Golgi-enriched fractions or isolated cell walls [22– 24]. Additionally, the labeling and imaging of sugars modified via click chemistry [25–29] can provide kinetic details of cell wall formation. Cell wall glycan-directed antibodies are an elegant option for the identification of plant cell carbohydrates in diverse tissues and species [30–34]. During glycome profiling, antibody libraries are paired with an automated large scale enzyme-linked immunosorbent assay (ELISA), enabling the fingerprinting of plant cell wall glycan content with both high sensitivity and specificity [31]. To interrogate the glycome of intracellular vesicles, we optimized our vesicle isolation protocols for this specific application. In previous studies, we established a methodology for the isolation of the syntaxin of plants 61 (SYP61) TGN/EE vesicle subpopulation to high purity levels, suitable for subsequent proteomic studies [18, 19]. The SYP61 vesicle population has been implied in postGolgi trafficking of the wall biosynthetic machinery, a notion supported by the analysis of the SYP61 proteome, which revealed

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Fig. 1 Structural polysaccharide transport and deposition, and a hybrid methodology for vesicle glycomic analysis. (a) Schematic representation of structural polysaccharide synthesis, transport and deposition. The structural polysaccharides xyloglucan and pectin are synthesized in the Golgi and transported via trans-Golgi Network/Early Endosome (TGN/EE) vesicles to the apoplast. The type of vesicles carrying specific polysaccharide cargo to the cell wall is unknown. Three different vesicle subpopulations, indicated with magenta, green and blue vesicle protein markers are depicted to illustrate the heterogeneity of the TGN/EE. (b) Schematic representation of vesicle isolation. Plant extracts derived from liquid-grown plantlets are sucrose fractionated. A heterogeneous TGN/EE vesicle population is isolated from the Golgi/TGN/EE enriched sucrose fractions after which the specific vesicle subpopulation of interest (magenta surface protein marker) is purified with the aid of an antibody against the target protein. (c) Vesicle cargo release and glycome analysis. Vesicle cargo is released by sonication for glycome analysis. An ELISA-based method of glycome detection is used and the resulting data are summarized in a heat map for analysis. mAb, monoclonal antibody; PM, plasma membrane

several cellulose synthase subunits and cell wall modifying enzymes as cargo of the SYP61 vesicles [19, 35]. Such findings prompted the question whether not only cell wall biosynthetic and modifying enzymes but also cell wall structural polysaccharides are transported in this specific TGN/EE vesicle compartment. Such information could help mapping the intracellular transport of polysaccharides. Toward answering this critical question, we designed an experimental approach combining an optimized protocol for SYP61 TGN/EE vesicle isolation with the large-scale profiling of TGN/EE vesicle through a polysaccharide carbohydrate antibody arraying technique investigating 155 carbohydrate epitopes [36]. The implementation of this hybrid approach revealed trafficking and sorting of diverse glycans of pectins, xyloglucans (XyGs), and structural cell wall glycoproteins through the SYP61 TGN/EE compartment in Arabidopsis [36]. Since TGN is a major intersection in post-Golgi trafficking, its comparison with the Golgi or

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sub-Golgi glycome(s) [37, 38] can offer major insights into polysaccharide biosynthesis and transport within the secretory pathway. This chapter describes in detail our (1) separation of vesicles by immunopurification, optimized for CFP-SYP61, combined with (2) large-scale automated carbohydrate antibody arraying methodology using an ELISA (Fig. 1b, c). Adopting this approach, different vesicle populations can be characterized, complementing our proteomic perspective of cellular pathways with glycomics.

2 2.1

Materials Plant Material

1. 50–100 transgenic Arabidopsis seeds for each flask, expressing CFP-SYP61 [19] (see Notes 1 and 2). 2. Seed sterilization solution: 75% ethanol, 0.1% Triton X-100, 24.9% autoclaved deionized water. 3. Liquid Murashige and Skoog (MS) medium: Full strength 1 MS medium, 1% (w/v) sucrose. Dissolve 4.26 g of MS minimal media and 10 g of sucrose in 1000 mL deionized water. Aliquot 200 mL of media into a 500 mL Erlenmeyer flask and autoclave (see Notes 3 and 4). 4. Flask shaker placed in a temperature- and photoperiodcontrolled environment (long day light cycle, 16 h of light at 22–24  C) (see Note 5).

2.2 Vesicle Fractionation Components

1. Vesicle immunoprecipitation extraction buffer (VIB): 50 mM HEPES, pH 7.5, 0.45 M sucrose, 5 mM MgCl2, 1 mM DTT, 0.5% PVP (w/v), protease inhibitors (cOmplete™ Protease Inhibitor Cocktail, ROCHE). 2. Sucrose gradient solutions: 38% (w/v) sucrose (1.1 M), 33% (w/v) sucrose (0.96 M), and 8% (w/v) sucrose (0.23 M) in 50 mM HEPES pH 7.5 (see Note 6). 3. Mortar and pestle. 4. Razor blades. 5. Miracloth. 6. Small funnel. 7. Refrigerated benchtop centrifuge. 8. 50 mL conical centrifuge tubes. 9. Ultracentrifuge (e.g., Optima L-90 K Beckman Coulter, or equivalent) with rotors SW28 and 70Ti. 10. Centrifuge tubes for SW28 rotor (thickwall, polyallomer, 32 mL tubes). 11. Centrifuge tubes for 70Ti (polycarbonate aluminum bottle with cap assembly).

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12. Disposable serological 5, 10, and 25 mL pipettes. 13. Pasteur pipettes. 2.3 Vesicle Immunoisolation Components

1. Resuspension buffer: 50 mM HEPES, pH 7.5, 0.25 M sucrose, 1.5 mM MgCl2, 150 mM NaCl, protease inhibitors (see Note 7). 2. Wash buffer: 50 mM HEPES, pH 7.5, 0.25 M sucrose, 1.5 mM MgCl2, 150 mM NaCl. 3. Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·H2O, KH2PO4, pH 7.4. 4. Protein-A agarose beads. 5. Anti-GFP rabbit IgG (2 mg mL 1) (e.g., Invitrogen A11122, anti-GFP IgG) (see Note 8). Alternatively, magnetic agarose beads covalently coupled to GFP antibodies (e.g., GFP-Trap®_MA ChromoTek) can be used for immunoisolation with non-coupled beads as controls. 6. Rabbit IgG (see Note 9). 7. Tube rotator. 8. Refrigerated benchtop centrifuge.

2.4 Material for Enzyme-Linked Immunosorbent Assay

1. Enzyme-linked immunosorbent assay (ELISA) acid-resistant 384 well flat bottom plates (e.g., Costar 3700 from Corning Life Sciences). 2. Automated platform mover (e.g., Orbitor RS Microplate Mover ORB2006 from Thermo Scientific). 3. Microplate sample processor (e.g., BioTek™ Precision™ XS from Biotek). 4. Washer Dispensers (e.g., MicroFlo™ and EL406™ Washer Dispensers from BioTek). 5. 0.1 M Tris-buffered saline (TBS), pH 7.6: 23.38 g of sodium chloride, 1.11 g of Tris-Base, 4.85 g of Tris–HCl, in 4 L of ultrapure water. Store at room temperature. 6. Blocking Buffer: 1.0% (w/v) milk in 0.1 M TBS, pH 7.6: 10 g of nonfat dry milk in 1 L of 0.1 M TBS. Store at 4  C. 7. Wash Buffer: 0.1% (w/v) milk in 0.1 M TBS pH 7.6. 8. Primary antibodies: CCRC series of antibodies generated in mouse; JIM, MAC, and LM series of antibodies generated in rat. A web-accessible database listing most of the available plant cell wall glycan-directed mAbs and providing information about their characteristics and suppliers can be found at WallMabDB (http://www.wallmabdb.net). The three main suppliers of plant glycan-directed antibodies are CarboSource (http://www.carbosource.net), PlantProbes (http://www. plantprobes.net), and BioSupplies (http://www.biosupplies. com.au/).

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9. Secondary antibody: anti-mouse or anti-rat IgG whole molecule goat antibody, conjugated with horseradish peroxidase diluted in wash buffer according to the manufacturer’s instructions. Secondary antibody stocks are stored at 20  C when not in use. 10. Substrate: TMB KPL 2-Component Microwell Peroxidase Substrate Kit (SeraCare Life Science Inc.). 11. Stop solution: 0.5 N sulfuric acid: 100 mL of 0.5 N sulfuric acid, 1 mL of 18 M sulfuric acid, 71 mL of deionized water. 12. Microplate spectrophotometer reader. 13. Probe sonicator, Branson 250–450 Sonifier or equivalent. 14. R-Console software.

3 3.1

Methods Plant Preparation

1. Two weeks before vesicle isolation, sterilize seeds and stratify them at 4  C overnight. 2. Grow 50–100 seeds in 200 mL liquid MS in each 500 mL Erlenmeyer flask while shaking at 150 rpm under a long day cycle at 22–24  C for ~10 days. For the analysis of TGN compartments, more than 12 g of tissue are required. Plants should be grown ~10 days in liquid media to yield sufficient root tissue.

3.2 Golgi/TGN Fractionation by Sucrose Density Gradient Ultracentrifugation

Plant extracts from liquid grown plantlets are fractionated using discontinuous sucrose gradient centrifugation to enrich for SYP61 vesicles. During vesicle isolation, maintain all buffers and rotors at 4  C. All steps after harvesting tissues should be performed on ice. Figure 2 illustrates the sucrose fractionation procedure. 1. Rinse plants carefully in deionized water and pat-dry with paper towels in a large petri dish. Weigh plants. 2. Slice plants with a razor blade in the petri dish set on ice. Transfer the finely sliced tissues into a cold mortar on ice. 3. Add ice-cold VIB to a final v/w ratio of 2:1 (e.g., 2 mL of VIB buffer for 1 g of plant tissue) and grind the plant tissue as gently as possible to a rough pulp (see Note 10). Place funnel with Miracloth over a 50 mL conical centrifuge tube to filter the plant extract and centrifuge at 1000  g at 4  C for 20 min. 4. Meanwhile, using a 10 mL pipette, add 8 mL of 38% sucrose to a thickwall 32 mL centrifuge tube. Load gently the supernatant from step 3 (S1 fraction) on top of the sucrose cushion (see

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Fig. 2 Enrichment of Golgi/trans-Golgi Network compartments by sucrose gradient ultracentrifugation. Schematic illustration of the sequential sucrose gradient ultracentrifugation to isolate Golgi/TGN compartments. Briefly, the Supernatant (S1) fraction of plant homogenates is loaded onto a 38% sucrose cushion and centrifuged at 100,000  g for 1.5 h (Subheading 3.2, step 4). The upper phase is removed and a discontinuous gradient is formed by adding the two 33 and 8% sucrose layers (Subheading 3.2, step 5). The interface between 8% and 33% sucrose fractions is collected (Subheading 3.2, step 6) and transferred onto a 30 mL centrifuge tube for centrifugation at 100,000  g, 1 h (Subheading 3.2, step 7). The resulting pellet is kept for vesicle immunoisolation. UP, upper phase; IF, interface. Note: Sucrose gradients can be adjusted for the isolation of different vesicle populations

Note 11). Centrifuge at 100,000  g at 4  C for 1.5 h using a SW28 rotor or equivalent. 5. Place the tube on ice, and remove the plant extract liquid above the green interface band, without disturbing it. Using a 25 mL pipette, carefully add 15 mL of 33% sucrose on top of the collected green band, and then add 5 mL of 8% sucrose. Centrifuge at 100,000  g at 4  C in SW28 rotor or equivalent for 2 h (see Note 12). 6. Using a 5 mL pipette, slowly remove and discard 4–5 mL of the top gradient layer (8% sucrose). Using a Pasteur pipette, collect the interface band between the 8% and 33% sucrose layers into an ice-chilled 30 mL centrifuge tube and add 0.5 the volume of 50 mM HEPES (pH 7.5).

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7. Centrifuge at 100,000  g at 4  C using a fixed angle 70Ti rotor or equivalent for 1 h. Decant the supernatant and keep the pellet at 4  C overnight (see Note 13). 3.3 Immunoisolation of SYP61 Vesicles

SYP61 vesicles are isolated by immunopurification from the fraction obtained in the previous section. 1. Couple the GFP antibody to protein-A agarose beads. First, mix the protein-A agarose beads and place 25 μL into a 1.5 mL microfuge tube. Add 500 μL of ice-cold PBS, mix well by inverting the tube, and centrifuge at 10,000  g for 30 s (see Note 14). Alternatively, use magnetic agarose beads directly coupled with GFP antibodies, for example, GFP-Trap®_MA ChromoTek. In this case, follow the protocol from Subheading 3.4, step 1. 2. Discard supernatant using a pipette and add 2 μL of the antiGFP antibody to the pellet. Add cold PBS containing protease inhibitors to a final volume of 100 μL, mix well by inverting the tube and incubate on a rotator for 2 h. Centrifuge at 10,000  g for 30 s and discard the supernatant. 3. Equilibrate the antibody coupled-agarose beads with 200 μL resuspension buffer on a rotator at 4  C for 20 min. Centrifuge at 1000  g for 30 s. Carefully discard the supernatant. 4. Meanwhile, resuspend the vesicle pellets from Subheading 3.2, step 7 in 400 μL of resuspension buffer and incubate with 25 μL of protein-A agarose beads (see Note 15). Gently mix the suspension for 20 min using a rotator at 4  C and then centrifuge at 1000  g for 30 s. Collect the supernatant. 5. Add 300 μL of the supernatant collected in step 4 to the antibody coupled agarose beads collected in step 3 and mix for 1 h on a rotator at 4  C. Centrifuge at 100  g for 1 min (see Note 16). Discard supernatant. 6. Wash the pellet with 1 mL of wash buffer under gentle agitation at 4  C for 2 min and centrifuge at 100  g for 1 min. Repeat this step three times (see Notes 17 and 18). Keep the pellet.

3.4 Proceed to the Following Steps if You Are Using Beads Covalently Bound to the Antibody

1. Vortex GFP-Trap®_MA beads and pipette 25 μL bead slurry into 500 μL resuspension buffer. Magnetically separate beads until supernatant is clear. Carefully discard the supernatant and repeat the bead wash/equilibration step twice. 2. Add 300 μL of the supernatant collected in subheading 3.3, step 4 to the equilibrated GFP-Trap®_MA beads collected in subheading 3.4, step 1 and mix for 1 h on a rotator at 4  C.

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3. Magnetically separate beads until supernatant is clear. If desired, save 50 μL supernatant for immunoblot analysis. Discard remaining supernatant. 4. Wash the beads with 1 mL of wash buffer under gentle agitation at 4  C for 2 min and magnetically separate until supernatant is clear. Discard supernatant. Repeat this step three times (see Notes 17 and 18). Keep the pellet. 3.5 Glycome Analysis

1. Dilute isolated vesicles to a final volume of 8 mL with double distilled water and sonicate on ice using a micro-tip with attachment, with output setting of 21.5 microns amplitude. Pulse-sonicate using 1s cycles followed by subsequent 1 s rest for a period of one min. 2. Repeat sonication three times to ensure complete disruption of vesicles. 3. Centrifuge for 15 min at 2200  g at 4  C. Collect the supernatant. 4. Coating of the ELISA plates: 15 μL per well of collected supernatant (after sonication and centrifugation) of each polysaccharide extract dilutions are added to the 384-well ELISA plates (with the number of coated wells equaling the number of mAbs to be tested plus controls) using a microplate sample processor. Evaporate to dryness overnight in a ventilated 37  C incubator. Handling of the acid-resistant flat-bottom 384-well plate and incubation times are performed by the automated platform microplate mover. 5. Blocking: Nonspecific sites in the coated ELISA plates are blocked by adding 15 μL of blocking buffer per well, with the aid of a washer dispenser, followed by incubation for 1 h at room temperature. 6. Addition of primary antibodies (mAbs): The blocking buffer is aspirated and 15 μL of primary mAb are dispensed into each well using different washer dispensers (e.g., the MicroFlo™ Washer Dispenser for CCRC anti-mouse series and EL406™ for JIM and MAC for anti-rat series). Incubate plates with the primary antibodies for 1 h at room temperature. 7. Washing the plates: Aspirate the primary antibodies using MicroFlo™ and EL406™ washer dispensers. Wash each well with 20 μL wash buffer. Completely aspirate the buffer after 5 s. Repeat washes three times. 8. Secondary antibodies: After washing, add 15 μL of secondary antibody per well. Dispense anti-mouse or anti-rat secondary antibodies (mixed at a 1:5000 dilution in wash buffer) using the MicroFlo and EL406 washer dispensers into the respective mouse (e.g., CCRC series, using the MicroFlo™ washer

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dispenser) and rat (e.g., JIM series, using the EL406™ washer dispenser) primary antibody-bound wells and incubate at room temperature for 1 h. 9. Washing secondary antibodies: Aspirate the secondary antibodies from each well after the incubation. Wash each plate well with 20 μL of wash buffer for 5 s and repeat four more times for a total of five washes using the washer dispensers. 10. Adding substrate and termination: Mix KPL TMB solution A with KPL TMB solution B in a 1:1 ratio (for 500 mL, mix 250 mL of Solution A with 250 mL of Solution B onto a separate container) without the stabilizer included in the kit. Dispense 10 μL of the prepared KPL TMB mix into each well. Allow each plate to incubate for precisely 30 min and stop the reaction with 10 μL of 0.5 N sulfuric acid per well using a microplate sample processor (see Note 19). 11. Quantitation: Immediately after termination, measure the net OD values of the color formation in the wells of the ELISA plates using a microplate spectrophotometer reader at 450 nm and subtract a background reading at 655 nm. Assemble the ELISA results into a heatmap using a modified version of the R-Console software [39] (Fig. 3) (see Note 20).

4

Notes 1. This protocol uses SYP61-CFP expressing plants for vesicle isolation. The CFP N-terminally fused to SYP61 interacts with the antibody during isolation, while the SYP61 C-terminus facilitates the attachment to the vesicle membranes. Special consideration should be given to the “bait” protein that will be used for isolation, in particular the accessibility to the antibody. 2. It is important to start with sufficient plant material. Only a selected fraction from the sucrose gradient will be used for vesicle isolation. Note that more than 12 g of plant tissues are required. 3. Growth in liquid media yields more root tissues. However, when the target protein is highly abundant in specific tissues or developmental stages, collect the appropriate tissues to obtain a higher yield. 4. The MS media used in this protocol are pH-adjusted. When other MS media are used, adjust the pH to 5.8–6.0. 5. Adjust growth conditions favoring the tissue expressing the bait protein.

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Strong Binding

mAbs

450-655 nm

1.3

Weak Binding

0.0

Fig. 3 Example of a vesicle glycome profiling showing diverse epitopes of pectic glycans in isolated SYP61 vesicles. The mean of three biological replicates is shown, after subtraction of negative controls. White to red scales indicates signal intensity in the ELISA assays, with white corresponding to no binding and intense red to strong binding

6. The sucrose gradient procedure is adapted from Drakakaki et al. (see Ref. 40). If necessary, the sucrose gradient can be adjusted with more layers or different sucrose densities to enrich for the specific target vesicles. 7. The composition of the resuspension buffer can be modified to enable the best binding of the bait protein. 8. Due to its ready availability and reactivity with CFP, the GFP antibody is used. 9. Rabbit IgG is used as a control, since the GFP antibody is raised in rabbit. Similarly, the control must be chosen according to the antibody host. 10. In this step, it is very important to keep the plant material cold, but not frozen. Ground tissue should be maintained as slurry. 11. A sharp interface between the sucrose cushion and supernatant should be visible. Carefully set up the sucrose layers and place the centrifugation tubes on the rotor without disturbing the sharp interface. 12. The extent of centrifugation might depend on the model of the centrifuge used. If the interfaces between the sucrose layers are not sharp, increase the centrifugation time.

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13. Keeping the pellet overnight at 4  C is fine. If time permits, the next steps can be performed immediately. 14. Instead of agarose beads, other types, such as magnetic beads, can be used. 15. This step is necessary to minimize nonspecific binding to the protein-A agarose beads. 16. After centrifugation, the supernatant can be stored and used to determine the vesicle purification efficiency by Western blot analysis. 17. Before the last washing step, keep a small fraction of ~10% of the suspension to test the isolation efficiency. Centrifuge at 100  g for 1 min. Resuspend in 50 μL PBS and mix with appropriate SDS protein loading buffer to prepare the sample for SDS-PAGE and Western blot analysis. 18. In addition to the sample collected in [17], we recommend that samples from the original enriched vesicle fraction (Subheading 3.3, step 4), the flow through (Subheading 3.3, step 5) and the immunoisolated fraction (Subheading 3.3, step 6) are analyzed by Western blot. The presence of CFP-SYP61 in those samples can be evaluated using a monoclonal antibody against GFP. In addition, antibodies against subcellular markers for the endoplasmic reticulum (ER) marker, BiP [41] and the pre vacuolar compartment (PVC) marker, SYP21 [42], can be used to test the purity of the isolated SYP61 vesicles. The physical integrity of the isolated vesicles can be assessed by transmission electron microscopy [19]. 19. The reproducibility and robustness of the data are superior when the ELISAs are performed with an automated platform, which minimizes human errors, particularly during the color development step. 20. Background noise from control data generated using no antigen (water) are subtracted from the mean values obtained, making the data more error resilient and statistically significant.

Acknowledgments This work was supported by the NSF MCB 1818219 award to GD, and the USDA Hatch CA-D-PLS-2132-H to G.D. G.R was partially supported by the China Scholarship Council. The generation of the CCRC series of the used plant cell wall glycan-directed antibodies was supported by the NSF (DBI-0423683 and IOS0923992) awards to MGH.

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(2008) High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles. Glycoconj J 25 (1):37–48. https://doi.org/10.1007/ s10719-007-9059-7 33. Pedersen HL, Fangel JU, McCleary B, Ruzanski C, Rydahl MG, Ralet MC, Farkas V, von Schantz L, Marcus SE, Andersen MC, Field R, Ohlin M, Knox JP, Clausen MH, Willats WG (2012) Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research. J Biol Chem 287 (47):39429–39438. https://doi.org/10. 1074/jbc.M112.396598 34. Pattathil S, Avci U, Zhang T, Cardenas CL, Hahn MG (2015) Immunological approaches to biomass characterization and utilization. Front Bioeng Biotechnol 3:173. https://doi. org/10.3389/fbioe.2015.00173 35. Worden N, Esteve VE, Domozych DS, Drakakaki G (2015) Using chemical genomics to study cell wall formation and cell growth in Arabidopsis thaliana and Penium margaritaceum. Methods Mol Biol 1242:23–39. https:// doi.org/10.1007/978-1-4939-1902-4_2 36. Wilkop T, Pattathil S, Ren G, Davis DJ, Bao W, Duan D, Peralta AG, Domozych DS, Hahn MG, Drakakaki G (2019) A hybrid approach enabling large-scale Glycomic analysis of postGolgi vesicles reveals a transport route for polysaccharides. Plant Cell 31(3):627–644. https://doi.org/10.1105/tpc.18.00854 37. Okekeogbu IO, Pattathil S, Gonzalez Fernandez-Nino SM, Aryal UK, Penning BW, Lao J, Heazlewood JL, Hahn MG, McCann MC, Carpita NC (2019) Glycome and proteome components of Golgi membranes are common between two angiosperms with distinct cell-wall structures. Plant Cell 31 (5):1094–1112. https://doi.org/10.1105/ tpc.18.00755 38. Parsons HT, Stevens TJ, McFarlane HE, VidalMelgosa S, Griss J, Lawrence N, Butler R, Sousa MML, Salemi M, Willats WGT, Petzold CJ, Heazlewood JL, Lilley KS (2019) Separating Golgi proteins from cis to trans reveals underlying properties of cisternal localization. Plant Cell 31(9):2010–2034. https://doi.org/ 10.1105/tpc.19.00081 39. R Development Core Team. (2006) R: a language and environment for statistical computing. R Foundation for Statistical Computing. http://www.R-projectorg 40. Drakakaki G, Zabotina O, Delgado I, Robert S, Keegstra K, Raikhel N (2006) Arabidopsis reversibly glycosylated polypeptides 1 and 2 are essential for pollen development.

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Chapter 14 Detection of Phosphorylation on Immunoprecipitates from Total Protein Extracts of Arabidopsis thaliana Seedlings Karin Vogel and Erika Isono Abstract Phosphorylation is a versatile posttranslational modification that can regulate the localization, stability, and conformation of proteins; protein–protein interactions; and enzyme activities. Phosphorylation of plasma membrane proteins, for example, can serve as recognition signals for ubiquitin ligases and hence can trigger its endocytic degradation. Key determinants of protein phosphorylation are kinases and phosphatases that are spatiotemporally regulated to phosphorylate or dephosphorylate specific target proteins. To understand the dynamics and regulatory mechanisms of protein phosphorylation, it is essential to analyze the phosphorylation status of the proteins and identify phosphorylation sites as well as the modifying enzymes. In this chapter, we describe methods that can be used for the detection of phosphoproteins that are immunoprecipitated from Arabidopsis total extracts. Key words Immunoprecipitation, Protein phosphorylation, Phosphostain, Phos-Tag™ gel

1

Introduction Posttranslational modifications (PTMs) of proteins such as phosphorylation, glycosylation and ubiquitylation regulate many fundamental cellular processes ranging from gene expression to protein degradation. Phosphorylation of plasma membrane receptors and transporters in plants is known to regulate their activity and protein stability and thus affect diverse signaling processes in which the modified protein is involved [reviewed in [1–5]]. PTMs can also depend on each other. For example, one of the best studied mammalian endosomal cargo, the epidermal growth factor receptor (EGFR), is first phosphorylated upon stimulation. Phosphorylated EGFR recruits the ubiquitin ligase c-Cbl, which then ubiquitylates EGFR with K63-linked ubiquitin chains [reviewed in [6]]. In Arabidopsis, a similar phosphorylation-dependent ubiquitylation was reported for the iron transporter IRT1 [7]. Phosphorylation is a dynamic process, which is determined by the activity of kinases and phosphatases. To unravel the molecular

Marisa S. Otegui (ed.), Plant Endosomes: Methods and Protocols, Methods in Molecular Biology, vol. 2177, https://doi.org/10.1007/978-1-0716-0767-1_14, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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details of cellular signaling processes, it is essential to understand where and when a given protein is phosphorylated. The addition of a phosphate group to serine and threonine residues increases the mass of the modified protein and slows the mobility of the protein in an SDS polyacrylamide gel. Using this characteristics, phosphorylation of proteins can be detected by the mobility shift of the phosphorylated protein in a polyacrylamide gel. The disadvantage with this method is that it is not clear whether the observed mobility shift is caused by phosphorylation or by other types of PTMs. To distinguish between these possibilities, an immunoblot with phospho-site specific antibodies such as anti-phosphoserineor anti-phosphothreonine antibodies can be used. Although these antibodies are commercially available, this approach has a number of important limitations: (a) one must know the identity of the residues that are phosphorylated, (b) the antibodies have to be highly specific, and (c) the phosphoproteins can only be detected if they are abundant in cell lysates or after immunoprecipitation. Custom made phospho-antibodies can be more specific, but are time and cost-intensive to generate. Another widely used method to detect phosphorylation is to analyze the mobility shift of the protein in an acrylamide gel containing Phos-tag™ [8, 9]. Phos-tag™ is a dinuclear metal complex that binds phosphate, and can be used for the detection of phosphoproteins in cell lysates, immunoprecipitates, and recombinant proteins. When used together with a total protein stain, the ratio of phosphorylated to nonphosphorylated forms can be determined [10]. Phos-tag™ enhances the mobility shift of the phosphorylated form of the protein, thereby enabling the detection of only moderately phosphorylated proteins and different phosphoisoforms. In addition, commercially available phosphoprotein staining dyes have the advantage that they can be directly applied after standard SDS-PAGE on the acrylamide gel and are thus a robust method to visualize phosphoproteins. For the determination of phosphorylation sites, publicly available phosphoproteome databases are useful starting points [11, 12], though verification by classical amino acid substitution approaches is still required. In this chapter, we describe the visualization of phosphoproteins using Phos-tag™ and a phosphoprotein staining fluorescent dye.

2

Materials

2.1 Plant-Related Material

1. Arabidopsis thaliana seeds expressing a tagged version of your protein of interest (see Note 1). 2. Seed sterilization solution: 1% (w/v) sodium hypochlorite, 0.1% (v/v) Silwet™ (BASF).

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3. Washing solution after seed sterilization: 0.05% (v/v) Silwet in autoclaved water. 4. Arabidopsis solid growth medium (GM): 4.33 g/L Murashige and Skoog (MS) medium including Gamborg B5 vitamins, 10 g/L sucrose, 0.5 g/L MES (2-(N-morpholino) ethanesulfonic acid), 8 g/L agar–agar (e.g., from Roth or Duchefa), adjust to pH 5.8 with KOH. To prepare the GM, weigh in MS, sucrose and MES, add water and adjust the pH to 5.8. Afterward prepare 1 L or 0.5 L bottles with the appropriate amount of agar and fill the liquid medium in the bottles. Autoclave the medium at 121  C for 20 min. 5. GM plates: Pour 75 mL of dissolved GM medium into each 12  12 cm square or Ø14 cm round petri dish under a sterile hood. Let the plates dry with open lids under the sterile hood for 20 min. The GM plates can be used directly or stored at 4  C. 6. Growth chamber at 120 μmol m2 s1 light intensity and 21  C. 2.2 Immunoprecipitation (IP)

1. Buffer A: 50 mM Tris [Tris(hydroxymethyl)aminomethane], 100 mM NaCl, 10% (w/v) glycerol; adjust pH to 7.5 with HCl and cool in a fridge or in a cold room overnight; adjust pH again to 7.5 with HCl. Store at 4  C. 2. 20% (w/v) Triton-X-100: 2 mL of 100% Triton-X-100 in 10 mL of distilled water. Store at room temperature. 3. 50 cOmplete™ EDTA-free Protease Inhibitor (Roche): dissolve 1 tablet in 1 mL of distilled water. Store at 20  C. 4. Extraction buffer: Buffer A supplemented with 0.2% Triton-X100 and 1 cOmplete™ EDTA-free Protease Inhibitor (Roche). Prepare directly before use. 5. Mortar and pestle. 6. Homogenizer (e.g., IKA-T25 Ultra TURRAX™) (see Note 2). 7. Liquid nitrogen. 8. Magnetic affinity beads (see Note 3). 9. Magnetic separation racks for 50 mL and 1.5 mL tubes (e.g., from New England Biolabs). 10. Rotator or shaker at 4  C suitable for 50 mL tubes. 11. Refrigerated centrifuge for 15 mL tubes. 12. Nylon mesh (mesh size 0.5 μm) or syringe filter (pore size 0.45 μm).

2.3 Phosphatase Treatment

1. Lambda Protein Phosphatase (400,000 units/mL). Store at 80  C.

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2. PeppermintStick™ Phosphoprotein Molecular Weight Standards (Thermo-Fisher). 3. Lambda Protein Phosphatase 10 reaction buffer: 50 mM HEPES, 100 mM NaCl, 0.1% Brij35, 2 mM DTT, pH 7.5. Store at 20  C. 4. 10 mM MnCl2 solution. Store at 4  C or room temperature. 5. 5 Laemmli buffer: 250 mM Tris–HCl pH 6.8, 10% (w/v) SDS, 50% (w/v) glycerol, 0.05% bromophenol blue (BPB), 5% β-mercaptoethanol. Store at room temperature. 6. Heating block for 1.5 mL tubes. 2.4 SDS– Polyacrylamide Gel Electrophoresis

1. Separation gel buffer: 1.5 M Tris–HCl pH 8.8 and 0.4% (w/v) SDS. Store at room temperature. 2. Stacking gel buffer: 0.5 M Tris–HCl pH 6.8 and 0.4% (w/v) SDS. Store at room temperature. 3. 30% acrylamide/bis-acrylamide solution (37.5:1), 10% (w/v) ammonium persulfate (APS), and N,N,N,N0 -tetramethyl ethylenediamine (TEMED). Store at 4  C. 4. 100% Isopropanol for overlaying the separation gel. 5. 10 SDS running buffer: 250 mM Tris, 1.92 M glycine, 0.4% (w/v) SDS. Dilute 10 stock in deionized water to prepare 1 running buffer. 6. 5 Laemmli Buffer: 250 mM Tris–HCl, pH 6.8, 10% (w/v) SDS, 50% (w/v) glycerol, 0.05% (w/v) bromophenol blue, 5% (v/v) ß-mercaptoethanol. Store at room temperature. 7. Prestained (e.g., PageRuler™ Plus) or unstained (e.g., Benchmark™ Protein ladder) molecular weight markers. 8. Minigel and electrophoresis system.

2.5 SDS– Polyacrylamide Gel Electrophoresis with Mn2+-PhostagTM-Gels

2.6 Total Protein Staining with SYPRO™ Ruby Protein Gel Stain

1. 5 mM Acrylamide-pendant Phos-tag™ AAL-107 (Fujifilm/ Wako) solution: dissolve 10 mg Phos-tag™ in 100 μL of methanol and dilute with 3.2 mL of distilled water. The solution can be stored at 4  C in the dark. 2. 10 mM MnCl2 solution (see Note 4). Store at 4  C or room temperature. 1. Plastic incubation box 12 cm  7.5 cm. 2. SYPRO™ Ruby Protein Gel Stain (see Note 5). Store at room temperature. 3. SYPRO™ Ruby fixing solution: 50% (v/v) methanol, 7% (v/v) acetic acid. Store at room temperature. 4. SYPRO™ Ruby destain solution: 10% (v/v) methanol, 7% (v/v) acetic acid. Store at room temperature.

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5. An imager that has a setting to visualize SYPRO™ Ruby stained gels (excitation maximum: 280/450 nm, emission maximum: 610 nm), (e.g., an Amersham™ Imager 600 with a Trans-UV illuminator or with blue (excitation 480 nm) or green epifluorescence (excitation 520 nm) options in combination with a Cy3 emission filter (570–613 nm)). 2.7 Phosphostaining with Pro-Q™ Diamond Phosphoprotein Gel Stain

1. Plastic incubation box 12 cm  7.5 cm. 2. Pro-Q™ Diamond Phosphoprotein Gel Stain. Store at 4  C in the dark. 3. Sodium acetate stock solution: 3 M sodium acetate, adjust pH to 4.0 with 100% acetic acid. Store at room temperature. 4. Pro-Q™ Diamond fixing solution: 50% (v/v) methanol, 10% (v/v) acetic acid. Store at room temperature. 5. Pro-Q™ Diamond destain solution: 20% (v/v) acetonitrile, 50 mM sodium acetate, pH 4.0. Store at room temperature. 6. PeppermintStick™ Phosphoprotein Molecular Weight Standards. Store at 20  C. 7. An imager capable of visualizing Pro-Q™ Diamond stained gels (excitation maximum: 555 nm, emission maximum: 580 nm) (e.g., a Typhoon FLA9200 equipped with an excitation wave length of 532 nm and an LPG filter (575 nm)) (see Note 6).

3

Methods

3.1 Plant-Related Material

1. Sterilize Arabidopsis thaliana seeds expressing the tagged version of the putative phosphoprotein of interest with the seed sterilization solution for 10 min on a rotary shaker at room temperature. After sterilization, wash the seeds five times with washing solution. Use sterile tips for washing and plating. 2. Plate the seeds under a sterile bench on GM plates. Stratify the seeds on plates at 4  C overnight, before growing the seedlings for 7–10 days at 21  C under continuous light or long day conditions (16 h light and 8 h dark) (see Note 7). 3. Collect 20 g of seedlings into a 15-mL tube and freeze them in liquid nitrogen. For 20 g of seedlings, seedlings are typically grown in four to five 12  12 cm square or Ø14 cm round petri dishes. Depending on the abundance of the protein of interest, plant materials between 1 g and 50 g can be used for the IP (see Note 8). Store samples at 80  C or continue directly with the IP.

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3.2 Immunoprecipitation (IP)

1. Transfer seedlings to a mortar and grind the sample under liquid nitrogen until the plant material is ground to fine powder. Transfer the powder with a liquid nitrogen frozen spoon to a 50 mL tube and add 1 mL of extraction buffer per g of seedlings. 2. Homogenize the suspension in a cold room or on ice until the sample is homogenized (e.g., two times 1 min pulses at 6.6  1000 1/min with the IKA-T25 Ultra TURRAX). 3. Centrifuge samples at 4  C and 13,000  g. Filter the supernatant with a nylon mesh or a 0.45 μm syringe filter to remove large cellular debris, and transfer the filtrate to a new 50 mL tube kept on ice (see Note 9). 4. Transfer 20 μL of magnetic affinity beads (see Note 10) to a 1.5 mL tube, remove the storage solution with magnetic separation rack for 1.5 mL tubes, and wash three times with 0.5 mL of ice-cold buffer A. 5. Dilute the plant extract with an equal volume of ice-cold buffer A. Add the beads and incubate at 4  C for at least 30 min (see Note 11). 6. After the incubation, remove the supernatant using a magnetic separation rack for 50 mL tubes on ice or in a cold room (see Note 12). Transfer the beads to a fresh 1.5 mL tube and wash three times with 0.5 mL of ice-cold buffer A and a magnetic separation rack for 1.5 mL tubes on ice. After the last washing step, spin down the tubes (6000  g, 4  C, 30 s), place the tubes in the magnetic separation rack and remove the remaining liquid from the tubes. Add 35 μL of buffer A and keep the samples on ice while preparing the phosphatase.

3.3 Phosphatase Treatment

1. To heat inactivate the lambda phosphatase mix 40 μL of water, 5 μL of 10 reaction buffer, 5 μL of 10 mM MnCl2, and 1 μL of lambda phosphatase in a 1.5 mL tube and incubate the mixture in a heating block at 96  C for 10 min. Cool the mixture on ice before use. 2. Prepare two 1.5 mL tubes with 40 μL of water, 5 μL of 10 reaction buffer and 5 μL of 10 mM MnCl2. Keep one tube as the buffer control and add 1 μL of lambda phosphatase to the other tube. Keep the phosphatase on ice during the preparation steps. 3. Mix the protein-decorated beads (see Note 13) thoroughly with the buffer A and transfer 10 μL of the slurry to three new 1.5 mL tubes for the buffer control, heat-inactivated and active phosphatase treatment (see Note 14). Remove buffer A with a magnetic separation rack for 1.5 mL tubes and add the phosphatase reaction mixtures to the beads.

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4. As a control for the phosphatase treatment, mix 1 μL of the PeppermintStick™ phosphoprotein molecular weight standards, 1 μL of 10x reaction buffer, 1 μL of 10 mM MnCl2, 7.8 μL of water, and 0.2 μL of lambda phosphatase. Prepare also a control without lambda phosphatase. 5. Incubate the samples for 30 min at 30  C in heating block. 6. Remove the supernatant with a 1.5 mL magnetic separation rack, wash three times with 0.5 mL of buffer A, spin down the tubes (6000  g, 4  C, 30 s), and remove the supernatant completely. Add 12.5 μL of 1 Laemmli Buffer to the beads and boil the beads for 3 min at 98  C. Store the samples at 20  C. 3.4 SDS– Polyacrylamide Gel Electrophoresis

1. Use a minigel system with 1-mm thick gels and combs for the SDS-PAGE (see Note 15). Clean the gel plates with ethanol before assembling. 2. Prepare 8 mL of 10% separation gel mixture by combining the following ingredients in a 50 mL tube (see Note 16): 3.4 mL of water, 2 mL of separation gel buffer, 2.6 mL of 30% acrylamide–bis-acrylamide, 50 μL of 10% APS, and 6 μL of TEMED. Pour the gel immediately after mixing the components and leave 2 cm space for the stacking gel. Overlay the separation gel with 100% isopropanol or carefully with water. Leave the gel at room temperature for 15–30 min to polymerize. 3. Discard the isopropanol or water overlay. Remove rests of the isopropanol solution or water with a paper towel. 4. Prepare the stacking gel (4 mL) by mixing 2.4 mL of water, 1 mL of stacking gel buffer, 0.6 mL of 30% acrylamide–bisacrylamide solution, 50 μL of 10% APS, and 6 μL of TEMED. Mix the components and pour the gel. Insert the comb immediately avoiding the formation of air bubbles. Leave the gel for 15 min at room temperature to polymerize. At this point the gels can be stored for several days at 4  C wrapped in wet paper towels and plastic bag. 5. Remove the sample comb carefully and assemble the gel apparatus. Fill the inner and outer chambers with 1 SDS Running buffer. 6. Use either unstained or prestained molecular mass markers. For the Pro-Q™ phosphostain use a phosphoprotein molecular size marker. Dilute 1 μL of the markers with 9 μL of 1 Laemmli buffer and load them on the gel or use the control samples prepared in Subheading 3.3, step 4. 7. Load 10 μL of the buffer control as well as the heat inactivatedand active phosphatase-treated samples.

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8. Run the gel at 25 mA (constant current) until the loading dye reaches the bottom of the gel in a 1 SDS Running buffer. 3.5 SDS– Polyacrylamide Gel Electrophoresis with Mn2+-PhostagTM-Gels

1. The overview of the whole procedure is shown in Fig. 1a. Prepare an acrylamide gel as a control (see Subheading 3.4, steps 2–4). 2. Prepare a Mn2+-Phos-tag™ separation gel (7.5% acrylamide, 20 μM Phos-tag™, 36 μM MnCl2) by mixing in a 50-mL tube (6 mL) (see Note 17): 3 mL of water, 1.5 mL of separation gel buffer, 1.5 mL of 30% acrylamide-bisacrylamide solution, 37.5 μL of 10% APS, 24 μL of 5 mM Phos-tag™ solution, 24 μL of 10 mM MnCl2, and 6 μL of TEMED. Pour the gel immediately after mixing the components and leave 2 cm space for the stacking gel. Overlay the separation gel with 100% isopropanol or water. Leave the gel at room temperature for 15–30 min to polymerize (see Note 18). 3. Discard the isopropanol or water overlay. Remove rests of the isopropanol solution or water with a paper towel. 4. Prepare the stacking gel for the Phos-tag™ gel as in Subheading 3.4, step 4. Mix the components and pour the gel. Insert the comb immediately avoiding the formation of air bubbles. Leave the gel for 15 min at room temperature to polymerize. Prepare Mn2+-Phos-tag™ gels directly before use, as the gels cannot be stored (see Note 19). 5. Load either unstained or prestained molecular mass markers (see Note 20). Load 10 μL of the buffer control, heatinactivated phosphatase, and active phosphatase sample on the acrylamide gel with and without Phos-tag™. 6. Run the gels at 25 mA (constant current) until the loading dye reaches the bottom of the gel in 1 SDS Running buffer (see Note 21). 7. The proteins can be stained using Coomassie Brilliant Blue, silver staining or fluorescent dyes such as SYPRO™ Ruby (Fig. 1b and c). The protocol for staining using SYPRO™ Ruby is described in Subheading 3.6. The proteins in the Phos-tag™ gels can be detected with total protein stains or by immunoblot (see Note 22).

3.6 Total Protein Staining with SYPRO™ Ruby Protein Gel Stain

1. Transfer the acrylamide gel or the Phos-tag™ acrylamide gel to a clean container with 50 mL SYPRO™ Ruby fixing solution, close the lid and incubate the gel for 30 min on an orbital shaker. All incubation steps are conducted at room temperature. Change the fixing solution and repeat the fixation step once (see Note 23). 2. Discard the fixing solution, and wash three times with water, for 10 min each.

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Fig. 1 Phos-tag™ electrophoresis of phosphorylated proteins (a) Workflow of the phosphatase treatment and Phos-tag™ electrophoresis. Total protein extracts are prepared from Arabidopsis seedlings expressing a tagged version of the putative phosphoprotein. Using an affinity matrix against the tag, the tagged protein was immunoprecipitated. After extensive washing, the immunoprecipitate was then subjected to lambdaphosphatase treatment. The immunoprecipitate was treated with heat-inactivated and active phosphatase and the reaction was stopped by adding Laemmli buffer and boiling at 96  C. The Laemmli buffer containing samples were resolved on a 7.5% acrylamide gel and a Mn2+-Phos-tag™ acrylamide gel with 7.5% acrylamide and 20 μM Phos-tag™. The gels were stained with the SYPRO™ Ruby total protein stain and the proteins were then visualized with a fluorescent imager (excitation maximum: 280 nm or 450 nm, emission maximum: 610 nm). (b) An immunoprecipitated phosphoprotein was treated with lambda phosphatase. In addition, heat-inactivated lambda phosphatase and buffer without lambda phosphatase were used as a control. The protein was incubated for 30 min at 30  C with the lambda phosphatase, the reaction was stopped with Laemmli buffer and boiling. The Laemmli buffer containing samples were resolved on a Mn2+Phos-tag™ gel with 7.5% acrylamide and 20 μM Phos-tag. In the lanes with the samples treated with heatinactivated lambda phosphatase and buffer without lambda phosphatase, there was a smear and a band at a higher molecular weight than the protein band in the lane with sample, that was treated with active phosphatase. The positions of the protein band in the phosphatase treated sample and the controls are indicated with arrowheads. The difference between the sample treated with active phosphatase and the controls suggests, that the immunoprecipitated protein is phosphorylated in the plant extract. (c) As in (b) an immunoprecipitated phosphoprotein was treated with active and heat-inactivated lambda phosphatase. The Laemmli buffer-containing samples were resolved on a 7.5% acrylamide gel. In the acrylamide gel there was no difference between the lambda phosphatase treated sample and the controls without phosphatase and with heat-inactivated phosphatase. In all lanes the immunoprecipitated protein formed one distinct band between 50 and 60 kDa, which indicates that the electrophoretic behavior of the protein in an acrylamide gel is not influenced by other factors than the phosphorylation status

3. Immerse the gel in 25 mL SYPRO™ Ruby Protein Gel Stain overnight at room temperature on a rotary shaker. Wrap the container in aluminum foil or another nontransparent material to protect the fluorescent dye from light. 4. Discard staining solution, transfer the gel to a clean container and wash 30 min in 50 mL SYPRO™ Ruby destain solution.

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5. Before imaging, wash the gel two times with water, for 5 min each. 6. Image the gel with a UV or other fluorescent light source with an appropriate filter set (excitation maximum: 280/450 nm, emission maximum: 610 nm). 3.7 Phosphostaining with Pro-Q™ Diamond Phosphoprotein Gel Stain

1. Run your samples on an acrylamide gel as described in Subheading 3.4. The overview of the procedure is shown in Fig. 2a. 2. Transfer the gel to a clean container with 50 mL of Pro-Q™ Dimond fixing solution, close the lid, and incubate the gel for 30 min on an orbital shaker. Change the fixing solution and incubate the gel overnight on a rotary shaker. All incubation steps are done at room temperature. 3. Discard the fixing solution and wash three times with water, for 10 min each. 4. Immerse the gel in 15 mL of Pro-Q™ Phosphoprotein Gel Stain solution and incubate the gel for 90 min on a rotary shaker. Wrap the container in aluminum foil or another nontransparent material to protect the fluorescent dye from light. 5. Discard the staining solution and wash the gel three times 30 min in 50 mL of Pro-Q™ Diamond destain solution. 6. Before imaging, wash the gel two times with water, for 5 min each. 7. Image the gel with a fluorescent imager with an appropriate filter set (excitation maximum at 555 nm and emission maximum at 580 nm) (Fig. 2b and c). 8. Poststain the gel with total protein staining (e.g., SYPRO™ Ruby) (Fig. 2b and c) (see also Note 23).

4

Notes 1. Tags that are commonly used for the immunoprecipitation of proteins from plant extract are HA, Strep, Flag, and Myc. GFP and other fluorophores can also be used as long as a suitable affinity matrix is available. If specific antibodies are available against the endogenous protein, untagged proteins can be directly immunoprecipitated. 2. Alternatively, other homogenizing methods or equipment can be used (e.g., TissueLyser II (Qiagen) with glass or ceramic beads). 3. If no magnetic beads for the specific tag are available, matrices based on agarose can be used in combination with a small spin column.

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Fig. 2 Staining of phosphorylated proteins using Pro-Q™ Diamond (a) Workflow of lambda-phosphatase treatment and phosphostaining. Total protein extracts are prepared from Arabidopsis seedlings expressing a tagged version of the putative phosphoprotein. Using an affinity matrix against the tag, the tagged protein was immunoprecipitated. After extensive washing, the immunoprecipitate was then subjected to lambdaphosphatase treatment. The immunoprecipitate was treated with heat-inactivated and active phosphatase and the reaction was stopped by adding Laemmli buffer and boiling at 98  C. The Laemmli buffer containing samples were resolved on a 10% acrylamide gel and after fixation, stained with the Pro-Q™ Diamond Phosphoprotein stain. The phosphoproteins were then visualized with a fluorescent imager (excitation maximum: 555 nm, emission maximum: 580 nm). The same gel was then stained with the SYPRO™ Ruby to visualize total proteins (excitation maximum: 280 nm or 450 nm, emission maximum: 610 nm). (b) PeppermintStick™ Phosphoprotein Molecular Weight Standard was used as a control for lambdaphosphatase treatment. 1 μL of PeppermintStick™ Phosphoprotein Molecular Weight Standard was incubated with lambda-phosphatase for 30 min at 30  C, and after mixing with Laemmli buffer, subjected to SDS-PAGE. The gel was then stained with Pro-Q™ Diamond Phosphoprotein stain (60 min, 15 mL) and visualized using a fluorescent imager. In the Pro-Q™ Diamond phosphostain, two bands of the PeppermintStick™ Phosphoprotein Standard at 45 kDa and 23.6 kDa, which contain phosphorylated proteins, are only visible in the untreated sample. With the total protein staining using SYPRO™ Ruby, the band at 45 kDa is stained in both treated and untreated samples. The band at 23.6 kDa seems to appear at a lower molecular weight after dephosphorylation. With these experiments, the activity of the lambda-phosphatase can be verified. (c) As in (b), an immunoprecipitated phosphoprotein was treated with lambda-phosphatase and stained using Pro-Q™ Diamond phosphostain and SYPRO™ Ruby. In addition, heat-inactivated lambdaphosphatase and buffer without lambda phosphatase was used as a control. In the lane with the active phosphatase treated sample, the protein band is less intensely stained by the Pro-Q™ Diamond phosphostain stain than the protein bands in the control samples. With SYPRO™ Ruby total protein stain there was no intensity difference between the samples, suggesting that the immunoprecipitated protein is phosphorylated in the plant extract

4. Depending on the phosphoproteins under analysis, the Mn2Phos-tag™ method might not give optimal results. If no protein shift can be observed, Zn2+-Phos-tag™ SDS-PAGE with a neutral-pH buffer system can be tried out. For this, prepare 10 mM ZnCl2, 4 Zn2+-Phos-tag™ stacking and resolving gel solution (1.4 M Bis-Tris/HCl, pH 6.8), 0.5 M NaHSO3

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solution, 5 Zn2+-Phos-tag™ running buffer (0.5 M Tris base, 0.5 M MOPS [3-(N-morpholino) propanesulfonic acid, 0.5% (w/v) SDS]. 5. To stain total proteins after the Pro-Q™ Diamond Phosphoprotein Gel stain, the fluorescent dye SYPRO™ Ruby is recommended. We also have tried the Krypton™ Protein Stain, which has approximately equal sensitivity (minimal amount of detectable protein: 0.25–1 ng) as SYPRO™ Ruby. If there is no UV- or fluorescent imager available with the appropriate filters, gels can be alternatively stained by silver staining (minimal amount of detectable protein: 0.3 ng protein), using for example the Silver Quest™ staining kit. If you have more than 0.5 μg of phosphoprotein, as it can be the case in experiments with recombinant proteins, you can also stain your acrylamide gels with Coomassie Brilliant Blue. 6. Visualization with a standard UV illuminator, albeit with lower sensitivity, is also possible. In our experiments, 1 μL of PeppermintStick™ Phosphoprotein Molecular Weight Standards (~0.5 μg) could be detected with the UV illuminator, however, the amount of immunoprecipitated phosphoproteins is in most cases too low for detection with this method. 7. It is recommended to grow the seedlings vertically on GM plates so that they can be easily removed from the agar afterward. 8. The optimal amount of plant material depends on the expression level of the transgene, the stability of the protein, and the efficiency of the IP. For transmembrane proteins, it might be necessary to establish additional solubilization steps prior to the IP. 9. At this point, the total protein concentration can be measured (e.g., with a Bradford reagent) and part of the lysate can be kept as an “input” sample. Add 5 Laemmli Buffer and keep the sample in the freezer until use. 10. The optimal bead volume for the IP depends on the capacity of the beads and the binding efficiency of the tagged protein. 11. The optimal incubation time depends on the stability of the protein under study and the efficiency of the binding. Incubation times between 30 min and 2 h are recommended but can be extended to overnight. 12. Wait until the magnetic beads are completely settled before removing the supernatant. This might take up to several minutes. 13. It is also possible to elute the protein prior to the lambda phosphatase treatment. If the proteins are eluted, it might be necessary to reduce the total reaction volume to 10 μL.

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Otherwise the phosphoprotein can be strongly diluted and therefore difficult to detect on the acrylamide gels. 14. It is important that the beads are homogenously mixed in the buffer to ensure equal amount of proteins in each sample. 15. The volumes given in the following steps are suitable for 1-mm thick acrylamide mini gels. It is also possible to use 0.75 mm or 1.5 thick mini gels with the corresponding combs. 16. The optimal acrylamide concentration depends on the size of the proteins to be analyzed. 17. Phosphorylated proteins migrate slower in the Phos-tag™ gels than nonphosphorylated proteins. The separation efficiency of phosphoprotein isoforms depends on the Phos-tag™ and acrylamide concentration, which must be optimized for every target protein. In our experiments with a 50 kDa protein, there was a clear difference between phosphorylated and nonphosphorylated isoforms in gels with 7.5% acrylamide and 18–50 μM Phos-tag™. 18. For the Zn2+-Phos-tag™ method, prepare the following separating gel (12% acrylamide, 20 μM Phos-tag™, 100 μM ZnCl2, 6 mL): 1.95 mL of water, 1.5 mL of 4 Zn2+-Phos-tag™ stacking and resolving gel solution, 2.4 mL of 30% acrylamide–bis-acrylamide solution, 37.5 μL of 10% APS, 24 μL of 5 mM Phos-tag™ solution, 24 μL of 10 mM ZnCl2, and 6 μL of TEMED. Phos-tag™ and acrylamide concentrations must be optimized for different phosphoproteins. 19. For the Zn2+-Phos-tag™ method, prepare the following stacking gel: 3.57 mL of water, 1.5 mL of 4 Zn2+-Phos-tag™ stacking and resolving gel solution, and 0.9 mL of 30% acrylamide–bis-acrylamide solution. Zn2+-Phos-tag™ gels can be stored up to 3 months at 4  C wrapped in wet paper towels and plastic bag. 20. Some of the protein bands of the molecular mass markers might be shifted or distorted in Phos-tag™ acrylamide gels. Often the standards will look very different than on the acrylamide gel without Phos-tag™ and it might be difficult to assign the correct molecular mass to the protein bands. 21. To prepare the running buffer for the Zn2+-Phos-tag™ gels, mix 100 mL of 5 Running buffer and 5 mL of 0.5 M NaHSO3 solution with 395 mL of water. Run the gels at 40 mA/gel with a maximum voltage of 90 V until the loading dye reaches the bottom of the gel. 22. If proteins should be detected by immunoblotting, Mn2+ and Zn2+ ions must be removed from the Phos-tag™ gel before the transfer to the PVDF or nitrocellulose membrane. Incubate the gel for 10 min with 1 mM EDTA dissolved in a transfer

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buffer and wash the gel afterward for 10 min in a transfer buffer without EDTA. 23. If you use SYPRO™ Ruby as poststain following the Pro-Q™ Diamond staining, the fixation step can be skipped. Wash the gel two times for 5 min with water before the SYPRO™ Ruby staining. References 1. Armengot L, Marques-Bueno MM, Jaillais Y (2016) Regulation of polar auxin transport by protein and lipid kinases. J Exp Bot 67 (14):4015–4037. https://doi.org/10.1093/ jxb/erw216 2. Mithoe SC, Menke FL (2018) Regulation of pattern recognition receptor signalling by phosphorylation and ubiquitination. Curr Opin Plant Biol 45(Pt A):162–170. https:// doi.org/10.1016/j.pbi.2018.07.008 3. Haruta M, Gray WM, Sussman MR (2015) Regulation of the plasma membrane proton pump (H(+)-ATPase) by phosphorylation. Curr Opin Plant Biol 28:68–75. https://doi. org/10.1016/j.pbi.2015.09.005 4. Yang W, Zhang W, Wang X (2017) Posttranslational control of ABA signalling: the roles of protein phosphorylation and ubiquitination. Plant Biotechnol J 15(1):4–14. https://doi.org/10.1111/pbi.12652 5. Yin X, Wang X, Komatsu S (2018) Phosphoproteomics: protein phosphorylation in regulation of seed germination and plant growth. Curr Protein Pept Sci 19(4):401–412. https://doi.org/10.2174/ 1389203718666170209151048 6. Nguyen LK, Kolch W, Kholodenko BN (2013) When ubiquitination meets phosphorylation: a systems biology perspective of EGFR/MAPK signalling. Cell Commun Signal 11:52. https://doi.org/10.1186/1478-811X-11-52 7. Dubeaux G, Neveu J, Zelazny E, Vert G (2018) Metal sensing by the IRT1

transporter-receptor orchestrates its own degradation and plant metal nutrition. Mol Cell 69 (6):953–964. e955. https://doi.org/10. 1016/j.molcel.2018.02.009 8. Kinoshita E, Kinoshita-Kikuta E (2011) Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced protein phosphorylation profiling. Proteomics 11(2):319–323. https://doi.org/10.1002/pmic.201000472 9. Kinoshita E, Kinoshita-Kikuta E, Takiyama K, Koike T (2006) Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol Cell Proteomics 5(4):749–757. https://doi. org/10.1074/mcp.T500024-MCP200 10. Kinoshita E, Kinoshita-Kikuta E, Koike T (2015) Advances in Phos-tag-based methodologies for separation and detection of the phosphoproteome. Biochim Biophys Acta 1854 (6):601–608. https://doi.org/10.1016/j. bbapap.2014.10.004 11. Heazlewood JL, Durek P, Hummel J, Selbig J, Weckwerth W, Walther D, Schulze WX (2008) PhosPhAt: a database of phosphorylation sites in Arabidopsis thaliana and a plant-specific phosphorylation site predictor. Nucleic Acids Res 36(Database issue):D1015–D1021. https://doi.org/10.1093/nar/gkm812 12. Gao J, Agrawal GK, Thelen JJ, Xu D (2009) P3DB: a plant protein phosphorylation database. Nucleic Acids Res 37(Database issue): D960–D962. https://doi.org/10.1093/nar/ gkn733

Chapter 15 Purification and Interaction Analysis of a Plant-Specific RAB5 Effector by In Vitro Pull-Down Assay Emi Ito, Seung-won Choi, and Takashi Ueda Abstract RAB GTPases regulate membrane traffic by interacting with effector proteins in the GTP-bound active form. RAB GTPases are highly conserved in a broad range of eukaryotic organisms, while land plants and some green algal species possess a plant-specific RAB5 group. A plant-specific RAB5 in Arabidopsis called ARA6 was shown to regulate a characteristic trafficking route, and participate in abiotic and biotic stress responses. The identification of ARA6 effectors is a powerful strategy to get insights into the molecular basis of ARA6 functions. Recently, we identified an ARA6 effector, PLANT-UNIQUE RAB5 EFFECTOR 2 (PUF2), and characterized its functions by biochemical means. PUF2 was hardly expressed as a recombinant protein in the bacterial system, but we solved this problem by optimizing the codon usage of PUF2 CDS to suite for expression in Escherichia coli. Here, we present the protocol we employed to purify PUF2 protein, and to test its nucleotide state-specific interaction with ARA6 by in vitro pull-down assay. This approach would be extended to analyze the molecular functions of other effector proteins of RAB GTPases. Key words RAB GTPases, Effector proteins, Protein Purification, In vitro pull-down assay

1

Introduction Eukaryotic cells carry membrane-bound organelles, which each bear characteristic sets of proteins and lipids to perform specialized functions. In order to fulfill particular functions, the specific composition of proteins and lipids must be properly maintained in each organelle. Meanwhile, single membrane-bound organelles constituting the endomembrane system and the plasma membrane actively exchange proteins and lipids via a membrane trafficking system. Cargo molecules are sorted into transport vesicles at the donor membrane and delivered to the target organelle. Some organelles also gradually change their characteristics, taking on a new role via the replacement of resident proteins, a process referred to as organelle maturation. A family of small molecular weight monomeric GTPases, RAB GTPases, plays essential regulatory roles in the membrane

Marisa S. Otegui (ed.), Plant Endosomes: Methods and Protocols, Methods in Molecular Biology, vol. 2177, https://doi.org/10.1007/978-1-0716-0767-1_15, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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trafficking system, conferring specificity to the tethering of transport vesicles to the target membrane, as well as regulating organelle dynamics and defining organelle identity [1–9]. RAB GTPases act as molecular switches by cycling between the GTP-bound active and GDP-bound inactive forms. When RAB GTPase is in the active form, it interacts with effector proteins that trigger various downstream reactions. For example, a mammalian RAB5 effector, Early Endosome Antigen 1 (EEA1), is a long coiled-coil protein that tethers transport vesicles to the early endosome membrane [10– 13]. The CORVET complex, consisting of six subunits, is another RAB5 effector also shown to act as a tether during early endosomal fusion [14–18]. In addition to these molecules, many other effector proteins have been identified for RAB5, indicating that RAB5 regulates multiple trafficking events by interacting with multiple effector proteins in animal cells [1, 3, 8, 19–21]. Multiple effector proteins with distinct structures and functions have also been identified for other RAB GTPases [3, 21–23], and identification and functional analyses of these effector proteins has been quite effective in unraveling the functions of RAB GTPases. RAB5 is also a key regulator of endosomal/vacuolar transport in plants, with unique features acquired during plant evolution [24, 25]. Arabidopsis thaliana has three RAB5 members: RHA1/ RABF2a, ARA7/RABF2b, and ARA6/RABF1 [26, 27]. RHA1 and ARA7 share high similarity with animal and yeast Rab5, and are therefore regarded to be canonical RAB5 in Arabidopsis. Conversely, ARA6 has a distinctive primary structure and close homologs have only been identified in land plants and some green algal species [26, 28, 29]. The functions of canonical RAB5s in endosomal/vacuolar transport have been clearly demonstrated [18, 26, 30–35]. ARA6 also resides on multivesicular endosomes, partly overlapping with canonical RAB5, but acts in distinct trafficking events underpinning a wide range of plant physiological responses such as abiotic stress responses, sugar metabolism, and immune responses against pathogens [26, 35–37]. To identify RAB effector proteins, yeast two-hybrid screening has been successfully employed in animal and plant systems [38– 45]. However, yeast two-hybrid assays frequently detect falsepositive interactions, and biochemical validation of these interactions is generally needed. Recently, we reported on the first ARA6 effector protein, PLANT-UNIQUE RAB5 EFFECTOR 2 (PUF2), which was also identified by yeast two-hybrid screening [41]. To meet the criteria for effector protein, PUF2 should interact with the active form of ARA6 but not with the inactive form, and this should be verified via biochemical assay. However, PUF2 protein was difficult to work with, and required repeated cycles of trial and error for successful expression in bacteria, preparation of the purified protein, and detection of its interaction with ARA6 in vitro. Here, we share the method we developed for biochemical detection

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of the interaction between the purified PUF2 protein and ARA6 by in vitro pull-down assay, which may be applicable to other proteins.

2

Materials

2.1 PUF2 Cloning and Expression in Bacteria

1. pGEX-6P-1 vector or similar for expression of GST-tagged proteins in bacteria. 2. Escherichia coli Rosetta-gami (DE3) competent cells. 3. Shaker incubator for bacterial cultures. 4. Luria broth (LB) agar plates with selective antibiotic (e.g., ampicillin for pGEX-6P-1). 5. Liquid LB medium containing 50 μg/mL ampicillin sodium salt and 0.1% glucose. 6. 2 YT broth containing 50 μg/mL ampicillin sodium salt and 0.1% glucose. 7. 1 M isopropyl-β-D()-thiogalactopyranoside (IPTG): 2.38 g IPTG in 10 mL of distilled water. Filter-sterilize with a 0.22 μm syringe filter.

2.2 Protein Purification

1. Ultrasonic homogenizer. 2. Glutathione Sepharose 4B. 3. PreScission Protease. 4. Lysis Buffer: 50 mM Tris–HCl pH 7.5, 150 mM NaCl, protease inhibitor cocktail (e.g., cOmplete EDTA-free protease inhibitor cocktail), 1% Triton X-100. 5. Wash Buffer: 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Triton X-100. 6. Desalination buffer: 20 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2. 7. Elution buffer: 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 30 mM glutathione (reduced form), adjusted to pH 7.5 by NaOH. 8. Poly-Prep® chromatography column. 9. Amicon® Ultra-15 centrifugation filter units with a nominal molecular weight limit of 50 kDa. 10. PD-10 desalinating column. 11. Coomassie Brilliant Blue solution: 0.25 g of Coomassie Brilliant Blue R-250 in a solution of 45 mL of methanol, 45 mL distilled water, and 10 mL of glacial acetic acid.

2.3 PUF2 and ARA6 Interaction Assay

1. Binding Buffer: 20 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 0.05% Tween 20.

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2. 0.5 M Ethylenediaminetetraacetic acid (EDTA) stock solution: add NaOH to adjust the pH to 8.0. 3. 10 mM GTPγS and GDP solutions in milliQ-grade water. 40 mM stock solutions are stored at 80  C, and diluted to 10 mM with milliQ-grade water. After dilution, the solutions are stored at 20  C until use. 4. 1 M MgCl2 stock solution. 5. 3 sample buffer: 0.5 M Tris–HCl pH 6.8, 9% sodium dodecyl sulfate, 30% glycerol, 15% 2-mercaptoethanol, 0.75 mg/mL bromophenol blue. 2.4 Western Blotting Analysis to Detect Interaction Between PUF2 and ARA6

1. Minigel and electrophoresis system. 2. 12.5% acrylamide gel or Precast polyacrylamide gels. 3. Running buffer: 25 mM Tris, 192 mM glycine, 0.1% sodium dodecyl sulfate. 4. PVDF membrane. 5. Semidry transfer system for minigels. 6. Plastic container prewiped with 100% ethanol. 7. Transfer buffer: 5 mM Tris, 38.4 mM glycine, 1% SDS, 5% methanol. 8. Blocking buffer: 10 mM Tris–HCl pH 8.0, 150 mM NaCl, 5% skimmed milk. 9. TBST: 10 mM Tris–HCl pH 8.0, 150 mM NaCl, 5% Triton X-100. 10. Anti-PUF2 antibody. 11. Hybridization bag or zip-lock bag. 12. Heat sealer. 13. Secondary antibodies conjugated to horseradish peroxidase to detect anti-PUF2 antibodies. 14. Chemiluminescent peroxidase substrate. 15. Chemiluminescence imaging system.

3

Methods

3.1 Plasmid Preparation

Here, we present an example of how to prepare a vector containing a codon-optimized gene for plants fused with a GST tag by restriction enzyme-based construction. Other cloning techniques are also applicable. The codon optimization substantially improved the expression of PUF2 in the E. coli expression system (Fig. 1). 1. Apply codon optimization tools to optimize the codon usage of the gene of interest (PUF2 in this case) for expression in E. coli (see Note 1).

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Fig. 1 Codon optimization improved the expression of PUF2 in E. coli. Codonoptimized PUF2 was subcloned into a BamH1 site of a pGEX-6p-1 vector, and transformed into the E. coli strain. A colony was inoculated to liquid LB broth supplemented with 50 μg/mL ampicillin, and grown to log phase. Then, IPTG was added to the culture at a final concentration of 1 mM, and cultured at 30  C overnight. For the negative control, an equivalent volume of sterilized distilled water was applied to the culture. The cells were collected by centrifugation, suspended in PBS buffer, and disrupted by handy ultrasonic disruptors (output of 3 W, 30 s each for five times on ice). Soluble and insoluble fractions were separated by centrifugation at 8000  g for 10 min at 4  C, and then analyzed by SDS-PAGE and immunoblotting using the anti-GST antibody (lane 5–8). The same procedure was performed for the E. coli strain transformed with pGEX6p-1 containing the native PUF2 CDS (lane 1–4). Leaky expression of PUF2 was observed under this experimental condition in the sample without IPTG (lane 5), hence transformants were cultured in LB liquid broth supplemented with 0.1% glucose for subsequent experiments

2. Digest the codon-optimized PUF2 by restriction enzyme (e.g., BamH1) according to the cloning strategy chosen for inserting the PUF2 CDS into the expression vector. 3. Purify the DNA fragment (e.g., by phenol–chloroform–isoamyl alcohol extraction, followed by ethanol precipitation). 4. Subclone the PUF2 DNA into the pGEX-6P-1 vector. 5. Confirm the insertion by using an appropriate sequence primer (see Note 2). 3.2 Protein Purification of PUF2

Day 1: 1. Transform E. coli strain Rosetta-gami (DE3) competent cells with the plasmid produced in Subheading 3.1 and grow cells overnight on LB plates with appropriate antibiotic. Day 2: 2. Inoculate a bacterial colony into 200 mL of liquid LB medium with 50 μg/mL ampicillin sodium salt and 0.1% glucose. Culture the bacteria at 37  C in a shaker incubator overnight.

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Day 3: 3. Transfer the culture to 12 L of liquid 2 YT broth with 50 μg/ mL ampicillin sodium salt and 0.1% glucose. Culture the bacteria at 18  C in an shaker incubator at 120 rpm for 24 h. Day 4: 4. Measure the optical density at 600 nm (see Note 3). 5. Add IPTG to a final concentration of 0.2 mM. Culture the bacteria at 18  C in a shaker incubator at 120 rpm for another 10 h. 6. Collect the cells by centrifugation at 750  g for 10 min at 4  C. 7. Freeze the pellet in the centrifugation bottles or in freeze tolerant bottles overnight at 80  C. Day 5: 8. Thaw the pellet by soaking the collection tube in a water bucket. Transfer the collection tube to an ice bucket immediately after the pellet has completely thawed. All steps hereafter are conducted either at 4  C or with the tubes in an ice bucket, unless otherwise specified. 9. Suspend the total pellet from 12 L of bacterial culture in 100 mL of ice-cold lysis buffer with gentle agitation (see Note 4). 10. Divide the bacterial suspension into four 50 mL conical tubes and disrupt the cells by using an ultrasonic homogenizer set at an output of 18 W, on/off rate of 0.5/0.5 s, and total duration of 5 min (see Note 5). 11. Centrifuge at 8000  g for 10 min at 4  C to roughly remove the debris. 12. Collect the supernatant and centrifuge again at 8000  g for 30 min at 4  C to further remove the debris. 13. Mix the supernatant with 4 mL of glutathione Sepharose 4B prewashed twice with 20 mL of wash buffer (5 min each wash with slow rotation). Incubate for 2 h at 4  C with slow rotation. 14. Pellet down glutathione Sepharose 4B bound to GST-PUF2 by centrifugation at 500  g for 5 min at 4  C. 15. Wash the resin three times with 40 mL of ice-cold wash buffer. Each time, incubate for 5 min with slow rotation and pellet down the resin by centrifugation at 500  g for 5 min at 4  C. 16. Add 5 mL of ice-cold wash buffer containing 100 μL of PreScission Protease. Incubate at 4  C with slow rotation overnight.

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Fig. 2 Purification of PUF2 expressed in E. coli. A pGEX-6p-1 vector containing codon-optimized PUF2 was introduced into the E. coli strain Rosetta-gami (DE3), and a colony was cultured at 18  C in 2YT liquid broth supplemented with 50 μg/mL ampicillin and 0.1% glucose. Then, IPTG was added at a final concentration of 0.2 mM when the culture reached the log phase. A portion of cells in the culture was collected at 10 h after induction, and lysed to confirm the expression of GST-PUF2 (lane 1). The sample was then centrifuged at 8000  g for 10 min at 4  C to separated soluble and insoluble fractions (lane 2 and 3, respectively). After procedure Subheading 3.2, step 14, a portion of the supernatant (lane 4) and the resin (lane 5) were collected to confirm binding of GST-PUF2 to the glutathione Sepharose 4B (G4B) resin. The bound GST-PUF2 was digested on the resin by PreScission protease. A portion of the resin (lane 6) and the flow-through (lane 7) were then analyzed to confirm the recovery of PUF2 after cleavage in the flow-through. PUF2 was then concentrated (lane 8) and used for the in vitro pull-down assay. Gray arrowhead; GST-PUF2, black arrowhead; PUF2, ∗; PreScission protease, ∗∗; GST

Day 6: 17. Pass the mixture through a Poly-Prep® chromatography column and collect the flow-through. 18. Concentrate the purified PUF2 protein in the flow-through fraction by passing the sample through Amicon® Ultra-15 centrifugation filter units (see Note 6). 19. Desalinate the sample by using a PD-10 desalinating column in ice-cold desalination buffer. 20. Measure protein concentration (e.g., Bradford assay), prepare aliquots, and store the samples at 80  C. 21. The expression and purity of the PUF2 protein can be checked by SDS-PAGE followed by Coomassie Brilliant Blue staining (Fig. 2). 3.3 Purification of GST and GST-Tagged ARA6

GST-tagged ARA6 exhibited sufficient expression in the E. coli expression system without codon-optimization. CDSs for Arabidopsis ARA6Q93L and ARA6S47N were cloned into the BamH1 site of pGEX-6P-1 vector [33].

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Day 1 1. Transform E. coli strain Rosetta-gami (DE3) competent cells with the plasmids containing CDSs for GST (e.g., pGEX6P-1), GST-ARA6Q93L, and GST-ARA6S47N and grow cells overnight on LB plates with appropriate antibiotic. Day 2 2. Inoculate a bacterial colony into 40 mL of liquid LB medium with 50 μg/mL ampicillin sodium salt and 0.1% glucose. Culture the bacteria at 37  C in a shaker incubator overnight. Day 3 3. Transfer the culture to 1.5 L of liquid 2 YT broths with 50 μg/mL ampicillin sodium salt and 0.1% glucose. Culture the bacteria at 20  C in an shaker incubator at 120 rpm for 5 h. 4. Measure the optical density at 600 nm (see Note 3). 5. Add IPTG to a final concentration of 0.2 mM. Culture the bacteria at 20  C in a shaker incubator at 120 rpm for another 16 h. Day 4 6. Collect the cells by centrifugation at 750  g for 10 min at 4  C. 7. Suspend the total pellet from 1.5 L of bacterial culture in 20 mL of ice-cold lysis buffer with gentle agitation (see Note 4). All steps hereafter are conducted either at 4  C or with the tubes in an ice bucket, unless otherwise specified. 8. Divide the bacterial suspension into two 15 mL conical tubes and disrupt the cells by using an ultrasonic homogenizer set at an output of 18 W, on/off rate of 0.5/0.5 s, and total duration of 5 min (see Note 5). 9. Centrifuge at 8000  g for 10 min at 4  C to roughly remove the debris. 10. Collect the supernatant and centrifuge again at 8000  g for 30 min at 4  C to further remove the debris. 11. Mix the supernatant with 1 mL of glutathione Sepharose 4B prewashed twice with 5 mL of wash buffer (5 min each wash with slow rotation). Incubate for 2 h at 4  C with slow rotation. 12. Pellet down glutathione Sepharose 4B bound to GST-PUF2 by centrifugation at 500  g for 5 min at 4  C. 13. Wash the resin three times with 10 mL of ice-cold wash buffer. Each time, incubate for 5 min with slow rotation and pellet down the resin by centrifugation at 500  g for 5 min at 4  C. 14. Add 3 mL of ice-cold elution buffer and incubate for 5 min on ice.

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15. Pass the mixture through a Poly-Prep® chromatography column and collect the flow-through. 16. Desalinate the sample by using a PD-10 desalinating column in ice-cold desalination buffer. 17. Measure protein concentration (e.g., Bradford assay), prepare aliquots, and store the samples at 80  C. 18. The expression and purity of the GST and GST-tagged ARA6 proteins can be checked by ordinary SDS-PAGE followed by Coomassie Brilliant Blue staining. 3.4 In Vitro Pull-Down Assay

We describe the protocol we used to detect the interaction between PUF2 and GST (negative control), GST-ARA6Q93L, and GST-ARA6S47N (Fig. 3). 1. Take 30 μL of glutathione Sepharose 4B in an autoclaved 1.5 mL Eppendorf tubes and wash twice with 500 μL of binding buffer (5 min each with slow rotation at room temperature). 2. Divide the suspension into three 1.5 mL Eppendorf tubes, and apply 200 pmol of GST or GST-fusion protein (either GST-ARA6Q93L or GST-ARA6S47N in this experiment) to prewashed resin in each test tube. 3. Add EDTA stock solution to a final concentration of 5 mM to all tubes, and then add GTPγS and GDP stock solutions to a final concentration of 100 μM to the tubes containing GST-ARA6Q93L and GST- ARA6S47N, respectively. 4. Increase the volume to 500 μL by adding binding buffer. Incubate at room temperature for 30 min with slow rotation. 5. Add 5 μL of 1 M MgCl2 (see Note 7). 6. Centrifuge at 500  g for 5 min at room temperature to pellet down the resin. 7. Wash the resin bound with GST or GST-ARA6 fusion proteins with 500 μL of binding buffer (5 min with slow rotation at room temperature). 8. Add 200 pmol of purified PUF2 (from procedure Subheading 3.2) to each tube. Add GTPγS and GDP stock solutions to a final concentration of 10 μM to the tubes containing GST-ARA6Q93L and GST- ARA6S47N, respectively. Increase the volume to 500 μL with binding buffer, and incubate at room temperature for 30 min with slow rotation. 9. Centrifuge at 500  g for 5 min at room temperature to precipitate the resin bound with the protein complexes. 10. Wash the resin twice with 500 μL of binding buffer (5 min with slow rotation at room temperature) (see Note 8).

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Fig. 3 Schematic representation of procedures of in vitro pull-down assay

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11. Add 30 μL of 3 sample buffer, and incubate for 5 min at 95  C. 12. Use 10 μL for SDS-PAGE and western blotting analysis. 3.5 Western Blotting Analysis of Coprecipitated PUF2 Proteins

1. Prepare a 12.5% acrylamide gel for SDS-PAGE. Precast polyacrylamide gels can also be used. 2. Load 10 μL of samples prepared in Subheading 3.4, step 11 and 10 μL of molecular weight markers into the wells of the SDS-PAGE gel (see Note 9). 3. Run the gel for 90 min at 20 mA. 4. Transfer the proteins from the gel to a PVDF membrane by using a semidry western transfer system. 5. Transfer the membrane into a plastic container prewiped with 100% ethanol, lay blocking buffer on the membrane, and incubate for 30 min at room temperature with shaking at 20 rpm. For a bottom area of 9  12 cm2 cases, 50 mL of blocking buffer is needed. 6. Insert the membrane into a hybridization bag or a zip-lock bag, and apply the anti-PUF2 antibody (primary antibody) diluted at 200 in blocking buffer to the bag (see Note 10). 7. Incubate the membrane overnight at 4  C. 8. Place the membrane into a plastic container prewiped with 100% ethanol, and wash the membrane for 10 min with about 15 mL of TBST three times. 9. Insert the membrane into a hybridization bag or a zip-lock bag, and apply the secondary antibody diluted at 10,000 in blocking buffer. 10. Incubate the membrane for 1 h at room temperature. 11. Place the membrane into a plastic container prewiped with 100% ethanol, and wash the membrane for 10 min with about 15 mL of TBST three times. 12. Drain TBST, and apply a chemiluminescent peroxidase substrate to the membrane. 13. Acquire images with an imaging device (e.g., ChemiDoc™ imaging system).

4

Notes 1. Codon optimization techniques are shown to ameliorate the yields of recombinant proteins from heterologous host cells [46, 47]. We used GENEius provided from the Gene Synthesis service of Eurofin Genomics LLC to synthesize the codonoptimized PUF2 CDS, and other codon-optimization tools

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would be also applicable [48, 49]. We designed the sequence in such a way as to attach the BanHI sites at 50 - and 30 -ends of the CDS. 2. The primer sequence we used in our study was as follows: 50 -CGCTCTTGATGTTGTTTTAT-30 . 3. The bacterial culture reaches the log phase in this protocol. 4. It is better not to vortex the sample to avoid excessive foam formation. We normally use an ethanol-sterilized paintbrush to dissolve the cell mass by gently stroking over the pellet. 5. We used UD-201 (TOMY SEIKO CO., LTD, Japan) at output of three, which is equivalent to output of 18 W, to disrupt the E. coli suspension. The intensity (amplitude) needs to be optimized prior to your experiment. An equal amount of water to the sample is put in the test tube that you may use in the experiment, and the intensity should be set at a point where you hear the cavitation. Also, check if the water is being mixed well in the tube without forming foams or overflowing. 6. For concentrating PUF2, we centrifuge at 7500  g for 20 min at 4  C by using a fixed angle rotor. 7. This step removes EDTA and stabilizes ARA6 in nucleotidebound conformations [19, 33]. 8. A 30 G needle was attached to the end of an aspirator to remove the supernatant without disturbing the resin. 9. We used molecular markers that do not bind with GST (e.g., Precision Plus Protein™ Dual Color Standards). 10. We generated anti-PUF2 antibodies for our experiment [41]. If an epitope tag is fused to the gene of interest, commercial antibodies would be also usable.

Acknowledgments This work was financially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T.U., 24114003 and 24370019, and E.I., 15K18527 and 17K15144), a Grant-in-Aid for JSPS fellows (E.I., 2010649), the Mitsubishi Foundation, Yamada Science Foundation, Kato Memorial Bioscience Foundation, NIBB Collaborative Research Program (16-339, 17-302, 18-302 to E.I.), and the Building of Consortia for the Development of Human Research in Science and Technology, MEXT, Japan. References

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INDEX A Abscisic acid receptors ..............................................35–47 Agrobacterium tumefaciens Agrobacterium-mediated transformation of Arabidopsis thaliana........................................ 28 Agrobacterium-mediated transformation of leaves 106 ALG-2 INTERACTING PROTEIN-X (ALIX)..................................................... 36, 49–57 ARA6 .......................................... 184–186, 188, 191, 194 Arabidopsis thaliana .......................................1, 3, 6, 7, 9, 16, 24, 36, 37, 49, 51, 52, 62, 71, 123, 145, 148, 169–182, 184

B Bimolecular fluorescence complementation (BiFC) ........................................ 36, 44–46, 50–53 BOR1............................................................1–3, 8, 11, 35 Boric acid/borate transporters................................... 1–12

C Cell-free protein translation ................................ 143–151 Clathrin-mediated endocytosis, see Endocytosis Co-localization analysis............................... 24, 32, 45, 88 Confocal microscopy ................................. 3, 4, 7, 29, 57, 86, 103, 112–114 Contact sites ..............................................................23, 29 Correlative light and electron microscopy (CLEM) .................................................. 60, 63–65

D Dot blots .............................................................. 145–147 DRP1A(K47A)..........................................................2, 3, 6 DYNAMIN-RELATED PROTEIN 1A (DRP1A)........................................................2, 3, 6

E Endocytosis ......................................1–12, 15, 16, 20, 35, 36, 41, 46, 60, 85, 119–121 Endomembranes ..................................23, 84, 85, 89, 95, 106, 110, 119, 120, 135, 153, 154, 183 Endoplasmic reticulum isolation ................................................................... 154 markers ........................................................... 121, 164

Endosomal intraluminal vesicles (ILVs) ................ 15, 16, 49, 50 Endosomal sorting complex required for transport (ESCRT) proteins .......................... 16, 36, 49–51, 84, 85, 91, 109, 111 Endosome markers ................................................................ 24–27 Enzyme-linked immunosorbent assay (ELISA)...........................154, 156, 157, 161, 162

F Fei-Mao (FM4-64) .......................... 4, 7, 16, 17, 19, 119 Fo¨rster resonance energy transfer (FRET).................................................... 24, 29, 31 Freeze-substitution ..................................... 61, 62, 70, 71 FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1) /FYVE DOMAIN-CONTAINING PROTEIN 1 (FYVE1) ...............................36, 37, 50, 85, 111 FYVE domains ...................................... 16, 36, 50, 83–93

G Glycome, see Glycomics Glycomics ............................................................. 153–164

H High-pressure freezing (HPF) .......................... 62, 70–73

I Immunofluorescence ................................................62–64 Immunoprecipitation (IP) ..................... 46, 47, 122–124, 126, 128, 130–132, 134–138, 171, 173, 174, 180

L Late endosome multivesicular bodies/multivesicular endosome/ prevacuolar compartments.................2, 15, 17, 24 Latrunculin B ............................................................27, 29 Lipid extraction ........................................................ 122, 131 profiling .......................................................... 121, 122 protein binding assays ............................................... 85 Lipid nitrocellulose strips ............................................. 145

Marisa S. Otegui (ed.), Plant Endosomes: Methods and Protocols, Methods in Molecular Biology, vol. 2177, https://doi.org/10.1007/978-1-0716-0767-1, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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PLANT ENDOSOMES: METHODS

200 Index

AND

PROTOCOLS

M

R

3-methyladenine (3-MA)....................110–112, 114, 115 Microsomal fractionation .................................. 51–53, 56

RAB GTPase effectors ................................................................... 184 RAB5 ....................................................................... 184 RING finger of seed longevity (RSL1).......................... 36

N Nicotiana benthamiana.................................. 24, 36, 51, 95–107 tabacum ..................................................................... 24 NIP5;1 .....................................................................1–3, 11

O Oligosaccharide mass profiling..................................... 154 Oryzalin .....................................................................27, 29 Osmiophilic thiocarbohydrazide (TCH) contrast enhancing................................................ 71–73, 80

P Phosphatase treatment........................171, 174, 175, 180 Phosphoinositides Phosphatidylinositol 3-phosphate (PI3P) ......................................................... 83, 110 Phosphatidylinositol 4-phosphate (PI4P) ..........................................................95–107 PI3P biosensor citrine-2FYVE ............................ 111 PI4P biosensor mCITRINE-P4MSidM .................. 111 PI4P depletion assay ......................... 97, 99, 102, 104 Phosphoproteins staining .................................................................... 170 Phos-tagTM acrylamide gel .................170, 172, 176, 181 PIN2-GFP .................................................................16, 19 Plant-unique rab5 effector 2 (PUF2)184–187, 189, 190, 193, 194 Polar localization................................................ 2, 6, 7, 11 Polysaccharides ...................................... 64, 153–156, 161 Protein expression in bacteria .............................................. 184 extraction .......................................38, 42, 87, 91, 138 purification from bacteria ....................................... 144 staining with SYPRO™ Ruby Purification from bacteria.....................................172, 173, 176, 177 PYRABACTIN RESISTANCE1/PYR1-LIKE/ REGULATORY COMPONENTS OF ABA Receptors (PYR/PYL/RCAR).....................35, 50

S SDS-Polyacrylamide Gel Electrophoresis ......................................... 172, 175 Serial block-face scanning electron microscopy .....................................................69–81 Small epitopes....................................................... 143–151 Software Amira ............................................................ 72, 74, 77 ImageJ/FIJI ..............................8–10, 31, 40, 46, 130 KNOSSOS ...........................................................72, 74 SYP61 ........................................120, 121, 130, 131, 134, 154, 155, 158, 159, 162, 164

T Three-dimensional electron microscopy, see Serial blockface scanning electron microscopy Trans-Golgi network (TGN) immuno-isolation.................................................... 121 Transmission Electron microscopy (TEM) ........................................ 60, 62–66, 69, 70 TUBBY .......................................................................... 144

U Ubiquitin detection by western blot........................91, 92

V Vacuolar protein sorting 23A (VPS23A).................36, 50 Vacuole markers ........................................................... 8, 16, 46 sorting........................................................2, 8, 15, 35, 36, 50, 85, 109 Vesicle fractionation ............................................. 156, 157 VHAa1-GFP....................................................... 61, 63–65

W Western blot ........................................................... 39, 164 Wortmannin treatment ............................... 86, 89, 91, 93