PLANT ENDOSOMES : methods and protocols.

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