Rab GTPases: Methods and Protocols (Methods in Molecular Biology, 2293)

✍ Scribed by Guangpu Li (editor), Nava Segev (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 307
• Category: Library

✦ Synopsis

This second edition volume expands on the previous edition with a discussion of new research and discoveries in the Rab field. Chapters in this book cover topics such as new information on Rab regulation and localization; interaction; function; and diseases. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and comprehensive, Rab GTPases: Methods and Protocols, Second Edition is a valuable resource for scientists working in the fields of Rab and other small GTPases, and beyond.

✦ Table of Contents

Preface
Contents
Reviewers and Contributors
Chapter 1: Newer Methods Drive Recent Insights into Rab GTPase Biology: An Overview
1 Introduction
2 Rab Regulation and Localization (Chapters 2-8)
3 Rab Interactions (Chapters 9-12)
4 Rab Function (Chapters 13-18)
5 Rab Dysfunction and Disease (Chapters 19-21)
6 Future Perspectives: How New Technologies Can Benefit the Rab Field?
References
Chapter 2: Rab29 Fast Exchange Mutants: Characterization of a Challenging Rab GTPase
1 Introduction
2 Materials
2.1 Purifying Rab29 and its Mutants
2.2 MANT-GDP Assay
3 Methods
3.1 Purification of Rab29 Proteins
3.2 MANT-GDP Assay
4 Notes
References
Chapter 3: High-Throughput Assay for Profiling the Substrate Specificity of Rab GTPase-Activating Proteins
1 Introduction
2 Materials
2.1 Reagents and Chemicals
2.2 Supplies
2.3 Proteins
2.4 Buffers
2.5 Chromatographic Columns
2.6 Instruments
2.7 Software
3 Methods
3.1 GTP Loading and Preparation of 2x Solutions
3.2 GAP Assay and Measurement
3.3 Kinetic Data Analysis
3.3.1 Initial Velocity Approach
3.3.2 Exponential Fit Approach
3.3.3 Integrated Michaelis-Menten Approach
4 Notes
References
Chapter 4: Detecting Endogenous Rab8 Activation
1 Introduction
2 Materials
2.1 Generation of GST-Fusion Probes
2.2 Pull-Down of Active Rab8 from Cell Lysates
2.3 SDS-PAGE and Immunoblotting
3 Methods
3.1 Molecular Cloning of GST-OCRL-RBD and GST-PI3K-RasBD
3.2 Bacterial Expression of GST-OCRL-RBD and GST-PI3K-RasBD
3.3 Rab8 Activation Assay and Immunoblotting
4 Notes
References
Chapter 5: Testing the Phenotypic Effects of a Rab Chimera that Resolves Exchange Factor Specificity from Effector Specificity
1 Introduction
2 Materials
2.1 Bgl2 Secretion Assay
2.2 Electron Microscopy Study
2.3 GFP-Snc1 Microscopy
2.4 Effector Localization
3 Methods
3.1 Bgl2 Assay (Fig. 1)
3.2 Electron Microscopy (Fig. 2)
3.3 Fluorescence Microscopy and Quantitative Localization of GFP-Snc1 (Fig. 3)
3.4 Effector Localization (Fig. 4)
4 Notes
References
Chapter 6: Profiling Structural Alterations During Rab5 Nucleotide Exchange by HDX-MS
1 Introduction
2 Materials
2.1 Protein Expression Bacteria
2.2 Protein Expression-Insect Cell
2.3 Protein Purification
2.4 HDX-MS
3 Methods
3.1 Protein Expression of Rab5
3.2 Purification of Rab5
3.3 Protein Expression of Rabex5:Rabaptin5
3.4 Protein Purification Rabex5:Rabaptin5
3.5 HDX-MS
4 Notes
References
Chapter 7: CLEM Characterization of Rab8 and Associated Membrane Trafficking Regulators at Primary Cilium Structures
1 Introduction
2 Materials
2.1 Cells and Culture Media
2.2 Chemicals, Buffers and Solutions
2.3 Equipment
2.4 Fluorescence and Electron Microscopies
3 Methods
3.1 Cell Culture and Stable Cell Line Generation
3.2 Light Microscopy
3.3 TEM Sample Preparation
3.4 Identifying the Target Cell in the Resin Block
3.5 Ultra-Thin Sectioning Using an Ultramicrotome
3.6 Electron Microscopy Imaging
3.7 Alignment and Processing of Fluorescence and EM Images
4 Notes
References
Chapter 8: Imaging of Spatial Cycling of Rab GTPase in the Cell
1 Introduction
2 Major Outcomes and Possible Uses
3 Materials
3.1 Construction of Plasmids
3.2 Mammalian Cell Culture and Transfection Reagents
3.3 Generation of PRA1 and TRAPPC4 Knockdown Stable Cell Lines
3.4 In Cellulo Prenylation Experiment
3.5 Live Cell Imaging
4 Methods
4.1 Design and Cloning of the Target Construct
4.2 Generation of PRA1 and TRAPPC4 Knockdown Cell Lines
4.3 Microscopy and Imaging
4.3.1 FRAP Experiments
4.3.2 FLAP Experiments
4.4 In Cellulo Prenylation Assay
5 Notes
References
Chapter 9: Deconvolution of Multiple Rab Binding Domains Using the Batch Yeast 2-Hybrid Method DEEPN
1 Introduction
2 Materials
2.1 Rab Expression Constructs
2.2 Data Processing
2.3 Plasmid Reconstruction
2.4 Validation
3 Methods
3.1 Data Processing
3.2 Plasmid Reconstruction
3.3 Validation
4 Notes
References
Chapter 10: Determination of the Rab27-Effector Binding Affinity Using a High-Throughput FRET-Based Assay
1 Introduction
2 Materials
2.1 PCR
2.2 Bacterial Expression Plasmids
2.3 Preparation of Polyhistidine-Tagged Recombinant Proteins
2.4 Preparation of 10% (v/v) SDS-PAGE and Protein Staining
2.5 Reagents Used for Western Blot Analysis
2.6 FRET-Based Assay Materials
3 Methods
3.1 PCR Reaction
3.2 Expression and Purification of Polyhistidine-Tagged Recombinant Proteins
3.3 SDS-PAGE and Protein Staining
3.4 Western Blot Analysis
3.5 Imaging
3.6 Determination of Protein Concentrations
3.7 FRET-Based Protein-Protein Interaction Assay Experiments
4 Notes
References
Chapter 11: Methods to Study the Unique SOCS Box Domain of the Rab40 Small GTPase Subfamily
1 Introduction
1.1 Origins and Evolution of the Rab40 Subfamily
1.2 Conservation and Function of the Rab40 SOCS Box
1.3 Known Functions of Rab40 Paralogs
1.3.1 Rab40a and Rab40al
1.3.2 Rab40b
1.3.3 Rab40c
1.4 The Rab40b/Cul5 Complex
1.5 Using the Rab40b SOCSAAAA Mutant to Uncover Function
2 Materials
2.1 Cross-Linking of FLAG Antibody to Protein G Sepharose
2.2 FLAG-Rab40b Immunoprecipitation
2.3 Proteomics
3 Methods
3.1 Cross-Linking of FLAG Antibody to Protein G Sepharose
3.2 FLAG-Rab40b Immunoprecipitation
3.3 Proteomics
3.4 Results and Conclusion
4 Notes
References
Chapter 12: Using GBP Nanotrap to Restore Autophagy in the Rab5/Vps21 Mutant by Forcing Snf7 and Atg17 Interaction
1 Introduction
2 Materials
3 Methods
3.1 Linearize ATG17-3GFP-PG5
3.2 Construct pHBKA81-SNF7-GBP-mCherry
3.3 Integrate ATG17-3GFP-PG5 and/or pHBKA81-SNF7-GBP-mCherry Plasmids into Wild-Type and vps21Delta Mutant Cells
3.4 Result Analysis
References
Chapter 13: Establishing Regulation of a Dynamic Process by Ypt/Rab GTPases: A Case for Cisternal Progression
1 Introduction
2 Project Strategy and Phases
3 Major Outcomes
4 Reagents, Methods and Precautions for Studying the Yeast Golgi Ypts
5 Suggestions for Studying Regulation of Dynamic Processes by Ypt/Rabs
References
Chapter 14: Methods for Assessing the Regulation of a Kinase by the Rab GTPase Ypt1
1 Introduction
2 Materials
2.1 Culture Media
2.2 Solutions
2.3 Reagents
3 Methods
3.1 An In Vitro Binding Assay for Rab1A and CK1δ (Mammalian Homologue of Hrr25)
3.1.1 Purification of Recombinant His6-Rab1A Q70L and His6-Rab1A S25N
3.1.2 Purification of Glutathione-Sepharose Beads Containing GST and GST Fusion Proteins
3.1.3 In Vitro Binding Assay with GST- CK1δ and His6-Rab1A (Q70L and S25N)
3.2 Differential Centrifugation Can Be Used to Assess if Ypt1 and Rab1 Regulate the Recruitment of Hrr25 and CK1δ to Membranes
3.2.1 Examining the Membrane Distribution of Hrr25 in WT and the ypt1-3 Mutant
3.2.2 The Distribution of CK1δ in HeLa Cells After Rab1A Depletion
3.3 Kinase Assays
3.3.1 Immunoprecipitation of Hrr25-HA from a Yeast Lysate
3.3.2 Kinase Activity Assay
3.3.3 Purification of His6-Tagged Ypt1 Q67L and WT.
3.3.4 Reconstitution Assays
3.4 The Analysis of Autophagosome Formation by Structured Illumination Microscopy (SIM)
4 Notes
References
Chapter 15: Qualitative and Quantitative Assessment of the Role of Endocytic Regulatory and/or Rab Proteins on Mitochondrial F...
1 Introduction
2 Materials
2.1 Major Equipment
2.2 Small Equipment
2.3 Cells
2.4 Reagents
2.5 Buffers
3 Methods
3.1 Qualitative Analysis of the Impact of Endocytic Regulatory Proteins on Mitochondrial Fusion and Fission
3.1.1 Experimental Strategies
3.1.2 Knock-Down of a Select Protein Using siRNA
3.1.3 Immunofluorescence
3.1.4 Imaging
3.1.5 Post-image Processing
3.1.6 Remarks
3.2 Quantitative Analysis of the Impact of Endocytic Regulatory Proteins on Mitochondrial Fusion and Fission
3.2.1 Experimental Strategies
3.2.2 Downloading Fiji and Installing the Mito-Morphology Macro
3.2.3 Using the Mito-Morphology Macro to Quantitatively Measure Mitochondria
3.2.4 Remarks
References
Chapter 16: Characterization of the Role of Rab18 in Mediating LD-ER Contact and LD Growth
Abbreviations
1 Introduction
2 Materials
2.1 Plasmids
2.2 Biological Material
2.3 Reagents
2.4 Buffers and Solutions
2.5 Equipment
2.6 Software
3 Methods
3.1 Characterization of the Function of Rab18 in Regulating LD Growth
3.1.1 Cell Preparation
3.1.2 Measurement of LD Sizes and Numbers
3.1.3 Representative Data
3.2 Characterization of the Function of Rab18 in Promoting the Formation of ER-LD Contacts
3.2.1 Sample Preparation and TEM Imaging
3.2.2 Representative Result
4 Notes
References
Chapter 17: Methods for Establishing Rab Knockout MDCK Cells
1 Introduction
2 Materials
2.1 MDCK II Cell Culture
2.2 Plasmid Construction
2.3 Plasmid Transfection and Selection of KO Cells
2.4 Immunoblotting
2.5 Genomic PCR and Electrophoresis
2.6 Direct Sequencing of Genomic PCR Products
3 Methods
3.1 MDCK II Cell Culture
3.2 Selection of KO Target Sites
3.3 Construction of sgRNA- and Cas9-Coding Plasmids
3.3.1 Digestion of the pSpCas9 Vector
3.3.2 Design of Sense and Antisense sgRNA-Coding-Oligonucleotides and Their Annealing
3.3.3 Preparation of pSpCas9 Vector Carrying sgRNA-Coding-Oligonucleotides
3.4 Primer Design for Genomic PCR
3.4.1 Check for a Genomic Location of the Target Sequence
3.4.2 Acquisition of a Genomic Sequence around the Target Sequence
3.4.3 Primer Design for Genomic PCR
3.5 Establishment of Rab-KO MDCK II Cell Lines
3.5.1 Transfection of sgRNA/Cas9-Coding Plasmids
3.5.2 Selection of Plasmid-Transfected Cells
3.5.3 Cloning of Plasmid-Transfected Cells
3.6 Check for Disruption of Targeted Genes
3.6.1 Check for Protein Expression by Immunoblotting
3.6.2 Check for Genomic DNA by Direct Sequencing
Preparation of Genomic DNA of MDCK II Cells
Genomic PCR
Direct Sequencing
Subcloning of Genomic DNA
4 Notes
References
Chapter 18: Generating Rab6 Conditional Knockout Mice
1 Introduction
1.1 Major outcomes
2 Materials
2.1 Mice
2.2 Tamoxifen
2.3 4-hydroxy (OH) Tamoxifen
2.4 Culture Media
2.5 RAB6 Antibodies
3 Methods
3.1 In Vitro Studies
3.1.1 Preparation of MEFs (Mouse Embryo Fibroblasts)
3.1.2 Preparation of Melanocytes
3.1.3 Preparation of Neurons
3.2 In Vivo Studies
3.2.1 RAB6 in the Melanocyte Lineage
3.2.2 RAB6 in the T cell Lineage
3.2.3 RAB6 in the Gut Epithelium
3.2.4 RAB6 in the Mammary Gland
3.2.5 RAB6 in the Brain
4 Notes
References
Chapter 19: Use of Immunohistochemistry to Determine Expression of Rab5 Subfamily of GTPases in Mature and Developmental Brains
1 Introduction
2 Materials
2.1 Human Autopsy Brain Tissue
2.2 Antibodies
2.3 Staining Materials
3 Methods
3.1 Human Brain Samples
3.1.1 Sample Collection
3.1.2 Processing
3.1.3 Hematoxylin-Eosin Stain
3.1.4 Immunohistochemistry (see Notes 1, 2, 4)
4 Comments
5 Notes
6 Conclusions
References
Chapter 20: Assessing Rab5 Activation in Health and Disease
1 Introduction
2 Materials
2.1 Fluorescence Recovery After Photobleaching (FRAP) Assay
2.2 Quantitative Measurement of Endosome Size Changes
2.2.1 Rab5 Positive Endosome Measurement in Human Fibroblasts
2.2.2 Rab5 Positive Endosome Measurement in Mouse Brain Sections
2.2.3 Electron Microscopy (EM) and Postembedding Immuno-EM (iEM) from Mouse Brain
EM General Method
Postembedding iEM
2.3 Detection of GTP-Rab5
2.3.1 In Situ Immunofluorescence In Vitro
2.3.2 In Situ Immunofluorescence Ex Vivo
2.3.3 Immunoprecipitation from Mouse Brain Tissue/Subcellular Fraction and Western Blot
2.3.4 Pull-Down with GTP-Agarose from Mouse Brain Tissue/Subcellular Fraction and Western Blot
2.4 Endosome Isolation by OptiPrep Density Gradient Centrifugation from Mouse Brain Tissue
3 Methods
3.1 Fluorescence Recovery After Photobleaching (FRAP) Assay
3.2 Quantitative Measurement of Endosome Size Changes
3.2.1 Rab5 Positive Endosome Measurement in Human Fibroblasts
3.2.2 Rab5 Positive Endosome Measurement in Mouse Brain Sections
3.2.3 EM and Postembedding Immuno-EM from Mouse Brain
General EM Method
Postembedding Immuno-EM
3.3 Detection of GTP-Rab5
3.3.1 In Situ Immunofluorescence In Vitro
3.3.2 In Situ Immunofluorescence Ex Vivo
3.3.3 Immunoprecipitation from Mouse Brain Tissue/Subcellular Fraction and Western Blot
3.3.4 Pull-Down with GTP-Agarose from Mouse Brain Tissue/Subcellular Fraction and Western Blot
3.4 Endosome Isolation by OptiPrep Density Gradient Centrifugation from Mouse Brain Tissue
4 Notes
References
Chapter 21: Quantitative Fluorescence Microscopy for Detecting Mammalian Rab Vesicles within the Parasitophorous Vacuole of th...
1 Introduction
2 Materials
2.1 Cell and Parasite Lines
2.2 Cell Culture and Transfection
2.3 Immunofluorescence Reagents
2.4 Imaging Equipment and Software
3 Methods
3.1 Propagation of Toxoplasma Gondii RH and Mutant Strains
3.2 Plate Mammalian Cells
3.3 Transient Transfection (HeLa Cells) (See Note 12)
3.4 Infection
3.5 Immunofluorescence
3.6 Imaging
3.7 Image Analysis
4 Notes
References
Index