Preface
Contents
Contributors
Chapter 1: Rab Family of GTPases
1 Introduction
2 Regulation of Rab GTPase Cycle
3 Rab Functions in Vesicular Transport
4 Other Rab Functions
5 Rabs and Disease
References
Chapter 2: Bioinformatic Approaches to Identifying and Classifying Rab Proteins
1 Introduction
2 Materials
3 Methods
3.1 Identifying Rabs
3.2 Classifying Rabs
4 Notes
References
Chapter 3: Rab-NANOPS: FRET Biosensors for Rab Membrane Nanoclustering and Prenylation Detection in Mammalian Cells
1 Introduction
1.1 The Emergence of Signaling Protein Nanocluster
1.2 Rab Protein Membrane Anchorage and Nanoclustering
1.3 Functioning and Design of Rab-NANOclustering and Prenylation Sensors (NANOPS)
2 Materials
2.1 Cell Culture
2.2 Flow Cytometer Settings and Sensitized Acceptor Emission FRET Channel Setup
2.3 Data Evaluation and Analysis Software
3 Methods
3.1 Preparation of Cells
3.2 Sample Preparation Before Flow Cytometry
3.3 Flow Cytometry and Data Acquisition
3.4 Flow Cytometer FRET Data Analysis
4 Notes
References
Chapter 4: 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 Chromatogra
2.6 Instruments
2.7 Software
3 Methods
3.1 GTP Loading and Preparation of 2× Solutions
3.2 GAP Assay and Measurement
3.3 Kinetic Data Analysis
3.3.1 Initial Velocity Approach
3.3.2 Exponential Fit Approach
4 Notes
References
Chapter 5: Measuring Rab GTPase-Activating Protein (GAP) Activity in Live Cells and Extracts
1 Introduction
2 Materials
2.1 Mammalian Expression Plasmids
2.2 Bacterial Expression Plasmids
2.3 Protein Purification
2.4 GAP Assays in Cells and Extracts
3 Methods
3.1 Expression and Purification of GST-RBDs
3.2 Expression and Purification of 6x-His-RUTBC1-C and 6xHis-RUTBC1-C R803A
3.3 GAP Assay in Cells
3.4 Biochemical GAP Assay in Cell Extracts
4 Notes
References
Chapter 6: Analysis of the Interactions Between Rab GTPases and Class V Myosins
1 Introduction
2 Materials
2.1 Buffers and Media
2.2 Preparation of Yeast Lysates
2.3 Yeast Transformation: Lithium Acetate Method
2.4 Yeast Transformation Protocol: Mating
2.5 Replica Plating
2.6 β-Galactosidase Assay
2.7 Cell Culture and Transfection
2.8 Coimmunop
3 Methods
3.1 The Yeast Two-Hybrid System
3.2 Generation of Prey Construct
3.3 Yeast Two-Hybrid “Living Chip” Assay
3.4 GFP-Rab Coimmunop
4 Notes
References
Chapter 7: Assaying the Interaction of the Rab Guanine Nucleotide Exchange Protein Sec2 with the Upstream Rab, a Downstream Effector, and a Phosphoinositide
1 Introduction
2 Materials
2.1 Expression and Purification of GST-Sec2p and GST Alone
2.2 Expression and Purification of HIS6-Ypt32p
2.3 Expression and Purification of HIS6-Sec15p
2.4 Expression and Purification of Lyticase
2.5 In Vitro Binding Assay
2.6 Immunopreci
2.7 Liposomes Preparation and Sedimentation Experiment
3 Methods
3.1 Expression and Purification of GST-Sec2p and GST Alone
3.2 Expression and Purification of HIS6-Ypt32p
3.3 Expression and Purification of HIS6-Sec15p
3.4 Expression and Purification of Lyticase
3.5 In Vitro Binding Assay (See Note 1)
3.6 Immunopreci
3.7 Liposomes Preparation and Sedimentation Experiment
4 Notes
References
Chapter 8: Kinetic Activation of Rab8 Guanine Nucleotide Exchange Factor Rabin8 by Rab11
1 Introduction
2 Materials
2.1 Protein Purification Reagents
2.2 GEF Activity Assay Reagents
2.3 Antibodies
3 Methods
3.1 Expression and Purification of Rab8a
3.2 Expression and Purification of the Constitutively Activated Rab11a, Rab5a, and Rab3a Mutants
3.3 Expression and Purification of Rabin8
3.4 Assay for the Activation of Rabin8 GEF Activity by Rab11
4 Notes
References
Chapter 9: Ypt1 and TRAPP Interactions: Optimization of Multicolor Bimolecular Fluorescence Complementation in Yeast
1 Introduction
2 Materials
3 Methods
3.1 Multicolor BiFC Principle
3.2 Optimized Plasmids for Use in Yeast
3.3 BiFC Localization in Red Strains
3.4 Examples of Using BiFC for Studying Ypt1, TRAPP II, and Beyond
3.5 Important Controls and Parameters
4 Notes
References
Chapter 10: Identifying a Rab Effector on the Macroautophagy Pathway
1 Introduction
2 Materials
2.1 Culture Media
2.2 Solutions
2.3 Plasmids
2.4 Reagents
3 Methods
3.1 Determination of the PAS Localization of Atg Proteins in the ypt1-2 Mutant by Fluorescence Microscopy to Identify a Candidate Ypt1 Effector on the Macroautophagy Pathway
3.2 In Vitro Binding Assay of Ypt1 and Atg1 to Determine If Atg1 and Ypt1 Physically Interact with Each Other
3.2.1 Purification of Ypt1-His6
3.2.2 Purification of Glutathione-Sepharose Beads Containing GST-Atg1 (1–500aa, 501–897aa) and GST
3.2.3 In Vitro Binding Assay with GST-Atg1 Constructs (1–500aa, 501–897aa) and Ypt1-His6
3.3 Coimmunoprecipitation of Atg1-3HA and Ypt1 Demonstrated That Atg1 Preferentially Interacts with the GTP-Bound form of Ypt1 In Vivo
4 Notes
References
Chapter 11: Functional Analysis of Rab27A and Its Effector Slp2-a in Renal Epithelial Cells
1 Introduction
2 Materials
2.1 Immunopre
2.2 Immunocytochemical Analysis of MDCK II Cells
2.3 Immunohistochemical Analysis of Mouse Kidney
3 Methods
3.1 Immunoprecipitation Analysis of Endogenous Interaction Between Slp2-a and Rab27A in MDCK II cells
3.2 Immunocytochemical Analysis of Slp2-a and Rab27A in MDCK II Cells
3.2.1 Localization of Slp2-a and Rab27A in a Single MDCK II Cell
3.2.2 Localization of Slp2-a and Rab27A in Polarized MDCK II Cells
3.2.3 Localization of Slp2-a and Rab27A in MDCK II Cysts
3.3 Immunohistochemical Analysis of Slp2-a and Rab27A in Mouse Kidney
4 Notes
References
Chapter 12: Small GTPases in Acrosomal Exocytosis
1 Introduction
2 Materials
2.1 Reagents
2.1.1 SLO Stock Solutions (See Note 1)
2.2 Plasmids
2.3 Recombinant Proteins
2.3.1 Expression and Purification of GST-Fused Proteins
2.3.2 Expression and Purification of His6-Tagged Proteins
2.4 Antibodies
2.5 Buffers
3 Methods
3.1 Human Sperm Samples
3.1.1 Sample Collection
3.1.2 Swim-Up (See Note 7) and Capacitation (See Note 8)
3.1.3 SLO-
3.2 In Vitro Solid Phase Isoprenylation and Activation of Rab Proteins
3.2.1 Geranylger
3.2.2 Immobilization of Recombinant Rabs
3.2.3 Solid Phase Isoprenylation Protocol
3.2.4 Loading Rabs with Guanine Nucleotides
3.3 Far-Immunofluorescence: Description of a New Method to Detect the Localization of Active Rabs
3.3.1 Far-
3.3.2 Sperm Treatment 1 for Standardization of Activity Probes Binding to Endogenous Active Rabs in Permeabilized Cells (Fig. 2a–d)
3.3.3 Sperm Treatment 2 for Analysis of Rab Activation in Response to Exogenous Stimuli (Fig. 2e)
3.3.4 Sperm Treatment 3 for Analysis of Rab3-GTP Levels After A23187 Treatment in Non-permeabilized Cells [24]
3.4 Pull-Down Assays for Active Rab27 and Rab3
3.4.1 Preparation of Human Sperm Lysates
3.4.2 Pull-Down Assay
3.5 Recruitment of a Human Sperm Rab3A GEF Activity by Immobilized Rab27A
3.5.1 Preparation of Human Sperm Extracts
3.5.2 Immobilization of Rab27A and Pull-Down of a Rab3A GEF Activity
3.5.3 Protein Precipitation
3.5.4 Analysis of His6-Rab3A-GTP Levels
4 Notes
References
Chapter 13: Rab Antibody Characterization: Comparison of Rab14 Antibodies
1 Introduction
2 Materials
2.1 Bacterial Strains
2.2 Cell Lines
2.3 Chemicals and Reagents
2.4 Buffers and Solutions
3 Methods
3.1 Plasmids
3.2 Transfections
3.3 Indirect Immunofluorescence
3.4 Protein Purification and Western Blotting
4 Notes
References
Chapter 14: Selective Visualization of GLUT4 Storage Vesicles and Associated Rab Proteins Using IRAP-pHluorin
1 Introduction
2 Materials
2.1 Coverglass Cleaning
2.2 Cell Culture and Transfection
2.3 TIRF Imaging
3 Methods
3.1 Coverglass Cleaning
3.2 Cell Culture
3.3 Transfection
3.4 TIRF Imaging
3.5 Image Analysis
4 Notes
References
Chapter 15: 3D Time-Lapse Analysis of Rab11/FIP5 Complex: Spatiotemporal Dynamics During Apical Lumen Formation
1 Introduction
2 Materials
2.1 3D Tissue Culture
2.2 Microscopy
3 Methods
4 Notes
References
Chapter 16: In Vitro and In Vivo Characterization of the Rab11-GAP Activity of Drosophila Evi5
1 Introduction
2 Materials
2.1 Effector Pull Down Using Copper-Inducible Stable Cell Lines Components
2.2 In Vivo Rab11-
3 Methods
3.1 Characterization of Evi5 GAP Activity Toward Rab11 In Vitro
3.1.1 Generation of GST-Rab11-Inducible Stable Cell Lines
3.1.2 Effector Pull-Down Assay
3.2 Characterization of Evi5 GAP Activity for Rab11 In Vivo
3.2.1 Preparation of Drosophila Ovaries to Monitor Rab11 Activity
3.2.2 Imaging Stage 9 Egg Chamber and Vesicle Analysis
4 Notes
References
Chapter 17: Characterization of the Role Rab25 in Energy Metabolism and Cancer Using Extracellular Flux Analysis and Material Balance
1 Introduction
2 Materials
2.1 Cell Lines and Constructs
2.2 Equipment
2.3 Materials for XF96 Extracellular Flux Assay (Seahorse)
2.4 Materials for YSI 2900 Biochemistry Analyzer
3 Methods
3.1 XF 96 Extracellular Flux Analyzer (Seahorse Bioscience)
3.2 Measuring Metabolite Turnover by YSI 2900
4 Notes
References
Chapter 18: Measurement of Rab35 Activity with the GTP-Rab35 Trapper RBD35
1 Introduction
2 Materials
2.1 Determining the Binding Specificity of RBD35 for 60 Rab Proteins
2.2 Measuring Rab35 Activity
2.3 Inhibiting the Function of Rab35
3 Methods
3.1 Assay to Determine the Binding Specificity of RBD35 for 60 Rab Proteins
3.2 Assay to Measure Rab35 Activity
3.3 Assay to Inhibit the Function of Rab35
4 Notes
References
Chapter 19: Analysis of Connecdenn 1–3 (DENN1A-C) GEF Activity for Rab35
1 Introduction
2 Materials
2.1 Cloning and DNA Purification
2.2 Protein Purification
2.3 GTPase Loading and GEF Assay
3 Methods
3.1 Expression Constructs
3.1.1 Connecdenn DENN Domain
3.1.2 Rab35
3.2 DNA Purification
3.3 Protein Expression and Purification
3.4 Rab35 Loading
3.5 GEF Assay
4 Notes
References
Chapter 20: Assay of Rab17 and Its Guanine Nucleotide Exchange Factor Rabex-5 in the Dendrites of Hippocampal Neurons
1 Introduction
2 Materials
2.1 Mouse Hippocampal Neuron Culture and Transfection
2.2 Immunostaining
2.3 Evaluation of Neurite Morphogenesis
2.4 Measurement of Filopodium Density
2.5 Anti-Syt I-N Antibody Uptake Experiment
2.6 Measurement of Dendritic Rab17 Signals
3 Methods
3.1 Mouse Hippocampal Neuron Culture and Transfection
3.2 Immunostaining
3.3 Evaluation of Neurite Morphogenesis
3.4 Measurement of Filopodium Density
3.5 Anti-Syt I-N Antibody Uptake Experiment
3.6 Measurement of Dendritic Rab17 Signals in Rabex-5-Expressing Neurons
4 Notes
References
Chapter 21: Methods for Analysis of AP-3/Rabin4′ in Regulation of Lysosome Distribution
1 Introduction
2 Materials
2.1 Bacterial Strains
2.2 Mammalian Cell Lines
2.3 Chemicals
2.4 Buffers
2.5 Plasmids
2.6 Primary Antibodies
2.7 Labeled Secondary Antibodies
3 Methods
3.1 Expression and Purification of GST-rabip4′(509–708) for Immunization
3.2 Preparation of Cell Lysate for Detection of rabip4′ by Western Blot
3.3 Cell Culture and Assays for Immunofluorescence Localization
3.4 Purification of GST-rabip4′(299–708) for Affinity Isolation of Effectors
3.5 Preparation of Pig Brain Cytosol
3.6 Affinity Isolation of Cytosolic Proteins on GST-rabip4′(299–708) Beads
3.7 Preparation of Cell Lysates and Co- immunoprecipitation
3.8 Localization of rabip4 and AP-3, Transfection and Morphological Assays
3.9 Morphological Analysis of AP-3 and rabip4/rabip4′ Function
4 Notes
References
Chapter 22: Determination of Rab5 Activity in the Cell by Effector Pull-Down Assay
1 Introduction
2 Materials
2.1 Preparation of GST Fusion Proteins
2.2 Expression of Rab5, MoRab5A, MoRab5B, and Their Mutants in Tissue Cultures and Preparation of Cell Lysates
2.3 SDS-PAGE and Coomassie Blue Staining
2.4 Immunoblot Assay with Bio-Rad Semi-dry Transfer Apparatus
3 Methods
3.1 Expression and Purification of GST-R5BD Fusion Proteins
3.2 Preparation of Mammalian Cell Lysates for the Pull-Down Assay
3.3 GST-R5BD Pull-Down Assay
3.4 SDS-PAGE and Immunoblot Analysis
4 Notes
References
Chapter 23: Identification of the Rab5 Binding Site in p110β: Assays for PI3Kβ Binding to Rab5
1 Introduction
2 Materials
2.1 Plasmids
2.2 Buffers for Bacterial Expression of GST-Rab5
2.3 Buffers for Mammalian Expression of p85/p110β
2.4 Buffers for Rab5 Nucleotide Loading
3 Methods
3.1 Production of Recombinant GST-Rab5 in Bacteria
3.2 Purification of GST-Rab5 (Adapted from Ref. 14)
3.3 Elution of GST-Rab5
3.4 Analysis of Protein Concentration
3.5 Preparation of Recombinant p85/p110 β
3.6 Nucleotide Loading of GST-Rab5A (Adapted from Ref. 14)
3.7 Analysis of Rab5-p85/p110 β Binding
4 Notes
References
Chapter 24: Role of the Rab5 Guanine Nucleotide Exchange Factor, Rme-6, in the Regulation of Clathrin-Coated Vesicle Uncoating
1 Introduction
2 Materials
2.1 Solutions and Reagents
2.2 Equipment
3 Methods
3.1 Culturing HEK293 Cells (See Note 2)
3.2 Setting Up Coverslips for an Experiment
3.3 Transfection of Cells with siRNA
3.4 Assay for Uncoating
3.5 Imaging
3.6 Image Processing
4 Notes
References
Chapter 25: Differential Effects of Overexpression of Rab5 and Rab22 on Autophagy in PC12 Cells with or without NGF
1 Introduction
2 Materials
2.1 Cell Culture/Transfection
2.2 Expression Plasmids
2.3 Antibodies
2.4 Staining Material
3 Methods
3.1 Plasmid Construction
3.2 Cell Culture and Transfection
3.3 Fixing/Mounting Cells
3.4 Autophagy Assay
3.5 Western Blot for Protein Expression
4 Notes
References
Chapter 26: Determining the Role of Rab7 in Constitutive and Ligand-Mediated Epidermal Growth Factor Receptor Endocytic Trafficking Using Single Cell Assays
1 Introduction
2 Materials
2.1 Cells and Cell Culture Reagents
2.2 RNAi
2.3 Transfection Reagent
2.4 Antibodies
2.5 Monitoring Ligand-Mediated Receptor Trafficking
3 Methods
3.1 siRNA-Mediated Transfection
3.2 Determining Changes in the Steady-State EGFR Distribution
3.3 Monitoring the Rate of Unliganded EGFR Endocytosis
3.4 Determine the Trafficking of Liganded Cell Surface EGFRs
4 Notes
References
Chapter 27: Visualizing Directional Rab7 and TrkA Cotrafficking in Axons by pTIRF Microscopy
1 Introduction
1.1 Functionality of Rab7 GTPase and Its Mutants in Neurodegeneration
1.2 Resolving Directional Axonal Transport by Compartmentalized Microfluidic Chambers
1.3 Pseudo-TIRF Microscopy for Tracking Axonal Transport with High Temporal Resolution
2 Materials
2.1 Pseudo-TIRF Microscope
2.2 DRG Cell Culturing
3 Methods
3.1 Setting Up a Pseudo-TIRF Microscope
3.2 DRG Transfection and Culturing in a PDMS Compartmentalized Chamber
3.3 Imaging Axonal Transport of Endosomes with Pseudo-TIRF Microscopy
3.4 Kymograph Data Analysis
4 Notes
References
Chapter 28: Quantitative Bead-Based Flow Cytometry for Assaying Rab7 GTPase Interaction with the Rab-Interacting Lysosomal Protein (RILP) Effector Protein
1 Introduction
1.1 Rab7 and Its Effector Protein RILP Are Disease Relevant Proteins
1.2 Rationale for a Bead-Based Flow Cytometry Assay for Quantitative Measurement of Rab7-Effector Protein Interactions
2 Materials
2.1 Reagent Sources
2.2 Synthesis of GSH Beads for Flow Cytometry Assays
2.3 Expression and Purification of GST, GST-RILP, GST-Rab7, His-Rab7, and His-SUMO-GFP-Rab7 in Bacterial Cells
2.4 Immobilization of GST, GST-Rab7, and GST-RILP on GSH Beads
2.5 Measurement of Specific Interaction Between Rab7 and RILP Effector Protein
2.6 Measurement of Time-Dependent Rab7 Interaction with RILP
2.7 Measurement of Temperature-Dependent Rab7 Interaction with RILP
2.8 Measurement of the Kinetics of BODIPY-GTP Displacement from Rab7 by Competitive Guanine Nucleotide-Binding Inhibitor (CID 1067700 or ML282)
2.9 Measurement of Rab7 Interaction with RILP in the Presence of a Competitive Guanine Nucleotide-Binding Inhibitor (CID 1067700 or ML282)
3 Methods
3.1 Experimental Design of a Bead-Based Flow Cytometry Assay and Its Application to Measurement of Protein–Protein Interactions
3.2 Reagent Preparation for Quantitative Protein–Protein Interaction Measurements
3.2.1 Synthesis of GSH Beads for Flow Cytometry Assays (see also Note 1)
3.2.2 Expression and Purification of Recombinant Proteins
3.2.3 Immobilization of GST, GST-Rab7, and GST-RILP on GSH Beads for Flow Cytometry
3.2.4 Preparation of Nucleotide-Bound, Active Rab7
3.2.5 Flow Cytometry Measurement of Bead-Associated Fluorescence
3.3 Quantitative Measurement of the Specific Interaction Between Rab7 and RILP
3.4 Measurement of the Time-Dependent Rab7 and RILP Interaction
3.4.1 Methods for Long-Term and Rapid Kinetic Measurements Using Flow Cytometry
3.4.2 Representative Kinetic Measurements of Rab7–RILP Interaction
3.5 Measurement of the Temperature Dependence of Rab7 and RILP Interaction
3.5.1 Methods for Measuring Temperature-Dependent Protein–Protein Interaction
3.5.2 Representative Temperature-Dependent Rab7 and RILP Interaction Measurements
3.6 Measurement of the Effect of a Novel Guanine Nucleotide-Binding Inhibitor on Rab7 and RILP Interaction
3.6.1 Measuring the Kinetics of Direct BODIPY-GTP Nucleotide Displacement by a Competitive Guanine Nucleotide-Binding Inhibitor (CID 1067700)
3.6.2 Assessment of Rab7 Interaction with RILP in the Presence of Competitive Guanine Nucleotide-Binding Inhibitor (CID 1067700) Under Equilibrium Binding Conditions
3.6.3 Measurement of the Effect of a Competitive Guanine Nucleotide-Binding Inhibitor on the Interaction Between Rab7 and RILP
3.6.4 Broader applications: Probing Other GTPases and Performing High Throughput Screens
3.7 Data Analyses
4 Notes
References
ERRATUM TO
Index