Ras Activity and Signaling: Methods and Protocols (Methods in Molecular Biology, 2262)

✍ Scribed by Ignacio Rubio (editor), Ian Prior (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 436
• Category: Library

✦ Synopsis

This volume compiles new experimental approaches and concepts focusing mostly, but not solely, on ways to manipulate and regulate Ras activity and its downstream signaling output. Chapters detail standard methodologies, biochemical methods, Ras processing trafficking and localization, Ras signaling and inhibition, and in vivo models for studying Ras function. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, application details for both the expert and non-expert reader, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and accessible, Ras Activity and Signaling: Methods and Protocols aims to provide support and guidance to lab workers in their work on Ras GTPases and in the design of new projects requiring novel methodologies.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Generic Ras Biology
Chapter 1: Ras Variant Biology and Contributions to Human Disease
1 Introduction
2 Ras Variants
3 Ras Contributions to Disease
3.1 RASopathies: Developmental Disorders
3.2 Oncogenic Ras
4 Challenges and Opportunities Ahead
5 Conclusions
References
Chapter 2: Regulation of the Small GTPase Ras and Its Relevance to Human Disease
1 Introduction
2 The Ras Superfamily
3 The Ras Cycle
4 Oncogenic RAS Mutations in Cancer
5 Targeting Oncogenic Ras
6 Ras Guanine Nucleotide Exchange Factors (RasGEFs)
7 RasGEFs in Cancer
8 Ras GTPase-Activating Proteins (RasGAPs) in Ras Activation Control
9 Regulation of RasGAPs by Extracellular Agonists
10 Challenges Faced by Research on RasGAPs
11 RasGAPs in Cancer
12 Subtly Altered Ras Signaling in Developmental Syndromes
13 Closing Remarks
References
Part II: Methods in Ras Biochemistry
Chapter 3: Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry
1 Introduction
2 Materials
2.1 SOP for KRAS4B Immunoprecipitation
2.2 KRAS4B Magnetic Bead Immunoprecipitation
2.3 LC-MS/MS
2.4 Data Analysis
3 Methods
3.1 SOP for KRAS4B Immunoprecipitation (Fig. 2)
3.2 KRAS4B Magnetic Bead Immunoprecipitation (Fig. 2)
3.3 LC-MS/MS Parameters for Targeted Top-Down Characterization of KRAS4B
3.4 Data Analysis (see Note 11; Figs. 3 and 4)
4 Notes
References
Chapter 4: Absolute Quantitation of GTPase Protein Abundance
1 Introduction
2 Materials
2.1 Production of Heavy Labeled His-Tagged Ras/Ral Standards
2.1.1 Expression of Heavy His-Ras/His-Ral Recombinant Protein in AT713 Bacteria
2.1.2 Affinity Purification Using His-Trap Columns
2.1.3 Purification Check: Confirm Purity and Distribution of His-Ras/His-Ral Protein
2.1.4 Size-Based Purification by Gel Filtration
2.1.5 Storage and Concentration Determination of the Standard
2.2 Mass Spectrometric Characterization of GTPases Such as Ras/Ral Isoforms
2.2.1 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)
2.2.2 In-Gel Digestion and C-18 Desalt
2.2.3 Mass Spectrometric Characterization of Ras Isoforms
2.2.4 Optimization of MRM Transitions and Quantitation of Ras and Ral Isoforms
2.3 Quantification of Endogenous Ras or Ral Levels in Cell Lysates
3 Methods
3.1 Production of Heavy Labeled His-Tagged Ras/Ral Standards
3.1.1 Expression of Heavy His-Ras/His-Ral Recombinant Protein in AT713 Bacteria
3.1.2 Affinity Purification Using His-Trap Columns
3.1.3 Purification Check: Sample Preparation for SDS-PAGE
3.1.4 Purification Check: Confirm Purity and Distribution of His-Ras/His-Ral Protein
3.1.5 Size-Based Purification by Gel Filtration
3.1.6 Storage and Concentration Determination of the Standard
3.2 Mass Spectrometric Characterization of GTPases Such as Ras/Ral Isoforms
3.2.1 Confirming Efficient Labeling of Recombinant His-Ras/His-Ral Protein
3.2.2 In-Gel Digestion
3.2.3 C-18 Desalting Using ZipTip with C18 Resin
3.2.4 Identification of Suitable Peptides and Transition Ions
3.2.5 Optimization of MRM Transitions
3.2.6 Calibration Curves to Determine Spike-In Concentrations
3.3 Quantification of Endogenous Ras or Ral Levels in Cell Lysates
3.3.1 Harvesting Cells
3.3.2 Collecting Cell Pellets
3.3.3 Cell Lysis and Spike-in
3.3.4 Mass Spectrometric Quantification of Endogenous Proteins and Spiked-In Standards
3.3.5 Calculation of Endogenous Protein Concentration from MRM Data
4 Notes
References
Chapter 5: Validation of Isoform- and Mutation-Specific RAS Antibodies
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Reagents, Chemicals
2.3 Equipment
3 Methods
3.1 Lysate Generation, Protein Quantification, and Normalization
3.2 Immunoblotting
3.3 Antibodies and Developing Blots
4 Notes
References
Chapter 6: Production and Membrane Binding of N-Terminally Acetylated, C-Terminally Farnesylated and Carboxymethylated KRAS4b
1 Introduction
2 Materials
2.1 Buffers
2.2 Baculovirus Protein Expression
2.3 Lysis
2.4 Protein Purification
2.5 Surface Plasmon Resonance (SPR)
3 Methods
3.1 Protein Expression
3.2 Lysis
3.3 Protein Purification
3.4 SPR: Anti-His Chip Build Using Amine Coupling Chemistry
3.5 SPR: Sample Preparation
3.6 SPR: Biacore T200 Setup
4 Notes
References
Chapter 7: Active GTPase Pulldown Protocol
1 Introduction
2 Materials
2.1 Bait Production
2.2 Active GTPase Pulldown
3 Methods
3.1 Bait Production Method
3.2 Active GTPase Pulldown Method
3.2.1 Cells in Suspension
3.2.2 Adherent Cells
3.2.3 Pulldown Procedure
3.2.4 Western Blotting
4 Notes
References
Chapter 8: Methods to Monitor Ras Activation State
1 Introduction
2 Materials
2.1 Ras Preloading
2.2 Nucleotide Exchange
2.2.1 Environment-Sensitive Nucleotide Analogs
2.2.2 QRET
2.2.3 FRET and TR-FRET
2.3 GTPase Cycling and GTP Hydrolysis
2.3.1 GTP Detection
2.3.2 GDP Detection
2.3.3 Phosphate Detection
2.4 Ras Loading State and Stability
2.4.1 FRET- and TR-FRET-Based GTP-Ras Assay
2.4.2 Ras Thermal Stability
3 Methods
3.1 Ras Preloading
3.2 Nucleotide Exchange
3.2.1 Environment-Sensitive Nucleotide Analogs
3.2.2 QRET
3.2.3 FRET and TR-FRET
3.3 GTPase Cycling and GTP Hydrolysis
3.3.1 GTP Detection
3.3.2 GDP Detection
3.3.3 Phosphate Detection
3.4 Ras Loading State and Stability
3.4.1 FRET and TR-FRET-Based GTP-Ras Assay
3.4.2 Ras Thermal Tability
4 Notes
References
Chapter 9: NMR Detection Methods for Profiling RAS Nucleotide Cycling
1 Introduction
2 Materials
2.1 Agar Plates and LB Medium
2.2 M9 Solutions for Protein Expression
2.3 His-Tagged RAS (and GEF/GAP) Purification and Nucleotide Stock Solutions
2.4 Mammalian Cell Lysis Buffer, Growth Media, and Transfection Reagent
3 Methods
3.1 Biochemical Methods
3.1.1 RAS GTPase Expression
3.1.2 RAS GTPase Purification
3.1.3 GTP Loading for GTP Hydrolysis Assays
3.1.4 GEF and GAP Preparation: Purified Recombinant Protein
3.1.5 GEF and GAP Preparation: Cell Lysates
3.2 NMR Methods to Study RAS Nucleotide Cycling
3.2.1 Intrinsic Nucleotide Exchange and Intrinsic GTP Hydrolysis Assays
3.2.2 Nucleotide Exchange Assay with GEFs and GTP Hydrolysis Assay with GAPs
3.2.3 Data Processing and Analysis
4 Notes
References
Part III: Methods in Ras Processing, Trafficking, and Localization
Chapter 10: Ras Diffusion and Interactions with the Plasma Membrane Measured by FRAP Variations
1 Introduction
2 Materials
2.1 Cells
2.2 Tissue Culture Medium
2.3 Plasmids
2.4 Transfection Reagent
2.5 Antibodies for Labeling and for Crosslinking HA Proteins
2.6 FRAP Equipment
2.7 Other Reagents
3 Methods
3.1 Cell Culture and Transfection
3.2 Cholesterol Depletion
3.3 Palmostatin B Treatment
3.4 Preparation of Fab′ Fragments
3.5 Fluorescent Labeling and Crosslinking of Cell-Surface HA Proteins
3.6 EGF Stimulation
3.7 FRAP Beam-Size Analysis and Patch/FRAP
4 Notes
References
Chapter 11: Understanding Ras Spatial Cycles Through Reaction-Diffusion Simulations
1 Introduction
2 Approximating Reaction-Diffusion Systems in OCTAVE
3 The KRas Spatial Cycle
4 Disrupting the KRas Localization Cycle
5 Discussion
6 Notes
References
Chapter 12: Super-Resolution Imaging and Spatial Analysis of RAS on Intact Plasma Membrane Sheets
1 Introduction
2 Materials
2.1 Solutions for Generating Gold Nanoparticles
2.2 Solutions for PM Sheet Preparation
2.3 Preparation of Copper and Gold EM Grids
2.3.1 Pioloform Coating
2.3.2 Poly-l-Lysine Coating
2.4 Preparation of Antibody-Coated Gold Nanoparticles of Univariate Spatial Analysis
2.5 Further Refining the Gold Particle Sizes for Bivariate Co-localization Analysis
3 Methods
3.1 Apical and Basolateral PM Rip-Off and Immunogold-Labeling
3.1.1 Apical PM Rip-Off
3.1.2 Basolateral PM Rip-Off
3.1.3 The Following Fixation and Labeling Procedures Are Identical for Both Apical and Basolateral PM Sheets Prepared Above an...
3.2 TEM Imaging and Spatial Analysis
4 Notes
References
Chapter 13: FLIM-FRET Analysis of Ras Nanoclustering and Membrane-Anchorage
1 Introduction
1.1 Targeting Ras Membrane Anchorage
1.2 Isoform-Specific Ras Nanocluster as Signaling Hubs
1.3 Ras-Nanocluster Detection Methods
1.4 Fluorescence Lifetime Imaging Microscopy (FLIM)-FRET to Detect Ras Membrane-Anchorage and Nanoclustering
2 Materials
2.1 Cell Culture
2.2 FLIM Setup
2.3 FLIM-Data Analysis Software
3 Methods
3.1 Cell Transfections and Treatments
3.2 Sample Preparation for FLIM
3.3 FLIM Data Acquisition
3.4 FLIM-FRET Data Analysis
4 Notes
References
Chapter 14: Assessment of Plasma Membrane Fatty Acid Composition and Fluidity Using Imaging Flow Cytometry
1 Introduction
2 Materials
2.1 Delivery of Fatty Acids
2.2 Determining Membrane Order
3 Methods
3.1 Preparation of BSA-FA
3.2 Treatment of Cells
3.3 Trypsinization and Labeling with Di-4-ANEPPDHQ
3.4 Imaging
3.5 Analysis
4 Notes
References
Chapter 15: Spatiotemporal Imaging of Small GTPase Activity Using Conformational Sensors for GTPase Activity (COSGA)
1 Introduction
2 Materials
2.1 Plasmids
2.2 Protein Expression and Purification
2.3 Protein Labeling and Ligation
2.4 Cell Culture and Microinjection
2.5 Confocal Microscopy and FLIM
3 Methods
3.1 Preparation of Recombinant K-Ras Thioester Proteins
3.2 Labeling and Native Chemical Ligation of K-Ras Thioester Proteins
3.3 Imaging of Ras Activity in Live Cells
4 Notes
References
Part IV: Methods in Ras Signaling and Inhibition
Chapter 16: Using BioID to Characterize the RAS Interactome
1 Introduction
2 Materials
3 Methods
3.1 myc-BirARAS Fusion Expression Constructs
3.2 Transfection of myc-BirA-RASG12V Constructs and Biotin Addition
3.3 Lysis of Cultured Cells for Immunoblot Analysis
3.4 Streptavidin Bead Preparation
3.5 Pull Down of Biotinylated Proteins
3.6 Confirming the Biotin-Labeling Activity of the myc-BirA-RASG12V Fusion Protein (s) by Immunoblot
3.7 Confirming Expression of the myc-BirA-RASG12V Fusion Protein (s) by Immunoblot
3.8 Quantitative LC MS/MS Analysis
3.9 Data Analysis
4 Notes
References
Chapter 17: Probing RAS Function with Monobodies
1 Introduction
2 Materials
2.1 Reagents and Cell Lines
2.2 Solutions and Buffers
3 Method
3.1 Isolation of RAS-Specific Mb
3.2 Subcloning Mb Sequences
3.3 Transfection
3.4 Co-immunoprecipitation (co-IP) Analysis of Mbs with RAS
3.5 Inhibition of RAS-Mediated Signaling
3.5.1 Analysis of Mb Effects on EGF-Stimulated ERK Activation
3.5.2 Analysis of Mb Effects on Oncogenic RAS Activation of ERK
3.6 Inhibition of RAS: Effector Interaction
3.6.1 RAS:RAF Interaction
3.6.2 RAS-Induced RAF Dimerization
3.6.3 RAF Activation
3.7 NIH/3T3 Focus Formation Assay
3.8 Chemically Regulated Mb Expression in Human Tumor Cells
3.8.1 Calcium Phosphate Transfections for Lentivirus Production
3.8.2 Generation of Stable Human Cell Lines with Regulated Mb Expression
3.9 Effects of Mb on Proliferation of RAS Mutant Oncogenic Lines
3.10 Effects of Mbs on Anchorage-Independent Growth
3.11 Xenograft Tumor Assays
3.12 Tumor Cell Line Generation
4 Notes
References
Chapter 18: Detection of Endogenous RASSF1A Interacting Proteins
1 Introduction
2 Materials
2.1 Immunoprecipitation
2.2 Immunoblotting
2.3 Sources of Proteins
3 Methods
3.1 Preparation of Lysates
3.2 Immunoprecipitation
3.3 Immunoblotting
4 Notes
References
Chapter 19: Mathematical Modeling to Study KRAS Mutant-Specific Responses to Pathway Inhibition
1 Introduction
2 Materials
2.1 RAS Model
2.2 Data on the Relevant Mutants
2.3 Software to Implement the Model and Hardware for Simulations
3 Methods
3.1 Simulate the Effects of an EGFR Inhibitor Dose Response
3.2 Simulate KRAS Mutant Chimeras to Evaluate the Contribution of Nucleotide Cycling Rate
3.3 Simulate KRAS Mutant Chimeras to Evaluate the Contribution of Impaired NF1 Binding
4 Notes
References
Chapter 20: A Facile Method to Engineer Mutant Kras Alleles in an Isogenic Cell Background
1 Introduction
2 Materials
2.1 Lentiviral Infection Components
2.2 CRISPR/Cas9-Mediated Genome Editing in Mouse Colon Epithelial Cells
2.2.1 Electroporation Components
2.2.2 Clonal Selection Components
3 Methods
3.1 Design and Generation of CRISPR/Cas9 Vectors
3.2 Establishment of Stable Cell Lines Expressing Cas9 and BFP
3.3 Electroporation of Mouse Colon Epithelial Cells with sgRNAs and ssODN
4 Notes
References
Chapter 21: RASless MEFs as a Tool to Study RAS-Dependent and RAS-Independent Functions
1 Introduction
2 Materials
2.1 Stable Transduction of Kraslox Cells via Retro- or Lentiviral Infection
2.2 Colony Assay in RASless MEFs
2.3 Confirmation of Kraslox Allele Excision
2.4 Cell Cycle Analysis in RASless MEFs
3 Methods
3.1 Stable Transduction of Kraslox Cells via Retro- or Lentiviral Infections
3.2 Colony Assay in RASless MEFs
3.3 Confirmation of Kraslox Allele Excision
3.4 Cell Cycle Analysis in RASless MEFs
4 Notes
References
Part V: Methods In Vivo Ras Biology
Chapter 22: Generation of Patient-Derived Colorectal Cancer Organoids for RAS Studies
1 Introduction
2 Materials
2.1 Reagents and Media
2.2 Materials
3 Methods
3.1 Acquisition of Tumor Tissue
3.2 Establishment of Primary Organoid Cultures
3.3 Maintenance and Passaging of Organoid Cultures
3.4 Cryopreservation of Organoids
3.5 Characterization of Tissue Architecture and Marker Expression in CRC Organoids
3.5.1 Preparation of Cell Blocks for Immunohistochemistry
3.5.2 Preparation of Organoid Cultures for Immunofluorescence Analysis
4 Notes
References
Chapter 23: Ras GEF Mouse Models for the Analysis of Ras Biology and Signaling
1 Introduction
1.1 Model Generation
1.2 Phenotypic Analyses of Ras GEF Mouse Models
1.3 Hematological Studies on Ras GEF Animals
1.4 Analyzing the CNS and Sensory Phenotypes of Ras GEF KO Mice
1.5 Ras GEF Animal Models for the Study of Human Illness
1.6 Preparing and Analyzing Cell and Organ Cultures from Ras GEF Mouse Models
2 Materials
2.1 Gene Targeting
2.2 Phenotypic Analyses of Ras GEF Animal Models
2.2.1 Morphological/Structural Analyses
2.2.2 Analysis of Hematological Phenotypes by Flow Cytometry
2.2.3 Bone Marrow Transplantation
2.2.4 Analyses of Behavioral Phenotypes
2.2.5 Electrophysiological Studies
2.2.6 Sensory Perception Studies
Electroretinogram Recordings (ERG)
Auditory Brainstem (ABR) Measurements
Structural Analysis of the Eye
2.2.7 Studies on Adult Neurogenesis
2.2.8 Ischemia Assays
2.2.9 Dyskinesia Assays and In Vivo Knockdown of RasGRF
Dyskinesia Induction
In Vivo RasGRF Knockdown
2.2.10 In Vivo Brain Microdialysis
2.2.11 Skin Carcinogenesis in GEF Animal Models
2.2.12 Echocardiographic Analysis
2.3 Isolation and Culture of Cell Lines from Mice
2.3.1 Culture of Mouse Embryonic Fibroblast (MEF)
2.3.2 Culture of Cells from Hematopoietic Lineage
T-Cell, B-Cell Isolation
Mast Cell Isolation and Culture
Neutrophil Isolation
2.3.3 Culture of Primary Neurons
2.3.4 Keratinocyte Cultures
2.3.5 Neural Stem Cell Cultures and Neurosphere Generation
2.3.6 Pancreatic Islet Cultures
2.3.7 Aortic Ring Explants
3 Methods
3.1 Gene Targeting
3.1.1 Modification of the Genomic DNA
3.1.2 Chimera Generation
3.2 Phenotypic Analyses of Ras GEF Animal Models
3.2.1 Morphological/Structural Analyses
3.2.2 Analysis of Hematological Phenotypes: Flow Cytometry
3.2.3 Analysis of Hematological Phenotypes: Bone Marrow Transplantation
3.2.4 Analyses of Central Nervous System and Sensory Phenotypes
3.2.5 Electrophysiological Studies
3.2.6 Sensory Perception Studies: Electroretinograms
3.2.7 Sensory Perception Studies: Auditory Brainstem (ABR)
3.2.8 Sensory Perception Studies: Structural Analysis of the Eye by Immunohistochemistry
3.2.9 Sensory Perception Studies: Structural Analysis of the Eye by Ultrastructure Studies
3.2.10 Studies on Adult Neurogenesis
3.2.11 Ischemia Assays
3.2.12 Dyskinesia Induction
3.2.13 In Vivo RasGRF Knockdown
3.2.14 In Vivo Microdialysis
3.2.15 Skin Carcinogenesis in GEF Animal Models
3.2.16 Echocardiographic Analysis
3.3 Isolation and Culture of Cell Lines from Mice
3.3.1 Culture of Mouse Embryonic Fibroblasts (MEFs)
3.3.2 Culture of Cells from Hematopoietic Lineage
T-Cell, B-Cell Isolation
Mast Cell Isolation and Culture
Neutrophil Isolation
3.3.3 Culture of Primary Neurons
3.3.4 Keratinocyte Cultures
3.3.5 Neural Stem Cell Cultures and Neurosphere Generation
3.3.6 Pancreatic Islet Cultures
3.3.7 Aortic Ring Explants
4 Notes
References
Chapter 24: Studying Metabolic Abnormalities in the Costello Syndrome HRAS G12V Mouse Model: Isolation of Mouse Embryonic Fibr...
1 Introduction
2 Materials
2.1 Mouse Embryonic Fibroblast (MEF) Isolation
2.1.1 Mice
2.1.2 Media
2.2 Adipocyte Differentiation Reagents
2.2.1 MEF Differentiation Media and Lipid Accumulation Staining
2.2.2 Total RNA Isolation and qPCR
3 Methods
3.1 MEF Isolation
3.2 Subculturing Primary MEFs
3.3 Freezing MEFs
3.4 Thawing MEFs
3.5 MEF Differentiation into Adipocytes
3.5.1 Differentiation
3.5.2 Oil Red O Staining
3.5.3 qPCR Analysis of Adipocyte Differentiation Markers
4 Notes
References
Chapter 25: Dissecting Oncogenic RAS Signaling in Melanoma Development in Genetically Engineered Zebrafish Models
1 Introduction
2 Materials
2.1 Generation of the Construct for Microinjection
2.2 Microinjection of the Plasmid
2.3 Screening and Raising Zebrafish Larvae
3 Methods
3.1 Generation of the Construct for Microinjection
3.2 Microinjection of the Plasmid
3.3 Screening and Raising Zebrafish Larvae
4 Notes
References
Chapter 26: Ras, Ral, and Rap1 in C. elegans
1 Introduction
2 Materials
3 Methods
3.1 Standard Growth and Assays
4 Notes
References
Index