Ras Activity and Signaling: Methods and Protocols

ISBN-13: 9781071611920 / Angielski / Miękka / 2022

Rubio, Ignacio

Opis

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.

Spis treści

Part I: Generic Ras Biology

1. Ras Variant Biology and Contributions to Human Disease

Ian A. Prior

 

2. Regulation of the Small GTPase Ras and its Relevance to Human Disease

Kayla R. Kulhanek, Jeroen P. Roose, and Ignacio Rubio

 

Part II: Methods in Ras Biochemistry

3. Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry

Lauren M. Adams, Caroline J. DeHart, and Neil L. Kelleher

 

4. Absolute Quantitation of GTPase Protein Abundance

Fiona E. Hood, Yasmina M. Sahraoui, Rosalind E. Jenkins, and Ian A. Prior

 

5. Validation of Isoform- and Mutation-specific RAS Antibodies

Andrew M. Waters and Channing J. Der

 

6. Production and Membrane Binding of N-terminally Acetylated, C-terminally Farnesylated and Carboxymethylated KRAS4b

Simon Messing, Constance Agamasu, Matt Drew, Caroline DeHart, Andrew G. Stephen,  and William K. Gillette

 

7. Active GTPase Pulldown Protocol

Martin J. Baker and Ignacio Rubio

 

8. Methods to Monitor Ras Activation State

Kari Kopra and Harri Härmä

 

9. NMR Detection Methods for Profiling RAS Nucleotide Cycling

Ryan C. Killoran and Matthew J. Smith

 

Part III: Methods in Ras Processing, Trafficking, and Localization

10. Ras Diffusion and Interactions with the Plasma Membrane Measured by FRAP Variations

Orit Gutman, Marcelo Ehrlich, and Yoav I. Henis

 

11. Understanding Ras Spatial Cycles through Reaction-Diffusion Simulations

Malte Schmick and Philippe I. H. Bastiaens

 

12. Super-Resolution Imaging and Spatial Analysis of RAS on Intact Plasma Membrane Sheets

Yong Zhou and John F. Hancock

 

13. FLIM-FRET Analysis of Ras Nanoclustering and Membrane-Anchorage

Hanna Parkkola, Farid Ahmad Siddiqui, Christina Oetken-Lindholm, and Daniel Abankwa

 

14. Assessment of Plasma Membrane Fatty Acid Composition and Fluidity Using Imaging Flow Cytometry

Natividad R. Fuentes, Michael L. Salinas, Xiaoli Wang, Yang-Yi Fan, and Robert S. Chapkin

 

15. Spatiotemporal Imaging of Small GTPase Activity using Conformational Sensors for GTPase Activity (COSGA)

Yao-Wen Wu

 

Part IV: Methods in Ras Signaling and Inhibition

16. Using BioID to Characterize the RAS Interactome

Hema Adhikari and Christopher M. Counter

 

17. Probing RAS function with Monobodies

Imran Khan and John P. O’Bryan

 

18. Detection of Endogenous RASSF1A Interacting Proteins

Howard Donninger, Desmond Harrell-Stewart, and Geoffrey J. Clark

 

19. Mathematical Modeling to Study KRAS Mutant-Specific Responses to Pathway Inhibition

Edward C. Stites

 

20. A Facile Method to Engineer Mutant Kras Alleles in an Isogenic Cell Background

Konstantin Budagyan and Jonathan Chernoff

 

21. RASless MEFs as a Tool to Study RAS-dependent and -independent Functions

Carmen G. Lechuga, Marina Salmón, Guillem Paniagua, Carmen Guerra, Mariano Barbacid, Matthias Drosten

 

Part V: Methods in vivo Ras Biology

22. Generation of Patient-derived Colorectal Cancer Organoids for RAS Studies

Dirk Schumacher, Joseph L. Regan, Dorothea Przybilla, and Reinhold Schäfer

 

23. Ras-GEF Mouse Models for the Analysis of Ras Biology and Signaling

Alberto Fernández-Medarde and Eugenio Santos

 

24. Studying Metabolic Abnormalities in the Costello Syndrome HRAS G12V Mouse Model: Isolation of Mouse Embryonic Fibroblasts and Their In Vitro Adipocyte Differentiation

Miray Fidan, Saravanakkumar Chennappan, and Ion Cristian Cirstea

 

25. Dissecting Oncogenic RAS Signalling in Melanoma Development in Genetically Engineered Zebrafish Models

Andrew P. Badrock and Adam Hurlstone

 

26. Ras, Ral, and Rap1 in C. elegans

Neal R. Rasmussen and David J. Reiner