Rho Gtpases: Methods and Protocols

ISBN-13: 9781617794414 / Angielski / Twarda / 2011 / 407 str.

Francisco Rivero

Opis

Although initially described as major regulators of cytoskeletal remodeling, Rho GTPases have been implicated in the establishment of polarity, endocytosis, vesicle trafficking, morphogenesis, cytokinesis, transcriptional activation, cell cycle progression, and apoptosis, to mention a few. In addition, Rho GTPases have acquired medical relevance because of their participation in tumorigenesis and metastasis, in cardiovascular conditions, and as targets of infectious agents. The field has broadened even more with the contribution of studies in model organisms (plants, amoebas, fungi, invertebrates), each adding a particular view to the complexity of the family and vastly enriching our perception of these important signaling components. Divided into five convenient sections, Rho GTPase: Methods and Protocols provides an historical overview of the field and an account of the phylogenetics of the Rho family, general biochemical methods, and functional assays that allow monitoring the consequences of manipulating Rho GTPases in a variety of contexts. Additionally, the volume devotes a section to advanced imaging methods and to recently developed high throughput methods, closing with techniques specifically designed for studies in selected non-mammalian model organisms. Written in the 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 protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Rho GTPase: Methods and Protocols provides techniques that are standard for researchers in the field but also includes protocols for those who, being already familiar with some of the techniques, wish to explore additional aspects.

Spis treści

Part I. History and Phylogenesis

1. Historical Overview of Rho GTPases   

                Anne J. Ridley

 

2. Rho GTPases: Deciphering the Evolutionary History of a Complex Protein Family

                Marek Eliáš and Vladimír Klimeš

 

Part II. Biochemistry of Rho GTPases

 

3. Biochemical Assays to Characterize Rho GTPases

                Mamta Jaiswal, Badri N. Dubey, Katja T. Koessmeier, Lothar Gremer, and Mohammad R. Ahmadian

 

4. How to Analyze Bacterial Toxins Targeting Rho GTPases

                Heike Bielek and Gudula Schmidt

 

5. Assessing Ubiquitylation of Rho GTPases in Mammalian Cells

                Anne Doye, Amel Mettouchi, and Emmanuel Lemichez

 

6. Posttranslational Lipid Modification of Rho Family Small GTPases

                Natalia Mitin, Patrick J. Roberts Emily J. Chenette, and Channing J. Der

 

7. Analysis of the Role of RhoGDI1 and Isoprenylation in the Degradation of Rho GTPases

                Etienne Boulter and Rafael Garcia-Mata

 

8. A Quantitative Fluorometric Approach for Measuring the Interaction of RhoGDI with Membranes and Rho GTPases

                Jared Johnson, Richard A. Cerione, and Jon W. Erickson

 

Part III. Functional assays

 

9. Rho GTPases and Cancer Cell Transendothelial Migration

                Nicolas Reymond, Philippe Riou, and Anne J. Ridley

 

10. Quantitative and Robust Assay to Measure Cell-cell Contact Assembly and Maintenance

                Sébastien Nola, Jennifer C. Erasmus, and Vania M. M. Braga

 

11. Rho GTPase Knockout Induction in Primary Keratinocytes from Adult Mice

                Esben Pedersen, Astrid Basse, Tine Lefever, Karine Peyrollier, and Cord Brakebusch

 

12. Rho GTPase Techniques in Osteoclastogenesis

                Roland Leung and Michael Glogauer

 

13. Assessment of Rho GTPase Signaling During Neurite Outgrowth

                Daniel Feltrin and Olivier Pertz

 

14. Assesment of the Role for Rho Family GTPases in NADPH Oxidase Activation

                Kei Miyano and Hideki Sumimoto

 

Part IV. Imaging and high throughput methods

 

15. Multiplex Imaging of Rho GTPase Activities in Living Cells

                Désirée Spiering and Louis Hodgson

 

16. FRET-Based Imaging of Rac and Cdc42 Activation During Fc-Receptor Mediated Phagocytosis in Macrophages

                Adam D. Hoppe

 

17. High Throughput Flow Cytometry Bead-Based Multiplex Assay for Identification of Rho GTPase Inhibitors

                Zurab Surviladze Susan M. Young, and Larry A. Sklar

 

18. Functional Analysis of RhoGTPase Activation and Inhibition in a Bead-Based Miniaturized Format

                Michael Schmohl, Stefanie Rimmele, Peter Gierschik, Thomas O. Joos, and Nicole Schneiderhan-Marra

 

19. Use of Phage Display For the Identification of Molecular Sensors Specific for Activated Rho

                Patrick Chinestra, Isabelle Lajoie-Mazenc, Jean-Charles Faye, and Gilles Favre

 

20. Identification of New Interacting Partners for Atypical Rho-GTPases: a SILAC-Based Approach

                Laura Montani, Ana Filipa Domingues, and João Bettencourt Relvas

 

Part V. Rho GTPases in Non-Mammalian Model Organisms

 

21. Using Zebrafish for Studying Rho GTPases Signaling in Vivo

                Shizhen Zhu and Boon Chuan Low

 

22. Analysis of Rho GTPase Function in Axon Pathfinding Using Caenorhabditis elegans

                Jamie K. Alan and Erik A. Lundquist

 

23. Protocol for ex vivo Incubation of Drosophila Primary Post-Embryonic Haemocytes for Real-Time Analyses

                Christopher J. Sampson and Michael J. Williams

 

24. Analysis of Rho GTPase Activation in Saccharomyces cerevisiae

                Gary Eitzen and Michael R. Logan

 

25. Assaying Rho GTPase-Dependent Processes in Dictyostelium discoideum

                Huajiang Xiong and Francisco Rivero

 

26. FRAP-Based Analysis of Rho GTPase-dependent Polar Exocytosis in Pollen Tubes

                An Yan and Zhenbiao Yang