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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 GTPases: 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 Rho GTPases: Methods and Protocols 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 GTPases: 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.
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 Elias and Vladimir Klimes
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 Sebastien 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 Desiree 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
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
