Rab GTPases, Softcover reprint of the original 1st ed. 2015
Methods and Protocols
Methods in Molecular Biology Series, Vol. 1298

This volume covers the latest technological advances in the characterization of the biosynthesis and functions of Rab GTPases and their regulation by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). The book consists of 28 chapters and starts with an overview of the Rab GTPase family. The next few chapters describe systematic approaches to the identification and classification of Rabs and Rab GAPs, as well as the detection of Rab isoprenylation and membrane distribution. The last few chapters examine the biochemical and functional properties of individual Rabs in the order of exocytic, recycling and endocytic Rabs. Written in the highly successful Methods of Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols and key tips on troubleshooting and avoiding known pitfalls.

Instructive and practical, Rab GTPases: Methods and Protocols approaches each topic with great detail and is a valuable resource for researchers and students interested in the field of Rab GTPases.

1. Rab Family of GTPases
Guangpu Li and M. Caleb Marlin

2. Bioinformatics Approaches to Identifying and Classifying Rab Proteins
Yoan Diekmann and Jose B. Pereira-Leal

3. Rab-NANOPS: FRET-Biosensors for Rab Membrane Nanoclustering and Prenylation Detection in Mammalian Cells
Arafath Kaja Najumudeen, Camilo Guzmán, Itziar M. D. Posada, and Daniel Abankwa

4. High Throughput Assay for Profiling the Substrate Specificity of Rab GTPase-Activating Proteins
Ashwini K. Mishra and David G. Lambright

5. Measuring Rab GTPase-Activating Protein (GAP) Activity in Live Cells and Extracts
Ryan M. Nottingham and Suzanne R. Pfeffer

6. Analysis of the Interactions Between Rab GTPases and Class V Myosins
Andrew J. Lindsay, Stéphanie Miserey-Lenkei, and Bruno Goud

7. Assaying the Interaction of the Rab Guanine Nucleotide Exchange Factor Rabin 8 by Rab11
Danièle Stalder and Peter J. Novick

8. Kinetic Activation of Rab8 Guanine Nucleotide Exchange Factor Rabin8 by Rab11      
Shanshan Feng, Bin Wu, Johan Perånen, and Wei Guo

9. Ypt1 and TRAPP Interactions: Optimization of Multi-Color Bimolecular Fluorescence Complementation in Yeast
Zhanna Lipatova, Jane J. Kim, and Nava Segev

10. Identifying a Rab Effector on the Macro autophagy Pathway
Juan Wang, Serena Cervantes, Saralin Davis, and Susan Ferro-Novick

11. Functional Analysis of Rab27 and its Effector Slp-2a in Renal Epithelial Cells
Takao Yasuda, Paulina Mrozowska, and Mitsunori Fukuda

12. Small GTPases in Acrosomal Exocytosis
Matias A. Bustos, Ornella Lucchesi, Maria Celeste Ruete, Luis S. Mayorga, and Claudia N. Tomes

13. Rab Antibody Characterization: Comparison of Rab14 Antibodies
Andrew J. Lindsay and Mary W. McCaffrey

14. Selective Visualization of GLUT4 Storage Vesicles and Associated Rab Proteins Using IRAP-pHluorin
Yu Chen and Jennifer Lippincott-Schwartz

15. 3D Time-Lapse Analysis of Rab11/FIP5 Complex: Spatiotemporal Dynamics During Apical Lumen Formation
Anthony Mangan and Rytis Prekeris

16. In Vitro and In Vivo Characterization of the Rab11-GAP Activity of Drosophila Evi5
Carl Laflamme and Gregory Emery

17. Characterization of the Role Rab25 in Energy Metabolism and Cancer Using Extracellular Flux Analysis and Material Balance
Shreya Mitra, Jennifer Molina, Gordon B. Mills, and Jennifer B. Dennison

18. Measurement of Rab35 Activity by the GTP-Rab35 Trapper RBD35
Hotka Kobayashi, Kan Etoh, Soujiro Marubash, Norihiko Ohbayashi, and Mitsunori Fukuda

19. Analysis of Connecdenn 1-3 (DENN1A-C) GEF Activity for Rab35
Patrick D. Allaire, Peter S. McPherson, and Brigitte Ritter

20. Assay of Rab17 and its Guanine Nucleotide Exchange Factor Rabex-5 in Dendrites of Hippocampal Neurons
Yasunori Mori and Mitsunori Fukuda

21. Methods for Analysis of AP-3/Rabin4’ in Regulation of Lysosome Distribution
Viorica Ivan and Peter van der Sluijs

22. Determination of Rab5 Activity in the Cell by Effector Pull-Down Assay
Yaoyao Qi, Zhimin Liang, Zonghua Wang, Guodong Lu, and Guangpu Li

23. Identification of the Rab5 Binding Site in p110b - Assays for PI3Kb Binding to Rab5
Rachel S. Salamon, Hashem A. Dbouk, Denise Collado, Jaclyn Lopiccolo, Anne R. Bresnick, and Jonathan M. Backer

24. Role of the Rab5 Guanine Nucleotide Exchange Factor, Rme-6, in the Regulation of Clathrin-Coated Vesicle Uncoating
Elizabeth Smythe

25. Differential Effects of Overexpression of Rab5 and Rab22 on Autophagy in PC12 Cells With or Without NGF
M. Caleb Marlin and Guangpu Li

26. Determining the Role of Rab7 in Constitutive and Ligand-Mediated Epidermal Growth Factor Receptor Endocytic Trafficking Using Single Cell Assays
Brian P. Ceresa

27. Visualizing Directional Rab7 and TrkA Co-Trafficking in Axons by pTIRF Microscopy
Kai Zhang, Praveen D. Chowdary, and Bianxiao Cui

28. Quantitative Bead-Based Flow Cytometry for Assaying Rab7 GTPase Interaction with the Rab-Interacting Lysosomal Protein (RILP) Effector Protein           
Jacob O. Agola, Daniel Sivalingam, Daniel F. Cimino, Peter C. Simons, Tione Buranda, Larry A. Sklar, and Angela Wandinger-Ness

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts