Beschreibung
In the last 10 years researchers have firmly established key roles for R- related GTPases in almost every aspect of cell biology. In the 1980s the pro- oncogene Ras itself was the focus of interest, though in the 1990s this shifted to the increasing variety of Ras-related proteins. In this new decade much yet needs to be done to establish the role for all the small GTPases now uncovered by the human genome project. In particular, these GTPases need to be und- stood in the appropriate biochemical and cellular contexts. In the process of trying to uncover the role of these versatile proteins, a variety of novel te- niques and methodologies has been developed. These now enable investi- tors to move easily within a diversity of fields ranging from structural studies to real-time in vivo analysis of a GTPase. In recognition of the need for access to key background methodologies, GTPase Protocols: The Ras Superfamily is devoted to techniques that are pr- ently widely used and that will continue to be the standard for researchers worldwide. Each chapter is aimed at supplying detailed methodologies to allow reproduction in any laboratory, while also providing the general pr- ciples on which the methods are based. Some of the techniques grouped in the first section apply broadly to small GTPases, whereas others in Part II are more applicable within each GTPase subfamily.
Inhaltsverzeichnis
Part I. General Protocols The GTPase Cycle: How Dominant Inhibitory Mutants Block the Biological Functions of Small GTPases Edward J. Manser Preparation of GTPases for Structural and Biophysical Analysis Susan J. M. Smith and Katrin Rittinger Using cDNA-Representational Difference Analysis (cDNA-RDA) in Combination with Microarrays to Identify Rac Regulated Genes Arndt A. P. Schmitz, Robert Lucito, and Linda Van Aelst Fluorescence Methods in the Study of Small GTP-Binding Proteins Mohammad Reza Ahmadian, Alfred Wittinghofer, and Christian Herrmann Part II. Specific Methods Ras Family GTPases Ras and Rac as Activators of Reactive Oxygen Species (ROS) Herbert Archer and Dafna Bar-Sagi Interfering with Ras Signaling Using Membrane-Permeable Peptides or Drugs Hiroshi Maruta, Hong He, and Thao Nheu A Ras-Based Module to Generate 32P-Labeled Fusion Proteins for Blot Overlays Zhoushen Zhao and Louis Lim Rho Family GTPases Determination of the Activity of Rho-Like GTPases in Cells Angeliki Malliri, Jean P. ten Klooster, Cristina Olivo, and John G. Collard Introduction of Dominant Inhibitory Proteins Directed Against ROK and MRCK Kinases Ivan Tan, Louis Lim, and Thomas Leung Effects of Rho Family GTPases on Cell-Cell Adhesion Masaki Fukata, Masato Nakagawa, Shinya Kuroda, and Kozo Kaibuchi Cell Motility and Invasion Assays Jackie Banyard and Marc Symons Rab Family GTPases Isolation of Regulatory Proteins for the Rab3 Subfamily GTPases Hiroyuki Nakanishi, Takuya Sasaki, and Yoshimi Takai Analysis and Preparation of Stable Complexes between Rab GTPases Rab Escort Protein, and Rab Geranylgeranyl Transferase Jose B. Pereira-Leal, Anita Q. Gomes, and Miguel C. Seabra Arf Family GTPases Preparation of Myristoylated Arf1 and Arf6 Proteins Paul A. Randazzo and Henry M. Fales ARF-Directed Guanine-Nucleotide-Exchange (GEP) Proteins Gustavo Pacheco-Rodriguez, Joel Moss, and Martha Vaughan Arf6 and its Role in Cytoskeletal Modulation Julie G. Donaldson ARF GTPase-Activating Protein 1 Irit Huber, Edna Cukierman, Miriam Rotman, and Dan Cassel Ran GTPases The Use of Permeabilized Cell Systems to Study Nuclear Transport Amy M. Brownawell, James M. Holaska, Ian G. Macara, and Bryce M. Paschal Analysis of Nuclear Protein Import and Export In Vitro Using Fluorescent Cargoes Ralph H. Kehlenbach and Larry Gerace Characterization of the Effects of RanGTP on the Microtubule Cytoskeleton Sofia B. Lizarraga, Yixian Zheng, and Andrew R. Wilde Index
