Synopses & Reviews
It is now clear that kinesin-like-proteins (KLP), generally thought to be responsible for the transport of cellular cargoes, are involved in many different cellular processes now being widely investigated. In Kinesin Protocols, Isabelle Vernos and a panel of hands-on experts present their most productive and reproducible techniques for the identification, purification, and characterization of the kinesin superfamily of microtubule-dependent motors. The methods range from the most basic to the most sophisticated and include step-by-step instructions and extensive cautionary notes to ensure experimental success. Among the approaches discussed are methods to express and purify kinesins in different systems, to characterize microtubule-enhanced ATPase activity and motility properties, and to test microtubule destabilizing activity. Detailed examples of how to address functional studies are also presented, along with some very new methods for studying the role of KLP in the organization of microtubules in three dimensions. There are also advanced methods for the study of kinesins at the structural level. Comprehensive and highly practical, Kinesin Protocols makes available all the key basic and cutting-edge methods needed successfully to study the multifaceted world of kinesin-like proteins and to explore their many functions.
"This is a concise but comprehensive book that would be a valuable reference for any researcher who wishes to study this important cytoplasmic motor protein. It would be a valuable book to have in the laboratory as well as a useful reference book in a life sciences library. "-Doody's Health Sciences Book Review Journal
By the end of the 1980s only two microtubule-dependent motors, the plus end-directed kinesin and the minus end-directed cytoplasmic dynein, had been identified. At the time, these two motors seemed almost sufficient to explain directional motility events on polar microtubule tracks in the cell. No- theless, shortly after, the tip of the iceberg began to emerge with the identi- cation of proteins containing in their sequences a domain found in kinesin. This domain, called the "motor domain," conferred on these proteins the essential property of moving on microtubules, using the energy derived from ATP hydro- sis. Since then, the identification of new proteins belonging to the kinesin superfamily of microtubule-dependent motors has gone at such a pace that nowadays more than 200 entries with motor domain sequences are deposited in the database. Kinesin family members are found in all eukaryotic org- isms tested. They present a wide range of domain organizations with a motor domain located at different positions in the molecule. Their motility prop- ties are also variable in directionality, velocity, and such other characteristics as bundling activity and processivity. Finally, and most important, they p- ticipate in a multitude of cellular functions. Our understanding of many cel- lar events, such as mitotic spindle assembly and neuronal transport, to cite only two, has progressed substantially in the last few years thanks to the id- tification of these motors.
Isabelle Vernos and a panel of hands-on experts present their most productive and reproducible techniques for the identification, purification, and characterization of the kinesin superfamily of microtubule-dependent motors. The methods range from the most basic to the most sophisticated and include step-by-step instructions and extensive cautionary notes to ensure experimental success. Among the approaches considered are methods to express and purify kinesins in different systems, to characterize microtubule-enhanced ATPase activity and motility properties, and to test microtubule destabilizing activity. Comprehensive and highly practical, Kinesin Protocols makes available all the key basic and cutting-edge methods needed to successfully study the multifaceted world of kinesin-like proteins and to explore their many functions.
Table of Contents
Purification of Kinesin from the Brain, Sergei A. Kuznetsov and Vladimir I. Gelfand. RT-PCR for the Identification of Developmentally Regulated Novel Members of the Kinesin-like Superfamily, Niovi Santama. Expression Cloning with Pan Kinesin Antibodies, Laura M. Ginkel and Linda Wordeman. Expression of Kinesin in Escherichia coli, Maryanne F. Stock and David D. Hackney. Plasmids for Expression of Chimeric and Truncated Kinesin Proteins, Kimberly W. Waligora and Sharyn A. Endow. Preparation of Recombinant Kinesin Superfamily Proteins Using the Baculovirus System, Nobutaka Hirokawa and Yasuko Noda. Assays for Kinesin Microtubule-Stimulated ATPase Activity, David D. Hackney and Wei Jiang. An Improved Microscope for Bead and Surface-Based Motility Assays, Nick Carter and Rob Cross. Use of Photonic Force Microscopy to Study Single-Motor-Molecule Mechanics, Sylvia Jeney, Ernst-Ludwig Florin, and J. K. Heinrich Hörber. Assays for Microtubule-Destabilizing Kinesins, Arshad Desai and Claire E. Walczak. Green Fluorescent Protein as a Tag for Molecular Motor Proteins, Sharyn A. Endow. In Vitro Reconstitution of Endosome Motility Along Microtubules, Erik Nielsen, Fedor Severin, Anthony A. Hyman, and Marino Zerial. Approaches to Study Interactions Between Kinesin Motors and Membranes, Gerardo Morfini, Ming-Ying Tsai, Györgyi Szebenyi, and Scott T. Brady. Microinjection Methods for Analyzing the Functions of Kinesins in Early Embryos, Robert L. Morris, Heather M. Brown, Brent D. Wright, David J. Sharp, William Sullivan, and Jonathan M. Scholey. The Use of Dominant Negative Mutants to Study the Function of Mitotic Motors in the In Vitro Spindle Assembly Assay in Xenopus Egg Extracts, Haralabia Boleti, Eric Karsenti, and Isabelle Vernos. A Dominant Negative Approach for Functional Studies of the Kinesin II Complex, Vladimir I. Gelfand, Nathalie Le Bot, M. Carolina Tuma, and Isabelle Vernos. Identification of Kinesin-Associated Proteins, Lisa C. Lindesmith, Janardan Kumar, and Michael P. Sheetz. Assaying Spatial Organization of Microtubules by Kinesin Motors, François Nédélec and Thomas Surrey. Crystallization of Kinesin, Manfred Thormählen, Jens Müller, and Eckhard Mandelkow. Structural Analysis of the Microtubule-Kinesin Complex by Cryo-Electron Microscopy, Fabienne Beuron and Andreas Hoenger. Index.