Kinesin Protocols

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.

Includes supplementary material: sn.pub/extras

Texte du rabat
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.

Contenu
Purification of Kinesin from the Brain.- RT-PCR for the Identification of Developmentally Regulated Novel Members of the Kinesin-like Superfamily.- Expression Cloning with Pan Kinesin Antibodies.- Expression of Kinesin in Escherichia coli.- Plasmids for Expression of Chimeric and Truncated Kinesin Proteins.- Preparation of Recombinant Kinesin Superfamily Proteins Using the Baculovirus System.- Assays for Kinesin Microtubule-Stimulated AT Pase Activity.- An Improved Microscope for Bead and Surface-Based Motility Assays.- Use of Photonic Force Microscopy to Study Single-Motor-Molecule Mechanics.- Assays for Microtubule-Destabilizing Kinesins.- Green Fluorescent Protein as a Tag for Molecular Motor Proteins.- In Vitro Reconstitution of Endosome Motility Along Microtubules.- Approaches to Study Interactions Between Kinesin Motors and Membranes.- Microinjection Methods for Analyzing the Functions of Kinesins in Early Embryos.- The Use of Dominant Negative Mutants to Study the Function of Mitotic Motors in the In Vitro Spindle Assembly Assay in Xenopus Egg Extracts.- A Dominant Negative Approach for Functional Studies of the Kinesin II Complex.- Identification of Kinesin-Associated Proteins.- Assaying Spatial Organization of Microtubules by Kinesin Motors.- Crystallization of Kinesin.- Structural Analysis of the MicrotubuleKinesin Complex by Cryo-Electron Microscopy.