Microtubules are highly dynamic structural filaments that spatially and temporally organize the cytoplasm of nearly all Eukaryotic cells. To adopt different spatial arrangements in functionally diverse and morphologically disparate cells, microtubule polymerization dynamics are subject to poorly understood physiological regulation based on external and internal cellular cues. In this study, we have designed a microfluidic approach capable of isolating biologically relevant volumes of Xenopus laevis cell-free extract. This approach allowed us to identify and characterize the relationship between the dynamic instability factor, microtubule polymerization rate, and the number of actively growing microtubule plus-ends in the cytoplasm. One function of this relationship might be to determine steady-state mass of the microtubule assembly, or to prevent sub-regions of microtubules from depleting limited precursor molecules. Our studies show a length scale for this relationship, with microtubules only a few microns apart exerting negative feedback on one another, which provides the first direct experimental evidence for a local component-limitation mechanism in regulating microtubule polymerization dynamics. Lastly, we have identified a role for component limitation in determining the microtubule mass of the interphase aster, as the addition of tubulin to our cell-free extracts was sufficient to drive changes in microtubule mass and microtubule polymerization rates.

There continues to be intense interest in the microtubule cytoskeleton; the assembly, structure and regulation of microtubules; and the numerous motors and accessory proteins that control cell cycle, dynamics, organization and transport. The field continues to grow and explore new aspects of these issues driven immensely by developments in optical imaging and tracking techniques. This volume (complemented by the forthcoming companion volume by Cassimeris and Tran) brings together current research and protocols in the field of microtoubules in vitro and will serve as a valuable tool for cell biologists, biophysicists and pharmacologists who study the microtubule cytoskeleton, as well as for researchers in the biomedical and biotechnology communities with interest in developing drugs that target microtubules, MAPS and motors. - Chapters reflect both experimental procedures and new developments in the field of microtubule in vitro research - Combines classical approaches and modern technologies - Presents easy-to-use protocols and thorough background information, compiled by leaders in the field

This volume presents the first comprehensive treatment of the wide range of uses for Xenopus laevis oocytes and embryos in cell and molecular biology. Each chapter includes background information, experimental protocols, and suggested applications. An extensive array of techniques is featured. The authors are experienced researchers who have written chapters that will be useful to both experienced researchers and to those new to Xenopus as an experimental system. Full-color plates and diagrams enhance the educational value of this book, which provides a valuable permanent resource for all laboratories that use Xenopus.* Features approximately twenty full-color plates illustrating experimental techniques and results and depicting embryonic development* Provides complete coverage of Xenopus laevis as an experimental system including* Embryonic development, genetics, and laboratory care* Up-to-date protocols for experimental techniques using oocytes and embryos* General information listing recipes, suppliers, sequences, codons, and clones

Microtubules are at the heart of cellular self-organization, and their dynamic nature allows them to explore the intracellular space and mediate the transport of cargoes from the nucleus to the outer edges of the cell and back. In Microtubule Dynamics: Methods and Protocols, experts in the field provide an up-to-date collection of methods and approaches that are used to investigate microtubule dynamics in vitro and in cells. Beginning with the question of how to analyze microtubule dynamics, the volume continues with detailed descriptions of how to isolate tubulin from different sources and with different posttranslational modifications, methods used to study microtubule dynamics and microtubule interactions in vitro, techniques to investigate the ultrastructure of microtubules and associated proteins, assays to study microtubule nucleation, turnover, and force production in cells, as well as approaches to isolate novel microtubule-associated proteins and their interacting proteins. Written in the highly successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Definitive and practical, Microtubule Dynamics: Methods and Protocols provides the key protocols needed by novices and experts on how to perform a broad range of well-established and newly-emerging techniques in this vital field.