Control of Actomyosin by Microtubules

University Of Wisconsin Madison, Madison WI

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Abstract

DESCRIPTION (Verbatim from the applicant's abstract): Microtubule-based control of the actin cytoskeleton is critically important for such fundamental cell processes as cell locomotion and cell division. Given the central importance of cell locomotion and cell division to both normal and pathological human physiology, understanding the mechanisms by which microtubules control actomyosin is of clear biological and clinical importance. And yet, in spite of three decades of intense interest in this question, microtubule-based control of actomyosin remains mysterious. The experiments described in this proposal are designed to exploit model systems that simplify analysis of microtubule-actomyosin interactions in vivo and in vitro. At the cellular level, two broad classes of microtubule-dependent actomyosin regulation will be investigated: "direct" control, by physical interaction and transport and "indirect" control by modulation of regulators of the actin cytoskeleton and directed membrane insertion. At the molecular level, the roles of several specific proteins in microtubule-dependent regulation of actomyosin will be investigated: the microtubule-based motors, kinesins and cytoplasmic dynein; the unconventional myosins, myosins-5 and -10; and the small GTPases rac and rho. Four distinct model systems will be employed to varying degrees: ectopic contractile ring assembly in Xenopus oocytes; Xenopus embryonic cytokinesis, inducible cortical flow ii Xenopus oocytes, and cell free Xenopus egg extracts. These model systems will be subjected to analysis using combination of high resolution microscopy, specific protein imaging and manipulation, biochemical fractionation, and recombinant DNA approaches. The Xenopus model systems we have developed for analysis of microtubule-actomyosin interactions are particularly well-suited to this kind of integrated approach. Thus, the proposed work will provide important and novel insights into the means by which microtubules control actomyosin in vivo.