Dissecting Mechanics of Contractile Ring Assembly

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Assembly of an actomyosin contractile ring that constricts the cell around the spindle equator is essential for cell cleavage. The molecular basis underlying the organization and constriction of the contractile ring is well documented, yet the mechanics of how actin filaments redistribute and assemble into the ring remains largely unknown. Accumulating evidence suggests that spindle microtubules may mediate equatorial distribution of actin filaments. However, it is unclear how microtubules interact with actin filaments in living cells. The long-term goal is to understand the mechanics of the contractile ring assembly by focusing on the interaction of microtubules with actin filaments and the incorporation of filaments into the ring. The hypothesis is that radiating spindle microtubules mediate transport of actin filaments and that pre-accumulated myosin II motors drive the incorporation of actin filaments into the contractile ring. The specific aims of this project are to mechanically create an optically clear environment in living cells and dissect the mechanism of microtubule-mediated transport of actin filaments and the mechanism of actin filament incorporation into the contractile ring. These aims will be accomplished by using a newly-developed approach that employs multimode micro-techniques to surgically remodel spindles and cells so as to mechanically dissect microtubule-mediated assembly of the contractile ring. This unique approach combines classic micromanipulation and microinjection with modern laser microbeam surgery using digital-enhanced polarization/fluorescence microscopy and spinning disc confocal microscopy. Thus, the approach permits direct micromanipulations of both non-labeled and fluorescently-labeled cytoskeleton in living cells and in vitro. This project will provide research training for undergraduate and graduate students.