Dissecting Mechanics of Contractile Ring Assembly

肌球蛋白收缩环的组装使细胞围绕纺锤体赤道收缩，这对细胞分裂是必不可少的。收缩环的组织和收缩的分子基础已经被很好地记录下来，但是肌动蛋白细丝如何重新分布和组装到环中的机制在很大程度上仍然不清楚。越来越多的证据表明，纺锤形微管可能调节肌动蛋白细丝的赤道分布。然而，目前尚不清楚活细胞中微管与肌动蛋白细丝是如何相互作用的。长期的目标是通过关注微管与肌动蛋白细丝的相互作用以及细丝的结合来了解收缩环组装的机制。假说是辐射纺锤形微管介导肌动蛋白细丝的运输，而预先积累的肌球蛋白II马达驱动肌动蛋白细丝并入收缩环。这个项目的具体目标是在活细胞中机械地创造一个光学清晰的环境，并剖析微管介导的肌动蛋白细丝运输机制和肌动蛋白细丝掺入收缩环的机制。这些目标将通过使用一种新开发的方法来实现，该方法使用多模式微技术来外科手术重塑纺锤体和细胞，从而机械地解剖微管介导的收缩环组装。这一独特的方法将经典的微操作和微注射与现代激光微束手术相结合，使用数字增强偏振/荧光显微镜和旋转圆盘共焦显微镜。因此，该方法允许在活细胞和体外对未标记和荧光标记的细胞骨架进行直接显微操作。该项目将为本科生和研究生提供研究性培训。

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.