Analysis of Actomyosin Ring Assembly and Contraction

描述（由申请人提供）： 细胞分裂是一个极其重要和复杂的过程。当细胞分裂不再受调节时，癌细胞就会出现。细胞分裂周期的最后一步是胞质分裂。在大多数真核生物中，胞质分裂涉及收缩环、肌动蛋白丝和肌球蛋白-II丝的赤道束的组装和收缩。我们的长期目标是了解肌球蛋白-II在胞质分裂中的作用。裂殖酵母为研究胞质分裂提供了一个优良的遗传学模型系统。我建议使用遗传学，生物化学和活细胞显微镜相结合，调查肌球蛋白-II在裂变酵母。最初，肌球蛋白-II功能所需的基因将通过克隆肌球蛋白-II温度敏感突变体的抑制子来鉴定。随后，将通过在已鉴定的胞质分裂组分中含有突变的裂变酵母细胞中可视化GFP标记的肌球蛋白-II，在活细胞中分析收缩环的组装和收缩。最后，将通过纯化肌球蛋白-II和抑制蛋白并在体外重建它们的相互作用来研究肌球蛋白-II功能的具体机制。这种实验策略的组合对于在全细胞和单个分子水平上研究胞质分裂是必不可少的。

DESCRIPTION (provided by applicant): Cell division is an extremely important and complex process. Cancer cells arise when cell division is no longer regulated. The final step in the cell division cycle is cytokinesis. In most eukaryotes, cytokinesis involves the assembly and constriction of a contractile ring, an equatorial bundle of actin filaments and myosin-Il filaments. Our long range goal is to understand the role of myosin-Il in cytokinesis. Fission yeast provides an excellent genetically tractable model system to study cytokinesis. I propose to use a combination of genetics, biochemistry and live-cell microscopy to investigate myosin-II in fission yeast. Initially, genes required for myosin-Il function will be identified by cloning suppressors of myosin-Il temperature-sensitive mutants. Subsequently, the assembly and contraction of the contractile ring will be analyzed in live cells by visualizing GFP-tagged myosin-Il in fission yeast cells that contain mutations in the cytokinesis components that were identified. Finally, the specific mechanism of myosin-Il function will be investigated by purifying myosin-Il and suppressor proteins and reconstituting their interaction in vitro. This combination of experimental strategies is essential for investigating cytokinesis at both the whole-cell and individual molecule level.