Myosin II Dynamics and Assembly in Mammalian Cells

描述（由申请人提供）：该提案的重点是在细胞分裂和极化细胞迁移和趋化反应过程中解读肌球蛋白II亚型的作用和调控。尽管肌球蛋白II是一种关键的、无处不在的产力酶，但人们对其在细胞收缩过程中的确切机械作用知之甚少，而且对其异构体的具体作用几乎一无所知。关于肌凝蛋白II丝的组装和定位是如何在体内调节的，存在多种相互矛盾的模型，目前流行的模型都没有在体内进行过测试。我们将利用GFP-myosin II报告蛋白的体内成像、光漂白后荧光恢复（FRAP）技术以及siRNA等方法，对myosin II调节的基本机制以及myosin MA和MB在细胞分裂和极化细胞迁移等过程中的亚型特异性作用产生重要的新认识。我们的研究还将确定在细胞分裂过程中负责肌球蛋白II组装和收缩激活的关键上游蛋白激酶和调节蛋白。

DESCRIPTION (provided by applicant): This proposal focuses on deciphering myosin II isoform roles and regulation during cytokinesis and during polarized cell migration and chemotactic responses. Although myosin II is a critical and ubiquitous force producing enzyme, its exact mechanical roles during cellular contractile events is poorly understood, and there is virtually no understanding of isoform specific roles. Multiple conflicting models exist regarding how myosin II filament assembly and localization is regulated in vivo, and none of the prevailing models have been tested in vivo. We will use in vivo imaging of GFP-myosin II reporter proteins, fluorescent recovery after photobleaching (FRAP) technology, as well as siRNA and other approaches to generate significant new understanding of fundamental mechanisms of myosin II regulation and new understanding of the isoform-specific roles of myosin MA and MB in processes such as cytokinesis and polarized cell migration. Our studies will also identify the key upstream protein kinases and regulatory proteins responsible for activation of myosin II assembly and contractility during cytokinesis.