Molecular Mechanisms of Regulation of Contraction

描述：（由申请人提供）：肌动蛋白细胞骨架的调节 细胞信号传导途径对于细胞运动和功能是重要的。的 一种Rho GT3，Cdc 42的激活，导致快速和短暂的 这是由Arp 2/3复合物成核的高度分支的肌动蛋白网络的聚合。 这一事件产生了一种不依赖肌球蛋白的力量， 在运动细胞的前缘。虽然我们对激活的了解很多 以及该过程的失活，重要的问题仍然存在。编队怎么样 的分支网络定位到细胞的前缘，而不是从 肌动蛋白丝在皮质和应力纤维中的位置因为能够保护 从切割和尖端的肌动蛋白丝选择群体 ADF/cofilin解聚？灯丝稳定性是否有层次结构？ 这项研究的目的是了解导致死亡的一连串事件。 在细胞中形成和维持稳定的肌动蛋白丝。研究 将探讨ADF/cofilin，Arp 2/3复合物，原肌球蛋白和 原调节蛋白，连同肌动蛋白核苷酸，在定义的途径， 新生的、分支的肌动蛋白丝到稳定的丝。有四个具体的 目标。1)有分支的纤维是如何转变成稳定的无分支的纤维的 长而不分枝的纤维又是如何保持不分枝的呢2)尖头怎么样 稳定的肌动蛋白丝的末端不被解聚？3)你长什么样 肌动蛋白丝在深层细胞皮层和应力纤维保护， 切断和尖端解聚的ADF/cofilin，而那些在 分支肌动蛋白网络是不是？4)确定原肌球蛋白的定位 Arp 2/3复合物、cofilin和动力学区域相关的同种型 肌动蛋白细胞骨架的重塑。 目的1-3的方法包括测量肌动蛋白聚合和 使用荧光探针标记的肌动蛋白以及 荧光标记的肌动蛋白丝的显微镜分析。目标4将 利用间接免疫荧光技术定位细胞中的蛋白质和游离丝末端， 免疫荧光这些结果将有助于了解细胞骨架是如何 在复杂的功能如白细胞趋化性和生长过程中重塑 因子刺激的细胞生长，在正常和 病理状态。

DESCRIPTION: (provided by applicant): Regulation of the actin cytoskeleton by cell signaling pathways is important for cellular movement and function. The activation of one Rho GTPase, Cdc42, leads to the rapid and transient polymerization of a highly-branched actin network nucleated by Arp2/3 complex. The event creates a myosin-independent force that pushes forward the membrane at the leading edge of motile cells. While much is known about the activation and deactivation of this process, important questions remain. How is formation of branched networks localized to the leading edge of the cell and not from actin filaments deeper in the cortex and in stress fibers? What protects selected populations of actin filaments from severing and pointed-end depolymerization by ADF/cofilin? Is there a hierarchy of filament stability? The goal of the research is to understand the cascade of events leading to the formation and maintenance of stable actin filaments in the cell. The research will explore the roles of ADF/cofilin, Arp2/3 complex, tropomyosin and tropomodulin, together with the actin nucleotide, in defining the pathway from nascent, branched actin filaments to stable filaments. There are four specific aims. 1) How are branched filaments converted to stable unbranched filaments and how do long, unbranched filaments remain unbranched?2) How are the pointed ends of stable actin filaments protected from depolymerization?3) How are long actin filaments in the deep cell cortex and stress fibers protected from severing and pointed end depolymerization by ADF/cofilin while those in the branched actin network are not?4) To determine the localization of tropomyosin isoforms in relation to Arp2/3 complex, cofilin, and regions of dynamic remodeling of the actin cytoskeleton. The methods for Aims 1-3 include measurement of actin polymerization and depolymerization using actin labeled with a fluorescent probe as well as microscopic analysis of fluorescently-labeled actin filaments. Aim 4 will localize the proteins as well as free filament ends in cells using indirect immunofluorescence. The results will help understand how the cytoskeleton is remodeled during complex functions such as leucocyte chemotaxis and growth factor-stimulated cellular outgrowth, processes important in both normal and pathological states.