Regulation of Microtubules by Rho GTPases

描述（由申请人提供）：该项目的总体目标是了解Rho家族的小GTPases如何在细胞极化过程中调节微管（mt）的稳定性和组织。MT的动力学特性使它们能够在细胞极化过程中对外部信号做出反应，但对于这些信号如何被转导到MT或参与MT重排的蛋白质，我们知之甚少。细胞迁移到体外创面是研究MT调控信号的一个模型系统，因为可溶性、基质和细胞相关因子的贡献可以被分解。在之前的研究中，我们发现伤口边缘迁移成纤维细胞中MT的两次重排，即异常稳定的MT亚群的形成和MT组织中心（MTOC）的重新定位，都是由血清溶血磷脂酸（LPA）触发的，但分别由Rho和Cdc42 gtpase调节。这两种重排都被认为涉及到MT末端与细胞皮层的相互作用，这一过程被称为MT捕获。我们发现mDia、EB1、APC、gsk3 β和新型PKCs是作用于Rho下游的因子，以及两条作用于Cdc42下游的独立通路；其中一个与维持细胞中心MTOC的Par6、动力蛋白和动力蛋白有关，另一个与MRCK、肌动蛋白和肌球蛋白II有关，它们在细胞核的一种新的奖励运动中起作用。目前的目标是进一步探索MT稳定的机制，通过探索mDia对MT和肌动蛋白的活性是如何分配的，通过测试mDia是否可以直接影响MT稳定，以及EB1和APC是否会影响mDia对MT的活性。我们还将测试mDia， EB1和APC之间的复合物是否受gsk3 β调节，并筛选可能有助于MT稳定的其他蛋白质。Par6、dynein和dynactin调节MTOC浓度的机制将通过确定Par6如何调节dynein和dynactin，是否有其他蛋白质调节dynein和dynactin，以及dynein和dynactin是否通过皮质MT捕获维持细胞中心的MTOC来探索。了解Rho gtpase及其刺激的通路如何调节MTs，将提供关于细胞在细胞迁移过程中传递信号以控制细胞骨架系统的基本途径的新信息，细胞迁移是一个对发育、伤口愈合和转移至关重要的过程。

DESCRIPTION (provided by applicant): The overall goal of this project is to understand how small GTPases of the Rho family regulate the stability and organization of microtubules (MTs) during cell polarization. The dynamics of MTs gives them the ability to response to external signals during cell polarization, yet little is known about how these signals are transduced to MTs or the proteins that are involved in MT rearrangements. Cells migration into an in vitro wound is a model system for studying the signals regulating MT as the contribution of soluble, matrix and cell-associated factors can be dissected. In the previous grant period, we found that the two rearrangements of MTs in wound edge migrating fibroblasts, formation of a subset of unusually stable MTs and reorientation of the MT organizing center (MTOC), are both triggered by serum lysophosphatidic acid (LPA), but are separately regulated by Rho and Cdc42 GTPases. Both of these rearrangements are thought to involve interactions of MT ends with the cell cortex, a process termed MT capture. We identified mDia, EB1, APC, GSK3beta and novel PKCs as factors working downstream of Rho and two separate pathways working downstream of Cdc42; one involving Par6, dynein and dynactin maintenance of the MTOC at the cell center, the other involving MRCK, actin and myosin II functioning in a novel reward movement of the nucleus. The current aims are to further explore the mechanism of MT stabilization by: exploring how mDia's activity toward MTs and actin is apportioned, by testing whether mDia can directly affect MT stabilization and whether EB1 and APC may affect mDia's activity toward MTs. We will also test whether complexes between mDia, EB1 and APC are regulated by GSK3beta and and screen for additional proteins that may contribute to MT stabilization. The mechanism of Par6, dynein and dynactin regulated MTOC centration will be explored by determining how Par 6 regulates dynein and dynactin, whether additional proteins regulate dynein and dynactin and whether dynein and dynactin maintain the MTOC at the cell center by cortical MT capture. Understanding how Rho GTPases and the pathways they stimulate act to regulate MTs will provide new information about the fundamental ways cells transduce signals to control cytoskeletal systems during cell migration, a process of importance for development, wound healing and metastasis.