Regulation of Microtubules by Rho GTPases

这个项目的总体目标是了解Rho家族的小GTP酶如何调节稳定性和 细胞极化过程中微管的组织。MTS的动态特性使他们能够 在细胞极化过程中对外界信号的反应，但对这些信号是如何传递的知之甚少 到MTS或参与MT重排的蛋白质。细胞迁移到体外创面是一种 研究作为可溶性、基质和细胞相关贡献的MT调控信号的模型系统 这些因素是可以剖析的。在之前的资助期间，我们发现创伤中MTS的两次重排 边缘迁移成纤维细胞、异常稳定的MT亚群的形成和MT的重新定位 组织中枢(MTOC)，均由血清溶血磷脂酸(LPA)触发，但分别 受Rho和CDC42 GTP酶调控。这两种重排都被认为涉及MT的相互作用 以细胞皮质结束，这一过程被称为MT捕获。我们鉴定了MDIA、EB1、APC、GSK30和NOVICE Pkcs作为Rho下游的因子和两条独立的路径在cdc42下游工作；一条 涉及细胞中心的MTOC的动力蛋白和动力肌动蛋白的维持，另一个涉及MRCK， 肌动蛋白和肌球蛋白II在核的一种新的奖赏运动中发挥作用。目前的目标是进一步 通过研究mdia对MTS和肌动蛋白的活性来探讨MT的稳定机制 通过测试MDIA是否可以直接影响MT的稳定以及EB1和APC是否可以 影响mdia对MTS的活性。我们还将测试MDIA、EB1和APC之间的复合体是否 受GSK3P和GSK3P调节，并筛选可能有助于MT稳定的其他蛋白质。这个 Par6、dynein和dynactin调节MTOC浓度的机制将通过确定PAR 6调节动力蛋白和动力蛋白，是否有额外的蛋白质调节动力蛋白和动力蛋白，以及动力蛋白是否 而dynactin通过捕获皮质MT来维持细胞中心的MTOC。了解Rho GTP酶是如何 它们刺激的调节MTS的途径将提供有关基本途径的新信息 细胞在细胞迁移过程中传递信号来控制细胞骨架系统，这一过程对 发展、伤口愈合和转移。

The overall goal of this project is to understand how small GTPases of the Rho family regulate the stabilityand 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 hi 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, GSK30 and novel PKCs as factors working downstream of Rho and two separate pathways working downstream of Cdc42; one involving Part), 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 GSK3P 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.