REGULATION OF MICROTUBULES BY RHO GTPASES

描述（由申请人提供）：本项目的总体目标是了解Rho家族的小GTP酶如何在细胞迁移极化期间调节微管（MT）的稳定性和组织。MT的动力学使它们能够在细胞极化期间对外部信号做出反应，并且已知Rho GTPase信号通路将这些信号传导至MT，从而改变其稳定性和组织。细胞迁移到体外伤口中是用于研究调节MT的信号的模型系统，因为可以剖析可溶性、基质和细胞相关因子的贡献。血清因子溶血磷脂酸（LPA）刺激单独的Rho和Cdc42信号通路，调节伤口边缘迁移成纤维细胞中MT的两个特征性重排：形成异常稳定的MT亚组和中心体的重定向。在以前的资助期间，我们确定了由这些GTP酶激活的途径中的许多因素，并发现这些途径改变了细胞皮质附近MT的动态，这一过程称为MT捕获。然而，尚不清楚这些信号通路激活的蛋白质如何介导MT捕获或捕获的MT如何影响细胞行为。目前的目标集中在MT捕获导致长寿命MT的机制，以及这些稳定的MT在迁移细胞的运动过程中的作用。除了它们在调节肌动蛋白细胞骨架中的良好表征的作用之外，formins已经成为MT的重要调节剂。我们将探讨的作用，在产生稳定的MTs通过定义其网站与MT的相互作用，确定其内在的能力，以调节MT的动态，研究如何相互作用的蛋白质，如MT +TIPs，改变其对MT的活动和测试是否与MT的相互作用改变其肌动蛋白聚合活性。我们将探讨mDia1和驱动蛋白马达蛋白，我们已经确定为一种新的调节MT稳定细胞之间的关系。将开发专门干扰迁移细胞中稳定MT生成的方法，以便确定它们对细胞运动过程的贡献。了解Rho GTPase信号通路如何调节MT，将提供有关细胞迁移过程中细胞将信号传导至细胞骨架系统的基本方式的新信息，细胞迁移是一个对发育、伤口愈合和转移很重要的过程。 公共卫生相关性：微管是动态的细胞骨架元件，有助于细胞对外部信号（如生长因子刺激）的反应。在迁移细胞中，微管有助于细胞活动的极化，使细胞以定向的方式迁移，但所涉及的分子机制尚不清楚。了解控制迁移细胞中微管的分子机制将有助于了解细胞在发育，伤口愈合和免疫反应过程中如何迁移的基本知识，并可能导致识别用于对抗涉及异常细胞迁移的疾病的新治疗靶点，如癌症转移和炎症。

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 polarization of cells for migration. The dynamics of MTs allows them to response to external signals during cell polarization and Rho GTPase signaling pathways are known to transduce these signals to MTs to bring about changes in their stability and organization. Cell 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. The serum factor lysophosphatidic acid (LPA) stimulates separate Rho and Cdc42 signaling pathways that regulate two characteristic rearrangements of MTs in wound edge migrating fibroblasts: formation of a subset of unusually stable MTs and reorientation of the centrosome. In previous grant periods, we identified many of the factors in the pathways that are activated by these GTPases and have found that the pathways alter the dynamics of MTs near the cell cortex, a process termed MT capture. Yet, it is unknown how the proteins activated by these signaling pathways mediate MT capture or how the captured MTs influence cellular behavior. The current aims focus on the mechanism by which MT capture leads to long-lived MTs and the role of these stabilized MTs in motile processes in migrating cells. In addition to their well-characterized roles in regulating the actin cytoskeleton, formins have emerged as important regulators of MTs. We will explore the role of the formin mDia1 in generating stabilized MTs by defining its sites of interaction with MTs, determining its intrinsic ability to regulate MT dynamics, examining how interacting proteins, such as the MT +TIPs, alter its activity toward MTs and testing whether its interaction with MTs alters its actin polymerizing activity. We will explore the relationship between mDia1 and a kinesin motor protein that we have identified as a novel regulator of MT stabilization in cells. Approaches will be developed to specifically interfere with the generation of stable MTs in migrating cells so that their contribution to motile processes in cells can be determined. Understanding how Rho GTPase signaling pathways act to regulate MTs will provide new information about the fundamental ways cells transduce signals to cytoskeletal systems during cell migration, a process of importance for development, wound healing and metastasis. PUBLIC HEALTH RELEVANCE: Microtubules are dynamic cytoskeletal elements that contribute to cellular responses to external cues, such as growth factor stimulation. In migrating cells, microtubules contribute to the polarization of cellular activities that allow the cell to migrate in a directional fashion but the molecular mechanisms involved are unclear. Understanding the molecular mechanism controlling microtubules in migrating cells will contribute to fundamental knowledge of how cells migrate during development, wound healing and immune response and may lead to the identification of novel therapeutic targets for combating diseases involving abnormal cell migration, such as cancer metastasis and inflammation.