Microtubule/Actin Interactions in Cell Motility

描述(申请人提供)：微管(MT)和丝状肌动蛋白(f-肌动蛋白)是定向组织细胞运动所必需的。F-肌动蛋白聚合和肌动球蛋白收缩的空间控制是产生前板脂膜突起和后部收缩的自我持久不对称以驱动细胞运动所必需的。MTS为这些基于肌动蛋白的事件提供了空间和时间上的协调，然而其分子基础却鲜为人知。MTS和f-肌动蛋白在迁移细胞中表现出两种机械性的相互作用。“结构相互作用”，其中f-肌动蛋白和MTs在物理上是交联的，因此直接影响彼此的组织和动态。“调控相互作用”是指Rho家族的小GTP酶信号通路的活动受MTS的组装和拆解的时空调控，同时Rho GTP酶在区域上共同调节MTS和f-肌动蛋白的动态。发现微管和肌动蛋白细胞骨架之间的分子连接，以及调节这些连接的分子机制和肌动蛋白和MT细胞骨架阵列之间的反馈，对于我们理解极化细胞运动的机制是至关重要的。具体目标：A.1。测试这样的假设，即MTS和f-肌动蛋白之间需要特定类别的结构相互作用来介导定向细胞运动。剖析了rac1 GTPase诱导MT生长的分子机制，并验证了rac1介导的促进微管生长是细胞运动所必需的假说。验证腺瘤性息肉病结肠蛋白(ARC)介导MT生长激活板脂突起和rac1 GTP酶的假设。A.4.开发自动化图像分析工具，以量化活细胞中大量MT的组装、拆卸和移动。这笔赠款中提议的实验将在我们理解细胞运动的基本机制方面取得重大进展，并提供重要的新技术，如果它们得到实现，将有助于生物科学其他领域的进步。

DESCRIPTION (provided by applicant): Microtubules (MTs) and filamentous actin (f-actin), are required for directed tissue cell motility. Spatial control of f-actin polymerization and actomyosin contraction is essential to generate a self perpetuating asymmetry of lamellipodial protrusion in the front and retraction in the rear to drive cell motility. MTs provide spatial and temporal orchestration of these f-actin-based events, however the molecular basis is poorly understood. MTs and f-actin exhibit two mechanistic categories of interactions in migrating cells. "Structural Interactions" in which f-actin and MTs are physically cross linked, and thus directly affect each other's organization and dynamics. "Regulatory Interactions" are those in which the activities of Rho-family small GTPase signaling cascades are spatiotemporally controlled by the assembly and disassembly of MTs, and at the same time, Rho GTPases regionally co-regulate the dynamics of MTs and f-actin. It is critical for our understanding of the mechanisms of polarized cell motility to discover the molecular linkages between the microtubules and actin cytoskeletons and the molecular mechanisms that regulate these linkages and the feedback between the dynamics of the actin and MT cytoskeletal arrays. Specific Aims:A.1. Test the hypothesis that specific classes of structural interactions between MTs and f-actin are required to mediate directed cell motility.A.2. Dissect the molecular mechanism of Rac1 GTPase-induced MT growth and test the hypothesis that Rac1-mediated promotion of microtubule growth is required for cell motility.A.3. Test the hypothesis that the adenomatous polyposis coli protein (ARC) mediates MT growth- activation of lamellipodial protrusion and Rac1 GTPase. A.4. Develop automated image analysis tools to quantitate the assembly, disassembly and movement of large populations of MTs in living cells. The experiments proposed in this grant will provide significant advances in our understanding of the basic mechanism of cell motility and provide important new technology that will aid in the advancement of other areas of biological science if they are achieved.