Regulation of Cell Migration by the APC-Microtubule Complex

描述（由申请人提供）：细胞向细胞外信号的定向迁移对于正常组织形态发生和修复至关重要，并且所涉及的调节途径的异常导致肿瘤转移期间的严重病理后果。该提议集中于腺瘤性结肠息肉病（APC）多蛋白复合物在响应刺激细胞迁移和粘附的细胞外信号而调节微管和肌动蛋白细胞骨架中的作用。APC最初被发现是一种控制β-连环蛋白水平的肿瘤抑制蛋白。APC是一种具有许多结合配偶体的大型支架蛋白，其还响应于细胞外信号调节微管和定向细胞迁移。虽然APC在不同形态学过程中的重要性已经得到了很好的证实，但是关于细胞外信号如何影响其与细胞骨架结合伴侣的相互作用和微管动力学的调节，或者APC支架复合物如何影响膜动力学和细胞迁移，我们的假设是APC通过充当细胞骨架调节剂的支架来协调微管和肌动蛋白的重组，并且直接响应于APC支架的细胞外信号传导组分，其被局部募集和激活以指定定向膜延伸。我们提出了一个严格的APC调控和功能的生化分析结合新的检测重组APC-微管动力学在体外和图像与高时空分辨率APC-微管动力学在细胞中。我们建议：1）。定义参与细胞外信号诱导的APC多蛋白复合物组成和功能的修饰的机制; 2）.解剖和重构APC多蛋白复合物组分对微管动力学和F-肌动蛋白结合的体外作用;和3）。表征APC介导的响应细胞外信号的微管和F-肌动蛋白动力学的局部变化。这些拟议研究的意义在于，它们将定义APC复合物中的蛋白质-蛋白质相互作用，该复合物在细胞延伸和接触形成期间局部调节细胞骨架重组，以及该复合物的功能如何通过在发育和疾病中重要的信号通路调节。

DESCRIPTION (provided by applicant): Directional cell migration towards extracellular signals is essential for normal tissue morphogenesis and repair, and abnormalities in the regulatory pathways involved result in severe pathological consequences during tumor metastasis. This proposal focuses on the role of the adenomatous polyposis coli (APC) multi- protein complex in regulating microtubule and actin cytoskeletons in response to extracellular signals that stimulate cell migration and adhesion. APC was originally discovered as a tumor suppressor protein that controls the level of beta-catenin. APC is a large scaffolding protein with many binding partners that also regulates microtubules and directional cell migration in response to extracellular signals. Although the importance of APC in diverse morphological processes is well established, very little is known about how extracellular signals affect its interaction with cytoskeletal binding partners and regulation of microtubule dynamics, or how the APC scaffolding complex influences membrane dynamics and cell migration Our hypothesis is that APC coordinates microtubule and actin reorganization by acting as scaffold for cytoskeletal regulators, and that in direct response to extracellular signaling components of the APC scaffold are locally recruited and activated to specify directional membrane extension. We propose a rigorous biochemical analysis of APC regulation and functions combined with novel assays to reconstitute APC- microtubule dynamics in vitro and to image with high spatiotemporal resolution APC-microtubule dynamics in cells. We propose to: 1). Define mechanisms involved in extracellular signal-induced modifications of APC multi-protein complex composition and functions; 2). Dissect and reconstitute effects of APC multi-protein complex components on microtubule dynamics and F-actin binding in vitro; and 3). Characterize APC- mediated localized changes in microtubule and F-actin dynamics in response to extracellular signals. The significance of these proposed studies is that they will define protein-protein interactions in the APC complex that locally regulate cytoskeleton reorganization during cell extension and contact formation, and how the function of this complex is regulated by signaling pathways important in development and disease.