Regulation of Cell Migration by the APC-Microtubule Complex

描述(申请人提供)：定向细胞向细胞外信号迁移对于正常的组织形态形成和修复是必不可少的，涉及的调节通路的异常会在肿瘤转移过程中导致严重的病理后果。这项建议侧重于结肠腺瘤性息肉病(APC)多蛋白复合体在调节微管和肌动蛋白细胞骨架中的作用，以响应刺激细胞迁移和黏附的细胞外信号。APC最初是作为一种肿瘤抑制蛋白被发现的，它控制β-连环蛋白的水平。APC是一种具有许多结合伙伴的大型支架蛋白，它还调节微管和细胞的定向迁移，以响应细胞外信号。尽管APC在不同的形态过程中的重要性已经得到了很好的证实，但关于细胞外信号如何影响其与细胞骨架结合伙伴的相互作用和微管动力学的调节，或者APC支架复合体如何影响膜动力学和细胞迁移我们的假设是，APC作为细胞骨架调节的支架来协调微管和肌动蛋白的重组，并且在直接响应细胞外信号的过程中，APC支架的成分被局部招募并激活以指定膜的定向延伸。我们建议对APC的调节和功能进行严格的生化分析，并结合新的方法来重建APC-微管动力学，并在细胞内以高时空分辨率成像APC-微管动力学。我们建议：1)。明确细胞外信号诱导APC多蛋白复合体组成和功能改变的机制；2)。解剖和重建APC多蛋白复合体成分对体外微管动力学和F-肌动蛋白结合的影响；表征APC介导的微管和F-肌动蛋白对细胞外信号反应的局部变化。这些拟议的研究的意义在于，他们将定义APC复合体中的蛋白质-蛋白质相互作用，该复合体在细胞延伸和接触形成过程中局部调节细胞骨架的重组，以及该复合体的功能如何通过在发育和疾病中重要的信号通路来调节。

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.