Dissection of the coordinated regulation of actin and microtubule dynamics by mDia1 and APC in reconstituted systems by advanced TIRF microscopy and single-molecule analysis

细胞具有独特的形状，可以通过细胞“细胞骨架”的动态重排来快速重塑，细胞骨架由动态蛋白质纤维网络组成。它的两个主要组成部分是肌动蛋白丝和微管（MT），它们具有不同的性质和动力学。多年来，肌动蛋白和MT的生理功能被认为是分开的，并由不同的机制和机制控制。然而，最近的证据表明，肌动蛋白和MT功能密切相关，必须在细胞形态发生和运动过程中紧密协调。该提案的目标是在这一领域获得新的机制见解。在古德实验室，我将在那里进行我的研究，最近的工作表明，结肠癌引起的腺瘤性结肠息肉病（APC）蛋白直接诱导肌动蛋白组装除了其在结合和稳定MT的既定作用。这使得APC成为第一个与癌症直接相关的肌动蛋白成核剂，也是解剖肌动蛋白-MT交叉调节的理想焦点。APC还表现出一种新的功能协同作用，在肌动蛋白装配与其在体内的结合伙伴，ADMDia 1，也结合肌动蛋白和MT。我将使用先进的体外TIRF显微镜技术来分析APC和mDia 1单独和一起对肌动蛋白丝和MT组装的影响，并采用荧光标记蛋白的单分子分析来剖析APC/mDia 1协同作用的基础。这些显微镜分析将补充mDia 1-APC相互作用的生化分析和肌动蛋白和MT聚合在这些蛋白质的存在下的动力学分析。我还将研究EB 1对这些活性的调节，EB 1是另一种MT相关蛋白，也是APC的体内结合伴侣。这项工作将有助于定义APC，mDia 1和EB 1之间的相互作用和活动的新网络，这些网络共同整合肌动蛋白和MT聚合物调节以管理关键的细胞过程。

Cells have distinct shapes that can be rapidly remodeled by dynamic rearrangements of the cell’s ‘cytoskeleton’, which consists of a network of dynamic protein fibers. Two of its primary components are actin filaments and microtubules (MT), which have distinct properties and dynamics. For many years, the physiological functions of actin and MT were considered to be separate and controlled by distinct machinery and mechanisms. However, more recent evidence suggests instead that actin and MT functions are closely intertwined and must be tightly coordinated during cell morphogenesis and movement. The goal of this proposal is to gain new mechanistic insights in this area. Recent work in the Goode lab, where I will perform my research, has shown that the colon cancer-causing Adenomatous polyposis coli (APC) protein directly induces actin assembly in addition to its established roles in binding and stabilizing MT. This makes APC the first actin nucleator with direct links to cancer, and an ideal focal point for dissecting actin-MT cross-regulation. APC also exhibits a novel functional synergy in actin assembly with its in vivo binding partner, the formin mDia1, which also binds actin and MT. I will use advanced in vitro TIRF microscopy techniques to analyze the effects of APC and mDia1, alone and together, on the assembly of actin filaments and MT, and employ single molecule analysis of fluorescently labeled proteins to dissect the basis of APC/mDia1 synergy. These microscopy assays will be complemented by biochemical analyses of the mDia1-APC interaction and kinetic analyses of actin and MT polymerization in the presence of these proteins. I will also study regulation of these activities by EB1, another MT associated protein and in vivo binding partner of APC. This work will be beneficial to define a novel network of interactions and activities among APC, mDia1, and EB1 that together integrate actin and MT polymer regulation to govern critical cellular processes.