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

细胞有不同的形状，可以通过细胞‘细胞骨架’的动态重排来快速重塑，细胞骨架由一个动态的蛋白质纤维网络组成。它的两个主要成分是肌动蛋白细丝和微管，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，受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的功能是紧密交织在一起的，在细胞形态形成和运动过程中必须密切协调。这项提议的目标是在这一领域获得新的机械性见解。我将在古德实验室进行我的研究，该实验室最近的工作表明，导致结肠癌的腺瘤性息肉病结肠(APC)蛋白除了在结合和稳定MT方面发挥既定作用外，还直接诱导肌动蛋白组装。这使得APC成为第一个与癌症有直接联系的肌动蛋白核仁，并成为解剖肌动蛋白-MT交叉调控的理想焦点。APC还与其体内结合伙伴Forin mDia1在肌动蛋白组装方面显示出新的功能协同作用，Forin mDia1也与肌动蛋白和MT结合。我将使用先进的体外TIRF显微镜技术分析APC和mDia1单独和共同对肌动蛋白细丝和MT组装的影响，并使用荧光标记蛋白质的单分子分析来剖析APC/mDia1协同作用的基础。这些显微分析将得到mDia1-APC相互作用的生化分析以及在这些蛋白质存在下肌动蛋白和MT聚合的动力学分析的补充。我还将研究另一种MT相关蛋白和APC的体内结合伙伴EB1对这些活性的调节。这项工作将有助于定义APC、mDia1和EB1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聚合物调节来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.