Cortactin function in lamellipodial protrusion

描述(由申请人提供)：这项建议在基础水平上研究细胞运动的机制。我们专注于阐明迁移细胞前沿的片脂动力学和焦点复合体形成之间的关系。我们在分子水平上进行这些研究是独一无二的，因为我们发表的和初步的数据揭示了Cortactin将片状脂膜持久性与焦点复合体组装结合的核心机制。在目标1中，我们将测试一种假设，即这种耦合实际上类似于移动细胞的“齿条和小齿轮”转向机构，它允许显性的片状脂膜形成并引导细胞向其方向移动。在目标2中，我们将定义如何产生和积极地调节片状脂体病的持久性。我们现有的数据已经确凿地表明，Cortactin与分支肌动蛋白的结合是持久性的基础，我们将测试Cortactin是稳定分支，还是超级激活Arp2/3复合体，或者两者兼而有之。在目标3中，我们将通过研究分别位于片状脂膜前部和后部的磷脂和Cofilin抑制皮质蛋白的分子机制，来定义持久性如何在片状脂膜活动区受到空间限制和负调控。我们研究的一个基本方面是，我们将生物化学与定量显微镜相结合，不仅是为了确定分子机制是否可能发生(例如，在含有纯化成分的试管中)，而且还为了确定它们是否确实发生在活细胞中。这些方法包括：趋化性和其他细胞运动性分析，活细胞片脂和黏附动力学的定量分析，以及对肌动蛋白结合蛋白突变体的仔细生化表征，然后对其产生的细胞表型进行量化。随着片状脂膜持续性研究的提出，我们将自己定位于黏附和肌动蛋白生物学领域的交界处，这两个领域的整合有望产生一门新的令人兴奋的学科。意义：我们的研究对基础细胞生物学和人类健康都具有重要意义。我们试图解决的分子机制不仅对细胞运动至关重要，而且对许多依赖于分支肌动蛋白组装的细胞功能也是至关重要的，包括囊泡运输和组织形态发生。就人类健康而言，这些研究与癌症转移特别相关，因为细胞运动是癌细胞侵袭的重要组成部分。更直接的是，皮质素通过基因扩增在许多癌症中过度表达，包括15%的乳腺癌和30%的头颈部鳞状细胞癌(HNSCC)。有趣的是，Cortactin的过度表达与不良的预后和降低的存活率相关。因此，该方案中的研究对于理解动态分支肌动蛋白组装的基本调节以及皮质肌动蛋白在癌细胞运动中的可能作用都是重要的。

DESCRIPTION (provided by applicant): This proposal examines mechanisms of cell motility at a fundamental level. We focus on clarifying the relationship between lamellipodial dynamics and focal complex formation, at the leading edge of a migrating cell. We are in a unique position to conduct these studies at the molecular level, because our published and preliminary data have unveiled a core mechanism whereby cortactin couples lamellipodial persistence with focal complex assembly. In Aim 1 we will test the hypothesis that this coupling is in effect akin to a "rack and pinion" steering mechanism for moving cells, which allows for a dominant lamellipodia to form and guide the cell in its direction. In Aim 2 we will define how lamellipodia persistence is generated and positively regulated. Our existing data already conclusively show that binding of cortactin to branched actin is the basis for persistence, and we will test whether cortactin stabilizes branches, super-activates Arp2/3 complex, or both. In Aim 3 we will define how persistence is spatially constrained and negatively regulated at areas of lamellipodia activity, by investigating molecular mechanisms of cortactin inhibition by phospholipids and cofilin, at the front and rear of the lamellipodia, respectively. An essential aspect of our studies is that we combine biochemistry with quantitative microscopy in order to determine not only whether molecular mechanisms can happen (e.g., in a test tube with purified components), but also whether in fact they occur in a living cell. These approaches include: chemotaxis and other cell motility assays, quantitative analyses of lamellipodial and adhesion dynamics in living cells, and careful biochemical characterization of actin binding protein mutants followed by quantification of cell phenotypes they produce. With the proposed studies on lamellipodial persistence, we are positioning ourselves at the interface of the adhesion and actin biology fields, whose integration will hopefully generate a new exciting discipline. Significance: Our studies are significant both for fundamental cell biology and human health. The molecular mechanisms we are attempting to solve are not only critical for cell motility, but also for the many cellular functions that depend on branched actin assembly, including vesicular trafficking and tissue morphogenesis. With respect to human health, these studies are particularly relevant to cancer metastasis, since cell motility is an essential component of cancer cell invasion. More directly, cortactin is well- documented to be overexpressed in a number of cancers via gene amplification, including 15% of breast and 30% of head and neck squamous cell cancer (HNSCC). Intriguingly, cortactin overexpression correlates with poor prognosis and decreased survival. Thus, the studies in this proposal are important for understanding both the fundamental regulation of dynamic branched actin assemblies and the possible role of cortactin specifically in cancer cell motility.