Cortactin function in lamellipodial protrusion

描述（由申请人提供）：该提案在基本水平上检查细胞运动机制。我们专注于澄清lamellipodial动力学和局灶性复合物的形成之间的关系，在迁移细胞的前沿。我们处于一个独特的位置，在分子水平上进行这些研究，因为我们发表的和初步的数据已经揭示了一个核心机制，即corneum夫妇lamelipodial持久性与焦点复合物组装。在目标1中，我们将测试这样的假设，即这种耦合实际上类似于用于移动细胞的“齿条和小齿轮”转向机制，其允许主导的板状伪足形成并引导细胞沿其方向运动。在目标2中，我们将定义如何产生和正调控板状伪足持久性。我们现有的数据已经决定性地表明，coronin与分支肌动蛋白的结合是持久性的基础，我们将测试coronin是否稳定分支，超级激活Arp 2/3复合物，或两者兼而有之。在目标3中，我们将定义如何持久性是空间上的限制和负调控领域的片状伪足的活动，通过研究皮质激素抑制磷脂和cofilin的分子机制，在前面和后面的片状伪足，分别。我们研究的一个重要方面是，我们将联合收割机生物化学与定量显微镜相结合，不仅为了确定分子机制是否会发生（例如，在含有纯化组分的试管中），而且还包括它们是否实际上发生在活细胞中。这些方法包括：趋化性和其他细胞运动性测定，活细胞中片状脂质体和粘附动力学的定量分析，以及肌动蛋白结合蛋白突变体的仔细生物化学表征，随后对其产生的细胞表型进行定量。随着对片状伪足持久性的拟议研究，我们将自己定位在粘附和肌动蛋白生物学领域的界面上，其整合有望产生一个新的令人兴奋的学科。意义：我们的研究对于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，而且对依赖于分支肌动蛋白组装的许多细胞功能也至关重要，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。更直接地，有充分的证据表明，通过基因扩增，coronin在许多癌症中过表达，包括15%的乳腺癌和30%的头颈部鳞状细胞癌（HNSCC）。有趣的是，corneum过度表达与预后不良和生存率降低相关。因此，在这个建议中的研究是重要的了解动态分支肌动蛋白组件的基本调节和corneumen具体在癌细胞运动的可能作用。

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.