Spatial Segregation of Cell Functioning During Motility

描述(由申请人提供)： 我们的长期目标是确定细胞如何在伤口修复过程中建立并维持渐进运动，以响应非方向性的外部信号。在伤口修复中，成纤维细胞和内皮细胞重新填充未成熟的基质，形成支持基质和再生组织结构所需的血管系统。最初的迁移是由来自缠绕床内的信号驱动的。然而，一旦进入伤口床，细胞必须经常在没有信号梯度的情况下分布。一个中心问题是细胞如何建立进步运动所需的不对称性。可溶性生长因子可诱导成纤维细胞和内皮细胞的迁移。这种运动需要细胞内生物物理细胞过程的不对称性。在前部，细胞必须伸展并形成新的粘连，而后部则需要去粘连和回缩。在这两个细胞区域之间，发生收缩以使细胞体向前移动。已经确定了调节每个生物物理过程的关键分子开关。目前尚不清楚的是，这些信号通路如何在其正确的空间方向上启动生物物理过程。虽然外部信号梯度是一个有吸引力的解释，但文献表明，对于真核细胞来说，关键的调节因素是细胞内。这对于化学动剂来说尤其如此，例如EGFR和VEGFR配体，即使在没有外部梯度的情况下，它们也可以诱导细胞向前移动。我们假设，生产运动所需的细胞不对称性是通过亚质粒限制关键生化开关的激活来实现的。我们建议测试以下分子机制： 一、由CdC42将PLC伽马活动限制在前导的拉姆椭圆轨道上。我们将使用成像和分子扰动来确定前端直接肌动蛋白重组所需的PLCGamma/-1在哪里以及如何与协调运动载体的小GTP酶CDC42相互作用。这些研究是基于初步数据。 M-calain驱动细胞体和后缘的脱粘，继而受到磷酸肌醇的限制。我们将根据生长因子激活m-calain所需的肌醇磷脂的初步发现，确定m-calain的活性如何限制在细胞体和尾部区域。 在EGF诱导的运动过程中，细胞内的收缩力量是不对称的。生物物理解构将确定一个可移动细胞的收缩能力在数量和质量上的差异。这些调查会的！定义信号和响应的空间限制的分子基础。这将使用于细胞和组织工程的智能支架的设计能够指导基质和血管床的合成，从而恢复Sunnort的组织功能。

DESCRIPTION (provided by applicant): Our long-term goal is to determine how cells establish and then maintain progressive motility in response to nondirectional external signals during wound repair. In wound repair, fibroblasts and endothelial cells repopulate the immature matrix to form both the supporting matrix and vasculature required to regenerate the tissue structures. The initial migration is driven by signals that arise from within the wound bed. However, once within the wound bed, the cells must distribute often m the absence of a gradient of signals. A central question is how cells establish the asymmetry required forprogressive motility. Soluble growth factors induce the migration of both the fibroblasts and endothelial cells. This motility requires asymmetry of biophysical cell processes within the cell. At the front, the cells must extend lamellipodla and form new adhesions, while rear de-adhesion and retraction is required. Between these two cell regions, contractility occurs to bring the cell body forward. Key molecular switches have been identified which regulate each biophysicalprocess. What remains unknown is how these signaling pathways initiate the biophysical processes in their correct spatial orientation. While an external signaling gradient would be an attractive explanation, the literature suggests that for eukaryotic cells, the key regulators are intracellular. This would be particularlz true for chemokinetic agents, such as EGFR and VEGFR ligands, which can induce progressive cell motility even in the absence of an external gradient. We hypothesize that cellular asymmetry needed for productive motility is accomplished by subeytoplasmie restriction of the activation of key biochemical switches. We propose to test the following molecular mechanisms: I. That PLCgamma activity is limited to the leading lamellipod by cdc42. We will use imaging and molecular perturbations to determine where and how PLCgamma/-1, required for front-direct actin reorganization, interacts with cdc42, the small GTPase that orchestrates the motility vector. These studies are based on preliminary data. II. That m-calpain drives de-adhesion in the cell body and trailing edge secondary to restricted availability of phospho-inositides. We will determine how m-calpain activity is restricted to the cell body and tail region, based on preliminary findings of phospho-inositides being required for m-calpain activation by growth factors. III. That contractile forces are asymmetric within the cell during EGF-induced motility. Biophysical deconstruction will identify quantitative and qualitative differences in contractility across a motile cell. These investigations wil! define molecular bases of the spatial restriction of signals and responses. This will enable the design of'smart scaffolds for cell and tissue engineering directing the synthesis of both the matrix and the vascular bed that is reauired to sunnort tissue function.