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建立，然后保持在伤口修复过程中响应于非定向外部信号的前向运动。在伤口修复中，成纤维细胞和内皮细胞重新填充未成熟基质以形成再生组织结构所需的支持基质和脉管系统。最初的迁移由伤口床内产生的信号驱动。然而，一旦在伤口床内，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前向运动所需的不对称性。可溶性生长因子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，细胞必须延伸板状伪足并形成新的粘附，而后部则需要去粘附和回缩。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药理学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，关键的调节因子是细胞内的。这对于即使在没有外部梯度的情况下也可以诱导进行性细胞运动性的化学动力学试剂（例如EGFR和VEGFR配体）尤其如此。我们推测，生产性运动所需的细胞不对称性是通过关键生化开关的激活的subeytoplasmie限制来完成的。我们建议测试以下分子机制： I. PLC γ的活性被cdc 42限制在主要的片足类中。我们将使用成像和分子扰动来确定前向肌动蛋白重组所需的PLC γ/-1在哪里以及如何与协调运动载体的小GTd 42相互作用。这些研究是基于初步数据。 二.该m-钙蛋白酶驱动细胞体中的去粘附和后缘继发于磷酸肌醇的受限可用性。我们将确定如何m-钙蛋白酶活性被限制在细胞体和尾部区域，基于磷酸肌醇被需要由生长因子激活m-钙蛋白酶的初步研究结果。 三.在EGF诱导的运动过程中，细胞内的收缩力是不对称的。生物物理解构将确定跨运动细胞的收缩性的定量和定性差异。这些调查将！定义信号和响应的空间限制的分子基础。这将使设计用于细胞和组织工程的智能支架成为可能，指导基质和血管床的合成，这是正常组织功能所需的。

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.