Spatial Segregation of Cell Functioning During Motility

描述（由申请人提供）：

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.