Clinical studies suggest that EGFR -mediated cell migration plays an important role in tumor proression. Our objective is to understand downstream biophysical processes by which EGFR signaling regulates cell migration. Our foundational hypothesis is that biochemical regulation of locomotion can be understood in terms of key biophysical determinants of cell movement: (a) lamellipod extension frequency, (b) cell contractile force, and (c) cell/substratum adhesion strength. We thus predict that EGFR-activated biochemical signals affect cell motility by altering one or more of these biophysical determinants. Cur primary hypothesis concerning molecular intermediaries connecting the signaling pathways with the physical determinants of locomotion is that PLC hydrolysis of PlP2 activates actin-modifying proteins which reorganize actin cytoskeleton to affect one or more of the key physical processes. Signaling-restricted motogenic and non-motogenic EGFR variants have been expressed in NR6 murine fibroblast lines devoid of endogenous EGFR; wild- type EGFR is expressed in Hs68 normal human fibroblasts. The effect of EGF on parameters characterizing the three physical determinants of movement will be evaluated in these cells on a range of concentrations of an extracellular matrix substratum, Amgel. We will test our hypotheses by the following Specific Aims: I. Determining EGF-induced changes in cell migration speed and/or directional persistence. II. Determining EGF-induced changes in cell/substratum adhesiveness. III. Determining EGF-induced changes in lamellipod extension frequency. IV. Determining EGF-induced changes in cell contractile force. V. Determining whether intracellular mobilization of gelsolin and/or profilin causes changes in these physical determinants of locomotion. Completion of these investigations will define the physical bases for EGFR-mediated cell migration and delineate an intracellular signaling pathway connecting to EGF-induced changes in physical function, helping identify novel targets for interventions aimed at preventing or minimizing tumor invasiveness and spread. This is a revised application, for which we have developed and demonstrated improved experimental procedures, and have included studies on normal fibroblasts, as suggested by the reviewers.
