Our objective is to understand how cell migration speed is affected by altering the linkage between integrin adhesion-receptors and the cytoskeleton. This work will provide a test of our bioengineering model for the relationship between migration and adhesion, and will provide understanding useful for modulation of cell migration by pharmacologic, genetic, or materials approaches.
Specific Aims are: I. Test effects of altering integrin/cytoskeleton linkage on cell migration speed versus substratum ligand density, and its correspondence with effects on overall cell/substratum adhesiveness and cell/substratum detachment at the cell rear during migration. a. Measure dependence of cell migration speed on substratum-ligand density for integrin variants exhibiting normal and altered cytoskeleton linkage. b,c. Measure dependence of short-time (30 minutes) and long-time (4 hours) cell adhesiveness on substratum-ligand density for the same integrin variants, and attempt to correlate each with migration speed dependence. d. Compare data from Parts Ia-c on the dependence of migration speed on ligand density and on short- or long-term adhesiveness to model predictions for how the ranges of density and adhesiveness over which migration occurs should vary with front-to-rear asymmetry in linkage. e. Measure relative amounts of integrin remaining on the substratum and with the cell following cell rear detachment for the same integrin variants. II. Extend the model by incorporating details concerning integrin/cytoskeleton linkage and applying it to the new experimental data from Aim I a. Incorporate integrin/cytoskeleton linkage dynamics explicitly into the model framework to predict dependence of migration speed on ligand density and adhesiveness, and compare predictions to data from Parts Ia-d. b. Incorporate alternative cell/substratum detachment modes explicitly into the model framework to predict fraction of integrin remaining on the substratum following cell rear detachment, and compare predictions to data from Part Ie. Our experimental system is NIH3T3 mouse fibroblasts transfected with variants of chicken beta1 integrin chains containing site-directed mutations in the cytoplasmic domain affecting cytoskeletal linkage. In order to focus on effects of the beta1 mutations we study migration and adhesion of these cells on substrata coated with an anti-chicken beta1 monoclonal antibody that does not compete with the endogenous mouse integrin ligand-binding site.
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