The investigators intend to elucidate design principles for how integrin adhesion receptors govern cell migration behavior. From the previous work, the investigators have already developed a mathematical model for cell migration that integrates integrins (e.g., expression level, ligand binding affinity) and ligand properties (e.g., concentration) with downstream physical processes underlying locomotion. They have also established that individual cell movement speed depends on integrin/ligand binding through cell/substratum adhesiveness, in accord with their model predictions. The focus of the next grant period would be on testing how this relationship is governed by integrin signaling targets (FAK and MAPK), regulators (calpain) and cytoskeletal linkage (talin), and how cell migration behavior and population distributions on ligand-coated substrata can be predicted from this relationship. The investigators will use genetically manipulated CHO cell lines having altered expression levels of integrin receptors, FAK, MAPK and calpain and also pharmacological agents to characterize basic principles governing cell migration on ligand coated substrata. The role of FAK and MAPK in modulating cell movement speed and cell/substratum adhesiveness will be studied by measuring the intercellular levels of activated FAK and MAPK in wild type CHO cells as a function of integrin number (by altering integrin expression levels), ECM ligand concentration and integrin/ligand binding affinity (cell/substratum adhesiveness), and by measuring the migration speed of mutant CHO cells with altered expression levels of FAK and MAPK activities (western blots & phosphorylation assays). The role of calpain will be addressed by measuring migration speed, adhesiveness and integrin release as a function of ECM ligand concentration using CHO cells with altered calpain activity, and also in wild type CHO cells and CHO cells with increased integrin/talin affinity. Fluorescently labeled integrin antibodies will be used to track and quantitate released integrins. An optical trap assay that employs integrin antibodies (anti-aIIbb3 or anti-a5) chemically conjugated to latex beads will be used to measure the front-vs-rear asymmetry in integrin/cytoskeleton linkage in CHO cells with altered calpain activity. The investigators will compute the basic physical and biochemical data obtained to extend their mathematical model to incorporate regulated release of integrin/cytoskeletal interactions at cell rear, and to predict cell migration behavior on spatially-patterned substrata. Finally the ability of the model to predict cell migration on patterned ligand-coated substrata will be tested by measuring cell distributions on substrata exhibiting discrete stripes of ECM ligand (low- and high-adhesive areas), and compare these to model predictions. The knowledge gained from these studies may help to control the cell migration in tissue engineering applications.
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