Cell migration requires the reversible linkage between the force- generating cytoskeleton and the extracellular matrix through the integrins. The integrins form a large family of transmembrane glycoproteins which bind matrix ligands and the cytoskeleton which are pivotal for cell migration. We propose to study the molecular and physical basis for fibronectin binding and linkage to the cytoskeleton through the alpha5beta1 integrin. Using single particle tracking (SPT) and the laser tweezers, we propose to measure the time course of ligand-receptor linkage to the cytoskeleton and force generation on attached ligand. Initially, we will determine the dependence of cell region, integrin tail sequence, force, ligand aggregate size and valency on the attachment of integrin, alpha5beta1 to the cytoskeleton in 3T3 and CHO cells. Because force generation is important for migration we will follow the course of force generation by the cell on dorsally applied beads with the laser tweezers and on ventral contacts with a newly designed force-measuring chip. From biochemical studies, integrins are recruited laterally into ligand contact regions. We will determine if that recruitment is active or passive and how it depends on contact size, force, ligand density and intracellular signaling. As the cell migrates past contacts integrins must release from matrix ligands in a time or regionally dependent manner. Our preliminary evidence suggests that integrin release from both ligand and cytoskeleton is regionally dependent. On the basis of these findings we hope to understand the molecular and physical basis of fibronectin-integrin- cytoskeleton complexes in migrating cells. In the future, we can hope to better understand which steps in cell migration are modified in chemotaxis, pathfinding and with regard to alteration of specific protein function.
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