The long-term goal of this research is to define the molecular basis of amoeboid movements. The biological system that will be utilized in this grant period will be the polarized migration of cultured Swiss 3T3 cells during wound healing. Two major experimental approaches are used: 1) the quantitation of temporal and spatial chemical and molecular processes in living cells, and 2) the reconstitution of a gel-sol-contract model in vitro. The projects take advantage of advances in molecular cytology, fluorescence spectroscopy, and quantitative light microscopy. The first specific aim is to define the temporal-spatial dynamics of the myosin II-based motor, the gel-sol transformations, and the regulatory chemistry in living Swiss 3T3 cells. The multimode light microscope workstation coupled with new fluorescent probes of calcium, calcium-binding to calmodulin and phosphorylation of the myosin II regulatory light chain will yield the dynamic information. The second specific aim directly tests the role of myosin III in cell locomotion by blocking the function with an injected antibody that blocks myosin II motor activity in vitro. Furthermore, the role of anterior-posterior gradients of gelation, phosphorylation of the myosin II regulatory light chain and calcium in polarized cell movement will be tested with reagents that perturb these conditions. The third specific aim is to reconstitute the gel-sol-contract dynamics in vitro in order to test the solation- contraction coupling hypothesis of amoeboid movement and to understand the molecular interactions that cause gelation, solation and contraction. The fourth specific aim is to define the role of myosin I in polarized cell movement. Results from this study will yield basic information about how amoeboid cells move and should aid our understanding of how wounds are repaired.
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