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.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Lymn, Richard W
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Carnegie-Mellon University
Schools of Arts and Sciences
United States
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Vanni, Steven; Lagerholm, B Christoffer; Otey, Carol et al. (2003) Internet-based image analysis quantifies contractile behavior of individual fibroblasts inside model tissue. Biophys J 84:2715-27
Lagerholm, B Christoffer; Vanni, Steven; Taylor, D Lansing et al. (2003) Cytomechanics applications of optical sectioning microscopy. Methods Enzymol 361:175-97
Abraham, V C; Krishnamurthi, V; Taylor, D L et al. (1999) The actin-based nanomachine at the leading edge of migrating cells. Biophys J 77:1721-32
Burton, K; Park, J H; Taylor, D L (1999) Keratocytes generate traction forces in two phases. Mol Biol Cell 10:3745-69
DeBiasio, R L; LaRocca, G M; Post, P L et al. (1996) Myosin II transport, organization, and phosphorylation: evidence for cortical flow/solation-contraction coupling during cytokinesis and cell locomotion. Mol Biol Cell 7:1259-82
Giuliano, K A; Post, P L; Hahn, K M et al. (1995) Fluorescent protein biosensors: measurement of molecular dynamics in living cells. Annu Rev Biophys Biomol Struct 24:405-34
Giuliano, K A; Taylor, D L (1995) Measurement and manipulation of cytoskeletal dynamics in living cells. Curr Opin Cell Biol 7:4-12
Post, P L; DeBiasio, R L; Taylor, D L (1995) A fluorescent protein biosensor of myosin II regulatory light chain phosphorylation reports a gradient of phosphorylated myosin II in migrating cells. Mol Biol Cell 6:1755-68
Giuliano, K A; Taylor, D L (1994) Fluorescent actin analogs with a high affinity for profilin in vitro exhibit an enhanced gradient of assembly in living cells. J Cell Biol 124:971-83
Post, P L; Trybus, K M; Taylor, D L (1994) A genetically engineered, protein-based optical biosensor of myosin II regulatory light chain phosphorylation. J Biol Chem 269:12880-7

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