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Spatiotemporal Regulation of Myosin II Dynamics during Cell Movement
Abstract
The goal of this research was to investigate how the phosphorylation of non-muscle myosin II (NMMII) by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) regulates cell motility. Cellular movement is important to both biological processes such as immune response, organism development, and axon guidance and to diseases such as cancer metastasis and hypertension. Movement requires a complex series of coordinated events involving the simultaneous buildup and tear down of the actomyosin cytoskeleton. The actomyosin cytoskeleton stabilizes cellular protrusions by joining with proteins in the extracellular matrix (EM), like fibronectin, and together they produce stress fibers that allow movement. NMMII regulatory light chain (RLC) phosphorylation at Serine 19 and Threonine 18 helps drive cell movement. Removing NMMII causes cells to lose structure and lose their migratory capabilities. Subsequently, phosphorylation allows NMMII to bind to actin filaments and create actomyosin crossbridges, the structural components, of the leading and lagging edges of moving cells. The dynamic activity of NMMII and MLCK at the leading edge remains undetermined in live cells, and it is also not well understood where MLCP influences cell movement on the motile edge. I investigated the moving edge of the cell using a multiparametric imaging approach with Förster resonance energy transfer (FRET) biosensors for NMMII, MLCK, and MLCP. Transfected NIH3T3 fibroblasts were imaged using fluorescence polarization microscopy. My results suggest that NMMII and MLCK activity are compartmentalized at the leading edge during cell motility and that there are differential phases of activity on a retracting membrane. We aim to understand the spatial relationship of NMMII phosphorylation with its regulators in different areas of the cell during movement. Taken together, this thesis work advances our understanding of non-muscle myosin II phosphorylation and regulation during random cell migration.Description
2019Physiology
University of Maryland, Baltimore
Ph.D.