Spatial Dynamics of Phosphorylation on the Regulatory Light Chains of Myosin II in Migrating Cells

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The broad scientific objective of this project is to understand how cells crawl during tissue development and homeostasis in mesozoans. Endothelial cells, which form the lining of blood vessels, will be studied because of the importance of their movements in the formation and repair of blood vessels. However, other tissue cells perform similar movements that are essential to a vast range of biological processes, including embryonic development, organ formation, tissue repair, the immune response, and other processes. The project will examine myosin II, a motor protein that plays an important role in coordinating crawling movements. Myosin II can contract cytoplasm, orient cell extensions, and generate forces to pull a cell forward. These activities must occur in precise locations and at appropriate times in order for directed cell movement to occur, but how myosin II behavior is controlled during cell movement is not well understood. In particular, the location and timing of the signals that control myosin II activity in living cytoplasm are unclear. Experiments will be performed to determine, in crawling cells, the kinetics and spatial distribution of phosphorylation on the regulatory light chains of myosin II, a modification known to affect the activity of isolated myosin II. Microscopic observations will be coupled with biochemical analysis to determine when and where myosin II is phosphorylated in bovine aortic endothelial cells as they crawl to heal small wounds in a cell culture model. Radioactive phosphate and antibodies that recognize phosphorylated myosin II will be used to monitor the kinetics and location of phosphorylation, and a modified form of myosin II that is labeled with a dye that changes in response to phosphorylation will also be constructed. The labeled myosin II will allow phosphorylation to be observed directly under a microscope in living cells, so that the timing and location of biochemical changes can be viewed in precise relationship to cell movements. Genetic alteration of the phosphorylation sites on myosin II, along with pharmacological agents that affect protein phosphorylation, will then be used to manipulate the kinetics of myosin II phosphorylation in migrating cells, in order to determine the extent to which phosphorylation affects myosin II behavior in vivo and, in turn, how myosin II regulation influences cell movement.Intellectual merit: By dissecting the dynamics of myosin II phosphorylation in the cytoplasm of living cells, this work will provide new and essential information about the molecular basis for regulation of the cellular locomotive machinery. Examination of phosphorylation as a potential regulatory mechanism of myosin II behavior will definitively test a widely invoked, but still largely uncertain, explanation for how cytoplasmic contractility is controlled. This will significantly advance our understanding of how cells crawl, which is a universal, fundamental biological process.Broader impacts: The interface between molecular biochemistry and cellular behavior is a growing and vital area of interest that has potential societal benefits in areas such as tissue engineering. This project will also contribute to the training of students in basic research.