The goal of our research is to understand the dynamic properties of actin and myosin in living cells and the mechanisms of the assembly and disassembly of contractile structures. In the present project, we will focus on the movement of actin and myosin filaments in living fibroblasts, ty examining fluorescently labeled exogenous or endogenous filaments with low light level video microscopy and digital image processing. We will first microinject fragments of actin filaments labeled with fluorescent phalloidin. Their movements will be studied both by directly tracking the position and by fluorescence recovery after photobleaching. Similar studies will be performed on fluorescently labeled endogenous myosin filaments, and on fluorescent polystyrene beads coated with myosin. The movement of these structures will be analyzed in different regions of the cell and will be correlated with cell locomotion. In addition, the effects of transformation and treatments mimicking transformation on these filaments will be examined. We will further study the intermixing of actin filaments after cell fusion, and the role of filament movement and sequestration in the formation of various contractile structures. These results will help us understand not only the normal functions of actin-containing structures, but also the disruptions caused by oncogenic transformation.
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