Nonmuscle myosin II molecules carry out a wide variety of functions within cells. There are three nonmuscle myosin II genes. We are using optical trapping nanometry to study the interaction of nonmuscle myosin IIB with actin. When phosphorylated this myosin has long attachment times as would be expected from such a slow myosin, but that it does not move processively in the optical trap and shows only attachments and detachments without stepping in a single molecule motility assay in which actin is bound to the surface and the interaction with fluorescently-labeled myosin is observed. We have expressed full length nonmuscle myosins IIA and IIB and have characterized their steady state MgATPase properties. We have examined the filament structure of these myosins using negative staining electron microscopy and find that both form short bipolar filaments of similar length and thickness. These short filaments move processively in the above mentioned motility assay. We will use a combination of solution studies and electron microscopy to study the assembly mechanism for these myosins and observe their interaction with actin. We have also expressed mutant forms of nonmuscle myosin IIA corresponding to naturally occuring, disease causing mutations that give rise to giant platelet disorders and will characterize the effect of these mutations on myosin function and filament assembly.
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