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. Preliminary results with nonmuscle myosin IIB indicate that it has long attachment times as would be expected from such a slow myosin, but that it does not move processively in the optical trap. We have expressed nonmuscle myosin IIA heavy meromyosin with a GFP-tagged regulatory light chain (RLC) with particular interest in whether the myosin would still adopt the off state when this light chain is not phosphorylated. The data show that the GFP tag does not interfere with regulation of the myosin. 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. We will use a combination of solution studies and electron microscopy to study the assembly mechanism for these myosins. 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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