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 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 find nearly identical attachment lifetimes with either single-headed or double headed nonmuscle myosin IIB fragments. Optical trapping shows that both single-headed and double-headed myosins give 6 nm power strokes. Superresolution light microscopic measurements show that no steps of the expected 5-7 nm distance are observed. It is likely that in cells, the functional unit for nonmuscle myosins is the bipolar filament. We find that nonmuscle IIB filaments do move processively along actin filaments in vitro and that they show multiple steps in the optical trap. Interestingly, in addition to the long actin attachment lifetimes we observe with phosphorylated nonmuscle myosin IIB, we also see numerous very short lived-interactions with actin that have a detachment rate constant 50 times that of the long-lived interactions. Analysis of the optical trap displacement records show no evidence for a power stroke associated with these interactions. We believe that these are transient, nonproductive interactions between the weakly bound state of myosin with actin. If we examine the interaction of unphosphorylated (i.e. inactive) nonmuscle myosin IIB with actin in the optical trap, we see only these short-lived interactions which also show a 0 nm power stroke. We are currently examining the mechanical properties of nonmuscle myosin IIA HMM in the optical trap. We have expressed full length nonmuscle myosins IIA , IIB and IIC 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 all form short bipolar filaments of similar length. NMIIC filaments are significantly thinner than those of NMIIA and NMIIB suggesting that they contain fewer molecules. We are using 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 have shown that these mutations have little or no effect on myosin filament structure. Preliminarly results suggests that NMIIA and NMIIB form co-polymers in vitro. We have examined the kinetics of ADP release from acto-myosin-ADP for NMIIA and smooth muscle myosin and compared these rates to the rates of in vitro motility and to the steady state actin activated MgATPase as a function of temperature. We find that the temperature dependence of the in vitro motility rate and ADP release are similar, but that of the actin activated MgATPase activity differs, suggesting that ADP release is the kinetic step that determines in vitro motility. We collaborated with the Korn lab at NHLBI to show that phosphorylation accelerates the phosphate release state of myosin II from Acanthamoeba.
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