Regulation Of Smooth and Nonmuscle Myosin

非肌肉肌球蛋白II分子在细胞内执行各种各样的功能。 有三个非肌肉肌球蛋白II基因。 我们正在使用光学捕获纳米技术来研究非肌肉肌球蛋白IIB与肌动蛋白的相互作用。 当磷酸化时，这种肌球蛋白具有长的附着时间，这是从这样一个缓慢的肌球蛋白所预期的，但它不会在光阱中向前移动，并且在单分子运动测定中仅显示附着和分离，而不会步进，其中肌动蛋白结合到表面，并且观察到与荧光标记的肌球蛋白的相互作用。 我们发现几乎相同的附着寿命与单头或双头非肌肉肌球蛋白IIB片段。 光学捕获显示，单头和双头肌球蛋白都产生6 nm的功率冲程。 超分辨光学显微镜测量表明，没有观察到预期的5-7 nm距离的台阶。 在细胞中，非肌肉肌球蛋白的功能单位很可能是双极丝。 我们发现，非肌肉IIB丝在体外确实沿着沿着肌动蛋白丝向前移动，并且它们在光阱中显示出多个步骤。 有趣的是，除了我们观察到的磷酸化非肌肉肌球蛋白IIB的长肌动蛋白附着寿命外，我们还看到许多非常短的与肌动蛋白的相互作用，其分离速率常数是长寿命相互作用的50倍。 光阱位移记录的分析表明，没有证据表明与这些相互作用的动力冲程。 我们相信，这些是短暂的，非生产性的相互作用之间的弱结合状态的肌球蛋白与肌动蛋白。 如果我们检查非磷酸化（即无活性）的非肌肉肌球蛋白IIB与肌动蛋白在光阱中的相互作用，我们只看到这些短暂的相互作用，它们也显示出0 nm的功率冲程。 我们已经表达了全长非肌肉肌球蛋白IIA，IIB和IIC，并表征了其稳态MgATPase特性。 我们用负染电子显微镜检查了这些肌球蛋白的细丝结构，发现两者都形成长度和厚度相似的短双极细丝。 我们正在使用溶液研究和电子显微镜相结合的方法来研究这些肌球蛋白的组装机制，并观察它们与肌动蛋白的相互作用。 我们也表达了非肌肉肌球蛋白IIA的突变形式，对应于自然发生的引起巨大血小板疾病的致病突变，并表明这些突变对肌球蛋白丝结构几乎没有影响。 我们已经研究了ADP释放的动力学从肌动蛋白-ADP的NMIIA和平滑肌肌球蛋白，并比较这些速率在体外运动的速率和稳态肌动蛋白激活MgATPase作为温度的函数。 我们发现，在体外运动率和ADP释放的温度依赖性是相似的，但肌动蛋白激活的MgATPase活性的不同，这表明ADP释放是决定在体外运动的动力学步骤。 非肌肉肌球蛋白II分子在细胞内执行各种各样的功能。 有三个非肌肉肌球蛋白II基因。 我们正在使用光学捕获纳米技术来研究非肌肉肌球蛋白IIB与肌动蛋白的相互作用。 当磷酸化时，这种肌球蛋白具有长的附着时间，这是从这样一个缓慢的肌球蛋白预期的，但它不会在光阱中向前移动，并且仅显示附着和分离，而不会在单分子运动测定中步进，其中肌动蛋白结合到表面，并观察到与荧光标记的肌球蛋白的相互作用。 我们已经表达了全长非肌肉肌球蛋白IIA和IIB，并表征了其稳态MgATPase特性。 我们用负染电子显微镜检查了这些肌球蛋白的细丝结构，发现两者都形成长度和厚度相似的短双极细丝。 这些短纤维在上述运动性测定中向前移动。我们将使用溶液研究和电子显微镜相结合的方法来研究这些肌球蛋白的组装机制，并观察它们与肌动蛋白的相互作用。 我们还表达了非肌肉肌球蛋白IIA的突变形式，对应于自然发生的引起巨大血小板疾病的致病突变，并将表征这些突变对肌球蛋白功能和细丝组装的影响。

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 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 both form short bipolar filaments of similar length and thickness. 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. 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. 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.