Regulation Of Smooth and Nonmuscle Myosin

平滑肌和非肌肉肌球蛋白的调节

• 批准号: 8557910
• 负责人: James Sellers
• 金额: $ 25.83万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8557910
• 关键词: Actins; Affect; Binding; Biological Assay; Blood Platelet Disorders; Cells; Dependence; Electron Microscopy; Filament; Genes; Giant Platelet; Head; In Vitro; Kinetics; Label; Length; Life; Light; Link; Measurement; Microfilaments; Microscopic; Mutation; Myosin ATPase; Myosin Type II; Negative Staining; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Phosphorylation; Power stroke; Property; Records; Regulation; Smooth Muscle Myosins; Solutions; Structure; Surface; Takeda brand of pioglitazone hydrochloride; Temperature; Thick; Time; base; cell motility; disease-causing mutation; human disease; in vivo; interest; mutant; non-muscle myosin; optical traps; single molecule

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.

非肌肉肌球蛋白II分子在细胞内执行各种功能。 有三个非肌肉肌球蛋白II基因。 我们正在使用光学诱捕纳米测定法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，这种肌球蛋白具有很长的固定时间，就像从如此缓慢的肌球蛋白中所期望的那样，但是它不会在光学陷阱中进行过程移动，并且仅显示附着和脱离而不在单个分子运动测定中踩踏而无需踩踏肌动蛋白与表面绑定到表面及其与蒸发肌蛋白的相互作用的相互作用。 我们发现单头或双头非肌球蛋白IIB片段几乎相同的固定寿命。 光学诱捕表明，单头和双头肌球蛋白都具有6 nm的动力中风。 上分辨率的光显微镜测量表明，未观察到预期的5-7 nm距离的步骤。 在细胞中，非肌肉肌球蛋白的功能单元是双极细丝。 我们发现，非肌IIB丝在体外确实沿肌动蛋白丝进行了过程，并且它们在光学陷阱中显示了多个步骤。 有趣的是，除了我们使用磷酸化的非肌肌球蛋白IIB观察到的长肌动蛋白附着寿命外，我们还看到许多与肌动蛋白的生活相互作用非常短，其分离率的恒定恒定是长期相互作用的50倍。 对光学陷阱位移记录的分析没有证据表明与这些相互作用相关的电动中风。 我们认为，这些是肌动蛋白弱结合的状态与肌动蛋白之间的短暂性，非生产性相互作用。 如果我们检查未磷酸化的（即无活性）非肌球蛋白IIB与肌动蛋白在光学陷阱中的相互作用，我们只会看到这些短寿命的相互作用，这些相互作用也显示出0 nm的功率中风。 我们已经表达了全长的非肌动物IIA，IIB和IIC，并表征了它们的稳态MGATPase特性。 我们已经使用负染色电子显微镜检查了这些肌动物的细丝结构，并发现两者都形成了相似长度和厚度的短双极丝。 我们正在使用溶液研究和电子显微镜的组合来研究这些肌动物的装配机制，并观察它们与肌动蛋白的相互作用。 我们还表达了与自然发生的非肌肉肌球蛋白IIA的突变形式，导致突变导致巨型血小板疾病，并表明这些突变对肌球蛋白细丝结构几乎没有影响。 我们已经检查了NMIIA和平滑肌肌球蛋白从Acto-Myosin-ADP中释放ADP的动力学，并将这些速率与体外运动速率以及稳态肌动蛋白激活的MGATPase作为温度的函数进行了比较。 我们发现体外运动速率和ADP释放的温度依赖性相似，但是肌动蛋白激活的MGATPase活性的依赖性有所不同，这表明ADP释放是决定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功能。 有三个非肌肉肌球蛋白II基因。 我们正在使用光学诱捕纳米测定法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，这种肌球蛋白具有很长的固定时间，就像从如此缓慢的肌球蛋白中所期望的那样，但是它不会在光学陷阱中进行过程移动，并且仅显示肌动蛋白与表面绑定到表面的单个分子运动测定法，并且观察到肌动蛋白的相互作用，而无需踏入单个分子运动测定。 我们已经表达了全长的非肌动物IIA和IIB，并表征了它们的稳态MGATPase特性。 我们已经使用负染色电子显微镜检查了这些肌动物的细丝结构，并发现两者都形成了相似长度和厚度的短双极丝。 这些简短的细丝在上述运动分析中进行了过程。我们将使用溶液研究和电子显微镜的组合来研究这些肌动物的组装机制，并观察它们与肌动蛋白的相互作用。 我们还表达了与自然发生的非肌肉肌球蛋白IIA的突变形式，导致突变引起巨大的血小板疾病，并将表征这些突变对肌球蛋白功能和细丝组装的影响。