Regulation Of Smooth and Nonmuscle Myosin

非肌肉肌球蛋白2(NM2)分子在细胞内具有广泛的功能。有三个NM2重链基因。我们在杆状病毒Sf9系统中表达了全长NM2。我们正在研究它们的细丝结构，以及重链和轻链的磷酸化如何影响细丝的形成。我们使用单丝运动分析系统，其中我们可以成像荧光标记的肌球蛋白细丝在固定在表面的肌动蛋白细丝上的运动。我们正在研究NM2A和NM2B形成的共聚在体外的共聚。 我们正在与NHLBI的Korn实验室合作，研究重链磷酸化对细丝组装的影响。我们用Sarah Hitchcock-DiGregori研究了不同的非肌原肌球蛋白亚型对NM2A、NM2B和NM2C HMM亚型的肌动蛋白激活的ATPase活性和体外运动的影响，发现一些TM亚型激活一个或多个HMM，而一些不激活。 光学俘获研究表明，NM2A和NM2B不是作为单分子进行的。含有约30个肌球蛋白分子的NM2B的双极细丝沿着附着在表面的肌动蛋白细丝连续移动。令人惊讶的是，在这些相同的条件下，NM2A的细丝不会连续移动，这可能是因为与NM2B相比，这种肌球蛋白的占空比更低。NM2A可以与NM2B分子共聚，如果存在足够的NM2B，这些异型细丝可以连续移动。在模拟细胞质粘度的0.5%甲基纤维素存在下，NM2A和NM2B细丝都进行了连续运动。 我们已经表达了果蝇非肌肉肌球蛋白2的S1、HMM和全长结构，并表明在酶的作用下，该蛋白具有低的占空比，磷酸盐的释放是速度限制的，并且HMM和全长蛋白的酶活性需要调节轻链的磷酸化。电子显微镜显示，肌球蛋白形成长约300 nm的双极细丝，每个细丝约14-16个肌球蛋白。我们用模拟磷酸化的调节轻链共表达这些分子，从而将可磷酸化的丝氨酸和苏氨酸残基替换为带负电荷的谷氨酸残基。研究了ATPase活性、体外运动能力和微丝形成情况。令人惊讶的是，没有一个模拟磷酸化的突变轻链复制了真正的磷酸化的激活效应。

Nonmuscle myosin 2 (NM2) molecules carry out a wide variety of functions within cells. There are three NM2 heavy chain genes. We are expressing full length NM2 in the baculovirus Sf9 system. We are studying their filament structure and how phosphorylation of both the heavy chain and light chain affects filament formation. We use a single filament motility assay system wherein we can image the movement of fluorescently labeled myosin filaments over actin filaments fixed to the surface. We are examining the copolymerization of NM2A and NM2B form co-polymers in vitro. We are collaborating with the Korn lab at NHLBI to study the effects of heavy chain phosphorylation on filament assembly. With Sarah Hitchcock-DiGregori we have examined the effect of various nonmuscle tropomyosin isoforms on the actin activated ATPase activity and in vitro motility of NM2A, NM2B and NM2C HMM isoforms and find that some TM isoforms activate one or more of the HMMs whereas some do not. Optical trapping studies reveal that NM2A and NM2B are not processive as single molecules. Bipolar filaments of NM2B containing about 30 myosin molecules move processively along actin filaments attached to the surface. Surprisingly, filaments of NM2A do not move processively under these same conditions which may be due to the lower duty ratio of this myosin compared to that of NM2B. NM2A can be co-polymerized with NM2B molecules and these heterotypic filaments move processively provided sufficient NM2B is present. In the presence of 0.5% methylcellulose which mimics the viscosity of the cytoplasm both NM2A and NM2B filaments move processively. We have expressed the S1, HMM and full length constructs for Drosophila nonmuscle myosin 2 and have shown that enzymatically the protein has a low duty ratio with phosphate release being rate limiting and that the enzymatic activity of the HMM and full length proteins require phosphorylation of the regulatory light chain. Electron microscopy shows that the myosin forms bipolar filaments of about 300 nm in length with about 14-16 myosins per filament. We co-expressed these molecules with phospho-mimetic regulatory light chains whereby the the phosphorylatable serine and threonine residues were replaced with negatively charged glutamic acid residues. ATPase activity, in vitro motility and filament formation was studied. Surprisingly, non of the phospho-mimetic mutant light chains replicated the activating effect of true phosphorylation.