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蛋白和这些肌球蛋白的片段。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。 我们正在与布达佩斯Eotvos Lorand大学的Laci Nyray实验室合作，研究重链磷酸化对细丝组装的影响。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。我们还可以使用iSCAT显微镜对未标记的NM2细丝的运动进行成像。我们重新检测了我们之前发表的NM2AN93K突变体的活性，该突变体在ATPase活性和体外运动能力方面是不活跃的。最近的研究表明，如果在Sf9细胞中正确表达，该蛋白具有相当大的ATPase活性。我们认为，之前确定的不活跃的原因可能与正确折叠分子的问题有关，这可能解释了携带这种突变的人类的一些疾病表型。 此外，我们与其他人合作，利用冷冻电子显微镜获得了在ADP存在和不存在的情况下与肌动蛋白结合的NM2A和NM2B的高分辨结构。在与Nyitray实验室的合作中，我们证明了GFP封顶的肌球蛋白尾部片段形成的双极细丝类似于全长肌球蛋白形成的双极细丝。S100a是一种小的钙结合蛋白，在转移细胞中上调。该蛋白以前被证明以高亲和力结合到NM2A的远端尾巴上。它对NM2B的亲和力要低3个数量级。将S100a添加到预制的NM2A/NM2B共细丝中，可以从这些细丝中提取NM2A。因此，在细胞中，NM2A可以在有钙存在的情况下有效地隔离NM2A，而使NM2B以丝状形式存在。

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 NM2proteins and fragments of these myosins 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 lab of Laci Nytray at Eotvos Lorand University in Budapest to study the effects of heavy chain phosphorylation on filament assembly. 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. We can also image the movement of unlabeled NM2 filaments using iSCAT microscopy. We have reexamined the activity of an N93K mutant of NM2A which we previously published was inactive with regards to ATPase activity and in vitro motility. Recent work suggests that if properly expressed in Sf9 cells, the protein has substantial ATPase activity. We believe that the reason for the previous determination of inactivity may be related to problems in properly folding the molecule and this may explain some of the disease phenotypes in humans bearing this mutation. In collaboration with others, In addition, we have obtained high resolution structures of both NM2A and NM2B bound to actin in the presence and absence of ADP using cryo-electron microscopy. In collaboration with the Nyitray lab we showed that GFP-capped myosin tail fragments formed bipolar filaments similar to those formed by full length myosin. S100a is a small calcium binding protein that is upregulated in metastatic cells. This protein has previously been shown to bind to the distal tail of NM2A with high affinity. Its affinity for NM2B is 3 orders of magnitude less. S100a addition to preformed NM2A/NM2B co-filaments results in the extraction of NM2A from these filaments. Thus in a cell, NM2A could effectively sequester NM2A in the presence of calcium while leaving NM2B in filamentous form.