The Role of Nonmuscle Myosins in Cardiac Development

先前的工作已经证明，在肌球蛋白II重链的ATP结合区附近的环1中存在选择性剪接的外显子，导致脊椎动物平滑肌和非肌肉重裂肌球蛋白（HMPs）II-B和II-C的肌动蛋白激活的MgATP酶活性和体外运动性增加。我们现在报告，在环2，这是附近的肌动蛋白结合位点的非肌肉肌球蛋白重链II-B的21个氨基酸的插入的存在下，在这些活动的损失的结果。这种选择性外显子通常在某些神经元细胞中表达，如小脑中的浦肯野细胞，并已显示影响小鼠的运动协调。令人惊讶的是，杆状病毒表达的HMM II-B2（插入的同种型）的Vmax小于未插入的HMM II-B 0的Vmax的7%（Vmax：HMM II-B2为0.01 +/- 0.0003 s-1，而HMM II-B 0为0.15 +/- 0.01 s-1;每种n=4），并且在HMM II-B2的体外运动试验中没有观察到显著的运动。共沉淀分析在ATP的存在和不存在下表明，HMM II-B的肌动蛋白的亲和力不受插入到环2的21个氨基酸残基。为了研究非肌肉肌球蛋白II-B2在细胞中的活性，我们研究了全长GFP-肌球蛋白II-B拯救多核化的能力。在COS-7细胞中，由于胞质分裂缺陷，使用siRNA减少非肌肉肌球蛋白II-B 0导致多核细胞。将GFP-肌球蛋白II-B 0引入II-B耗尽的细胞中显著减少多核化。相反，GFP-肌球蛋白II-B2转染到II-B siRNA处理的细胞未能挽救多核化。因此，将这21个氨基酸插入非肌肉肌球蛋白II-B的环2中会导致体外和细胞中活性的丧失。

Previous work has demonstrated that the presence of an alternatively-spliced exon in loop 1 near the ATP-binding region of the myosin II heavy chain results in an increase in both the actin-activated MgATPase activity and in vitro motility of vertebrate smooth muscle and nonmuscle heavy meromyosins (HMMs) II-B and II-C. We now report that the presence of a 21 amino acid insert in loop 2, which is near the actin-binding site of nonmuscle myosin heavy chain II-B, results in the loss of these activities. This alternative exon is normally expressed in certain neuronal cells, such as the Purkinje cells in the cerebellum and has been shown to affect motor coordination in mice. Surprisingly, the Vmax of baculovirus-expressed HMM II-B2 (inserted isoform) was less than 7% of that of the noninserted HMM II-B0 (Vmax: 0.01 +/- 0.0003 s-1 for HMM II-B2 compared to 0.15 +/- 0.01 s-1 for the HMM II-B0; n=4 for each) and no significant movement was observed in the in vitro motility assay for HMM II-B2. Cosedimentation assays in the presence and absence of ATP showed that the affinity of HMM II-B for actin is not affected by the insertion of the 21 amino acid residues into loop 2. To study the activity of nonmuscle myosin II-B2 in cells, we investigated the ability of full-length GFP-myosin II-B to rescue multinucleation. In COS-7 cells, decreasing nonmuscle myosin II-B0 using siRNA results in multinucleated cells due to a defect in cytokinesis. Introducing GFP-myosin II-B0 into the II-B depleted cells significantly reduced multinucleation. In contrast, transfection of GFP-myosin II-B2 into the II-B siRNA-treated cells failed to rescue multinucleation. Therefore, insertion of these 21 amino acids into loop 2 of nonmuscle myosin II-B causes a loss of activity both in vitro and in cells.