The Role of Nonmuscle Myosins in Cardiac Development

以前的工作已经证明，在肌球蛋白II重链的ATP结合区附近的环1中存在一个可选择剪接的外显子，导致脊椎动物肌肉和非肌肉重肌球蛋白II-B和II-C的肌动蛋白激活的镁ATPase活性和体外运动性增加。我们现在报道，在非肌肉肌球蛋白重链II-B的肌动蛋白结合部位附近的环2中插入了21个氨基酸，导致这些活性的丧失。这种可选择的外显子通常在某些神经细胞中表达，如小脑中的浦肯野细胞，并已被证明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(插入的异构体)的Vmax不到未插入的HMM II-B0的7%(Vmax：0.0003+/-0.0003 S-1；HMM II-B0的Vmax为0.15+/-0.01 S-1；n=4)，在体外运动试验中没有观察到明显的运动。在ATP存在和不存在情况下的共沉积分析表明，HMM II-B对肌动蛋白的亲和力不受环2中21个氨基酸残基插入的影响。为了研究非肌肉肌球蛋白II-B2在细胞内的活性，我们研究了全长GFP-肌球蛋白II-B拯救多核的能力。在COS-7细胞中，由于胞质分裂的缺陷，使用siRNA减少非肌肉肌球蛋白II-B0会导致多核细胞。将GFP-肌球蛋白II-B0引入II-B耗竭的细胞中，显著减少了多核。相反，将GFP-肌球蛋白II-B2导入II-B siRNA处理的细胞未能挽救多核。因此，将这21个氨基酸插入到非肌肉肌球蛋白II-B的环2中会导致体外和细胞内活性的丧失。

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.