Dynamics of actin assembly during myofibrillogenesis and troponin

在此之前，我们报道了cofilin、ADF和profilin参与肌肉发育过程中肌动蛋白组装的调控。在这项研究中，在哺乳动物中区分了两种cofilin变体，肌肉型和非肌肉型，并在肌肉发育过程中观察到从非肌肉型到肌肉型的转变。用PCR方法克隆了肌型cofilin异构体cDNA，并测定了cDNA序列。Northern blot分析显示，肌cofilin的表达在肌肉分化过程中受到诱导。此外，从鸡胚胎骨骼肌cDNA文库中克隆了profilin cDNA，并确定了其序列。微注射cofilin或转染cofilin cDNA均可提高肌原细胞中cofilin的含量，并可改变胞浆中肌动蛋白（actin）的含量。将大肠杆菌中产生的cofilin引入培养的肌肉细胞后，肌动蛋白微丝被迅速分解，胞质中产生标记的肌动蛋白/cofilin棒。但24 h内杆状结构消失，微丝恢复，提示cofilin活性逐渐减弱。另一方面，在转染cofilin cDNA的肌肉细胞中，尽管cofilin过表达，肌动蛋白丝仍未受到影响。热休克或dmso刺激引起这些细胞中肌动蛋白丝的剧烈解体。这些结果表明，cofilin在细胞质中被调节以改变其功能活性。结果表明，磷酸化是调控途径之一。3) cofilin在退行性肌肉细胞中的表达升高。在失神经或营养不良的鸡退行性骨骼肌中，cofilin的表达明显升高。这一事实表明，在肌肉变性过程中，cofilin可能参与肌动蛋白分子的动态再分配或周转。少

1) Further characterization of actin-binding proteins which regulate actin assembly in developing muscle cells Previously, we reported that cofilin, ADF and profilin is involved in the regulation of actin assembly in developing muscle. In this study, two cofilin variants, muscle-type and non-muscle-type, were distinguished in mammals, and the transition from the non-muscle-type to the muscle-type was observed during muscle development. The cDNA for the muscle-type cofilin isoform was cloned by a PCR method and the cDNA sequence was determined. Northern blot analysis showed that expression of the muscle cofilin is induced as muscle differentiates. In addition, cDNA of profilin was cloned from the cDNA library of embryonic chicken skeletal muscle and the sequence was determined. 2) Functional analysis of cofilin in cultured muscle cells Cofilin amount in myogenic cells was increased either by 1) microinjection of cofilin or 2) transfection of cofilin cDNA, and change in cytoplasmic actin … More filaments was examined. When cofilin produced in E. Coli was introduced into cultured muscle cells, actin microfilaments were disassembled quickly and marked actin/cofilin rods were generated in the cytoplasm. However, the rod structures disappeared and microfilaments were recovered within 24 hrs, suggesting that activity of cofilin was weakened gradually. On the other hand, in the muscle cells with transfection of cofilin cDNA, actin filaments were remained unaffected regardless of over-expression of cofilin. Heat-shock or DMSO-stimulation caused drastic disassembly of actin filaments in these cells. These results suggest that cofilin is modulated in the cytoplasm to alter the functional activity. Evidence was obtained which suggests that phosphorylation is one of the way of modulation. 3) Increased expression of cofilin in degenerating muscle cells Marked increase in the expression of cofilin was detected in denervated or dystrophic degenerating chicken skeletal muscle. This fact suggests that cofilin may be involved in dynamic redistribution or turnover of actin molecules during muscle degeneration. Less