Dynamics of actin assembly during myofibrillogenesis and troponin

1)肌动蛋白结合蛋白调控肌动蛋白组装的进一步研究以前，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粘连蛋白变体，肌肉类型和非肌肉类型，并观察到在肌肉发育过程中从非肌肉类型到肌肉类型的转变。用聚合酶链式反应方法克隆了肌型粘连蛋白亚型的基因，并对其进行了序列测定。Northern印迹分析表明，肌胶原蛋白的表达是在肌肉分化过程中诱导的。此外，还从鸡胚胎骨骼肌的cDNA文库中克隆了profilin基因，并进行了序列测定。2)培养的肌细胞中cofilin的功能分析：1)微量注射cofilin或2)转染cofilin cdna可增加肌源性细胞中cofilin的含量，并改变胞浆肌动蛋白…。检查了更多的细丝。将大肠杆菌产生的粘连蛋白导入培养的肌肉细胞后，肌动蛋白微丝被迅速分解，在细胞质中产生明显的肌动蛋白/粘连蛋白杆状结构。但在24小时内，杆状结构消失，微丝恢复，表明Cofilin的活性逐渐减弱。另一方面，在转导cofilin基因的肌肉细胞中，无论cofilin过表达与否，肌动蛋白细丝都不受影响。热休克或DMSO刺激导致这些细胞中肌动蛋白细丝的急剧解体。这些结果表明，cofilin在细胞质中受到调节，从而改变了功能活性。有证据表明，磷酸化是一种调节方式。3)退行性肌细胞中Cofilin的表达增加在失神经或营养不良的变性鸡骨骼肌中Cofilin的表达显著增加。这一事实表明，粘连蛋白可能参与了肌肉退行性变过程中肌动蛋白分子的动态重新分布或周转。较少

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