Nagaoka R, Kusano K, Abe H, Obinata T
Department of Biology, Faculty of Science, Chiba University, Japan.
J Cell Sci. 1995 Feb;108 ( Pt 2):581-93. doi: 10.1242/jcs.108.2.581.
The previous investigation (Abe et al. (1989) J. Biochem. 106, 696-702) suggested that cofilin is deeply involved in the regulation of actin assembly in developing skeletal muscle. In this study, to examine further the function of cofilin in living myogenic cells in culture, recombinant cofilin having extra Cys residues at the N terminus was produced in Escherichia coli and was labeled with tetramethylrhodamine-iodoacetamide (IATMR). When the cofilin labeled with IATMR (IATMR-cofilin) was introduced into myogenic cells, actin filaments in the cytoplasm or nascent myofibrils were promptly disrupted, and many cytoplasmic rods which contained both IATMR-cofilin and actin were generated. Sarcomeric myofibrillar structures were not disrupted but tropomyosin was dissociated from the structures by the exogenous cofilin, and the IATMR-cofilin became localized in I-band regions. 24 hours after the injection, however, the actin-cofilin rods disappeared completely and the IATMR-cofilin became diffused in the cytoplasm as endogenous cofilin. Concomitantly, actin filaments were recovered and tropomyosin was re-associated with sarcomeric I-bands. At this point, the IATMR-cofilin in the cells still retained the functional activity to form intranuclear actin-cofilin rods in response to stimulation by DMSO just as endogenous cofilin. FITC-labeled actin introduced into myogenic cells at first failed to assemble into filamentous structures in the presence of the exogenous cofilin, but was gradually incorporated into myofibrils with time. The drastic effects of the exogenous cofilin on actin assembly were suppressed by phosphatidylinositol 4,5-bisphosphate (PIP2). These results indicate that the exogenous cofilin is active and alters actin dynamics remarkably in muscle cells, but its activity in the cytoplasm gradually becomes regulated by the action of some factors including PIP2-binding.
先前的研究（Abe等人，(1989) J. Biochem. 106, 696 - 702）表明，丝切蛋白在发育中的骨骼肌肌动蛋白组装调节中起重要作用。在本研究中，为了进一步研究丝切蛋白在培养的活肌原细胞中的功能，在大肠杆菌中产生了在N端具有额外半胱氨酸残基的重组丝切蛋白，并用四甲基罗丹明 - 碘乙酰胺（IATMR）进行标记。当用IATMR标记的丝切蛋白（IATMR - 丝切蛋白）被引入肌原细胞时，细胞质或新生肌原纤维中的肌动蛋白丝迅速被破坏，并产生了许多同时含有IATMR - 丝切蛋白和肌动蛋白的细胞质杆状物。肌节肌原纤维结构未被破坏，但原肌球蛋白通过外源性丝切蛋白从结构上解离，并且IATMR - 丝切蛋白定位在I带区域。然而，注射24小时后，肌动蛋白 - 丝切蛋白杆状物完全消失，IATMR - 丝切蛋白像内源性丝切蛋白一样在细胞质中扩散。与此同时，肌动蛋白丝恢复，原肌球蛋白重新与肌节I带结合。此时，细胞中的IATMR - 丝切蛋白仍然保留了响应二甲基亚砜刺激形成核内肌动蛋白 - 丝切蛋白杆状物的功能活性，就像内源性丝切蛋白一样。最初引入肌原细胞的异硫氰酸荧光素（FITC）标记的肌动蛋白在存在外源性丝切蛋白的情况下未能组装成丝状结构，但随着时间的推移逐渐整合到肌原纤维中。磷脂酰肌醇4,5 - 二磷酸（PIP2）抑制了外源性丝切蛋白对肌动蛋白组装的显著影响。这些结果表明，外源性丝切蛋白具有活性，并在肌肉细胞中显著改变肌动蛋白动力学，但其在细胞质中的活性逐渐受到包括PIP2结合在内的一些因素作用的调节。
