Recovery of flagellar dynein function in a Chlamydomonas actin/dynein-deficient mutant upon introduction of muscle actin by electroporation
Recovery of flagellar dynein function in a Chlamydomonas actin/dynein-deficient mutant upon introduction of muscle actin by electroporation
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DOI：
10.1002/cm.1028
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发表时间：
2001-07-01
影响因子：
--
通讯作者：
Kamiya, R
中科院分区：
文献类型：
--
作者：
Hayashi, M;Hirono, M;Kamiya, R
鞭毛和纤毛内臂动力蛋白含有肌动蛋白作为一个亚基，然而，这个肌动蛋白亚基的功能仍然未知。作为对肌动蛋白在动力蛋白中的实验操作的第一步，我们开发了一种通过电穿孔将外源性肌动蛋白引入衣原体细胞的方法。在兔骨骼肌肌动蛋白的存在下，电穿孔一个非运动突变体，ida 5 oda 1，缺乏内臂动力蛋白，由于缺乏常规肌动蛋白。在最佳条件下，约20%的电穿孔细胞恢复运动。此外，通过利用它们的趋光行为，可以集中被拯救的细胞。运动也恢复与荧光标记的肌动蛋白，在这种情况下，轴丝成为荧光电穿孔后，表明肌动蛋白实际上是作为一个动力蛋白亚基。通过电穿孔掺入大量大分子的可行性不仅可用于研究肌动蛋白功能，而且可用于使用衣原体的各种研究，其中尚未开发出表达或引入外源蛋白和其他大分子的有效方法。(C)2001 Wiley-Liss，Inc.
Flagellar and ciliary inner-arm dyneins contain actin as a subunit; however, the function of this actin subunit remains unknown. As a first step toward experimental manipulation of actin in dynein, we developed a method for introducing exogenous actin into Chlamydomonas cells by electroporation. A non-motile mutant, ida5oda1, lacking inner-arm dyneins due to the absence of conventional actin, was electroporated in the presence of rabbit skeletal muscle actin. About 20% of the electroporated cells recovered motility under optimal conditions. In addition, by taking advantage of their phototactic behavior, the rescued cells could be concentrated. Motility was also recovered with fluorescently labeled actin; in this case, axonemes became fluorescent after electroporation, suggesting that actin was in fact incorporated as a dynein subunit. The feasibility of incorporating a substantial amount of macromolecules by electroporation will be useful not only for studying actin function, but also for a variety of studies using Chlamydomonas in which no efficient methods have been developed for expressing or introducing foreign proteins and other macromolecules. (C) 2001 Wiley-Liss, Inc.