Vertebrate nonmuscle myosin II Isoforms rescue small interfering RNA-induced defects in COS-7 cell cytokinesis
Vertebrate nonmuscle myosin II Isoforms rescue small interfering RNA-induced defects in COS-7 cell cytokinesis
复制标题
DOI：
10.1074/jbc.m501573200
复制
发表时间：
2005-05-20
影响因子：
4.8
通讯作者：
Adelstein, RS
中科院分区：
文献类型：
--
作者：
Bao, JJ;Jana, SS;Adelstein, RS
对猴 COS-7 细胞（一种缺乏非肌肉肌球蛋白重链 II-A (NMHC II-A) 但含有 NMHC II-B 和 II-C 的细胞系）进行 RNA 干扰 (RNAi) 处理，用于研究 NMHC 亚型在胞质分裂中的参与。我们使用 21 核苷酸小干扰 RNA (siRNA) 双链体特异性抑制 NMHC II-B 或 II-C 的表达。与对照细胞相比，RNAi 处理的细胞中 NMHC II-B 蛋白表达下调至 10.2 +/- 0.7% 可抑制 COS-7 细胞增殖 50%。此外，转染72小时后，8.7+/-1.0%的对照细胞是多核的，而62.4+/-8.8%的NMHC II-B RNAi处理的细胞是多核的。 RNAi 处理的细胞表面积增加，并且与对照细胞不同，缺乏肌动蛋白应力纤维。与内源性 II-C 含量相比，用 NMHC II-C siRNA 处理 COS-7 细胞，NMHC II-C 表达降低至 5.2 +/- 0.1%；然而，NMHC II-C 的下调并不会导致多核增加。使用泛肌球蛋白抗体的免疫印迹分析表明，NMHC II-C 的含量低于 NMHC II-B 含量的二十分之一，从而解释了对 II-C siRNA 缺乏反应的原因。将绿色荧光蛋白 (GFP) 标记的 NMHC II 亚型引入 II-B siRNA 处理的细胞中，导致多核化从 62.4 +/- 8.8% 减少到使用 GFP-NMHC II-B 的 17.8 +/- 2.2%，使用 GFP-NMHC II-A 减少到 29.8 +/- 7.4%，使用 NMHC 减少到 34.1 +/- 8.6% II-C-GFP。这些研究表明，COS-7细胞中内源性NMHC II-C的表达不足以进行正常的胞质分裂，而外源性NMHC II-A和NMHC II-C可以至少部分地挽救由于NMHC II-B丢失而导致的胞质分裂缺陷。
RNA interference (RNAi) treatment of monkey COS-7 cells, a cell line that lacks nonmuscle myosin heavy chain II-A (NMHC II-A) but contains NMHC II-B and II-C, was used to investigate the participation of NMHC isoforms in cytokinesis. We specifically suppressed the expression of NMHC II-B or II-C using 21 nucleotide small interfering RNA (siRNA) duplexes. Down-regulation of NMHC II-B protein expression to 10.2 +/- 0.7% inhibited COS-7 cell proliferation by 50% in the RNAi-treated cells compared with control cells. Moreover, whereas 8.7 +/- 1.0% of control cells were multinucleated, 62.4 +/- 8.8% of the NMHC II-B RNAi-treated cells were multinucleated 72 h after transfection. The RNAi-treated cells had increased surface areas and, unlike control cells, lacked actin stress fibers. Treatment of the COS-7 cells with NMHC II-C siRNA decreased NMHC II-C expression to 5.2 +/- 0.1% compared with the endogenous content of II-C; however, down-regulation of NMHC II-C did not cause increased multinucleation. Immunoblot analysis using a pan-myosin antibody showed that the content of NMHC II-C was less than one-twentieth the amount of NMHC II-B, thereby explaining the lack of response to II-C siRNA. Introducing green fluorescent protein (GFP)-tagged NMHC II isoforms into II-B siRNA-treated cells resulted in reduction of multinucleation from 62.4 +/- 8.8% to 17.8 +/- 2.2% using GFP-NMHC II-B, to 29.8 +/- 7.4% using GFP-NMHC II-A, and to 34.1 +/- 8.6% using NMHC II-C- GFP. These studies have shown that expression of endogenous NMHC II-C in COS-7 cells is insufficient for normal cytokinesis and that exogenous NMHC II-A and NMHC II-C can, at least partially, rescue the defect in cytokinesis due to the loss of NMHC II-B.