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J. Biol. Chem., Vol. 280, Issue 20, 19594-19599, May 20, 2005

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Vertebrate Nonmuscle Myosin II Isoforms Rescue Small Interfering RNA-induced Defects in COS-7 Cell Cytokinesis^*

Jianjun Bao, Siddhartha S. Jana, and Robert S. Adelstein

From the Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

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.

Received for publication, February 10, 2005 , and in revised form, March 8, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: National Institutes of Health, Bldg. 10, Rm. 8N202, 10 Center Dr. MSC 1762, Bethesda, MD 20892-1762. Tel.: 301-496-1865; Fax: 301-402-1542; E-mail: AdelsteR{at}NHLBI.NationalInstitutesofHealth.GOV.

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