Abstract
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.
Highlights
The three isoforms are well conserved throughout the whole molecule with a 64 – 80% identity in amino acids among the various isoforms (5), suggesting that they might share some cellular functions
We have shown that NMHC II-A and II-C can partially rescue a defect in cytokinesis induced by down-regulation of NMHC II-B
We provided evidence that the total noblotting of the total cell lysates from nonsense (Control) siRNAtreated cells, NMHC II-B small interfering RNA (siRNA) ϩ green fluorescent protein (GFP)-transfected cells, and NMHC II-B siRNAϩ NMHC II-C-GFP-transfected cells using an antibody against human NMHC II-C
Summary
Vol 280, No 20, Issue of May 20, pp. 19594 –19599, 2005 Printed in U.S.A. Vertebrate Nonmuscle Myosin II Isoforms Rescue Small Interfering RNA-induced Defects in COS-7 Cell Cytokinesis*. 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. Nonmuscle myosin II plays a major role in cleavage furrow formation and ingression by assembling with actin to form an actomyosin-based contractile ring This important function of nonmuscle myosin II in cytokinesis has been studied extensively (20 –23). To investigate the participation of NMHC II isoforms in regulating cytokinesis, we down-regulated NMHC II-B or II-C expression by using small interfering RNA (siRNA) in COS-7 cells, a cell line that lacks NMHC II-A but contains NMHC II-B as well as II-C. This defect in COS-7 cells can be rescued by NMHC II-B and to a lesser extent by NMHC II-A and II-C
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