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PMID:15774463
| Citation |
Bao, J, Jana, SS and Adelstein, RS (2005) Vertebrate nonmuscle myosin II isoforms rescue small interfering RNA-induced defects in COS-7 cell cytokinesis. J. Biol. Chem. 280:19594-9 |
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| 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. |
| Links |
PubMed Online version:10.1074/jbc.M501573200 |
| Keywords |
Actins/metabolism; Animals; Base Sequence; COS Cells; Cercopithecus aethiops; Cytokinesis; Gene Expression; Myosin Type II/antagonists & inhibitors; Myosin Type II/genetics; Myosin Type II/metabolism; Nonmuscle Myosin Type IIA/antagonists & inhibitors; Nonmuscle Myosin Type IIA/genetics; Nonmuscle Myosin Type IIA/metabolism; Nonmuscle Myosin Type IIB/antagonists & inhibitors; Nonmuscle Myosin Type IIB/genetics; Nonmuscle Myosin Type IIB/metabolism; Protein Isoforms/antagonists & inhibitors; Protein Isoforms/genetics; Protein Isoforms/metabolism; RNA Interference; RNA, Small Interfering/genetics; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Transfection |
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Significance
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