Vertebrate nonmuscle myosin II isoforms rescue small interfering RNA-induced defects in COS-7 cell cytokinesis.
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.
Bao
Jianjun
J
Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Jana
Siddhartha S
SS
Adelstein
Robert S
RS
D000199:Actins / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D048749:Cytokinesis; D015870:Gene Expression; D024462:Myosin Type II / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D024507:Nonmuscle Myosin Type IIA / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D024501:Nonmuscle Myosin Type IIB / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000199:Actins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D024462:Myosin Type II; D024507:Nonmuscle Myosin Type IIA; D024501:Nonmuscle Myosin Type IIB