Conditional expression of a truncated fragment of nonmuscle myosin II-A alters cell shape but not cytokinesis in HeLa cells.
A truncated fragment of the nonmuscle myosin II-A heavy chain (NMHC II-A) lacking amino acids 1-591, delta N592, was used to examine the cellular functions of this protein. Green fluorescent protein (GFP) was fused to the amino terminus of full-length human NMHC II-A, NMHC II-B, and delta N592 and the fusion proteins were stably expressed in HeLa cells by using a conditional expression system requiring absence of doxycycline. The HeLa cell line studied normally expressed only NMHC II-A and not NMHC II-B protein. Confocal microscopy indicated that the GFP fusion proteins of full-length NMHC II-A, II-B, and delta N592 were localized to stress fibers. However, in vitro assays showed that baculovirus-expressed delta N592 did not bind to actin, suggesting that delta N592 was localized to actin stress fibers through incorporation into endogenous myosin filaments. There was no evidence for the formation of heterodimers between the full-length endogenous nonmuscle myosin and truncated nonmuscle MHCs. Expression of delta N592, but not full-length NMHC II-A or NMHC II-B, induced cell rounding with rearrangement of actin filaments and disappearance of focal adhesions. These cells returned to their normal morphology when expression of delta N592 was repressed by addition of doxycycline. We also show that GFP-tagged full-length NMHC II-A or II-B, but not delta N592, were localized to the cytokinetic ring during mitosis, indicating that, in vertebrates, the amino-terminus part of mammalian nonmuscle myosin II may be necessary for localization to the cytokinetic ring.
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Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Adelstein
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D008841:Actin Cytoskeleton / Q000502:physiology / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism; D000818:Animals; D002455:Cell Division / Q000502:physiology*; D048429:Cell Size / Q000502:physiology*; D005982:Glutathione Transferase / Q000235:genetics; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D000199:Actins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D018995:Myosin Heavy Chains; D009218:Myosins