The tip of the coiled-coil rod determines the filament formation of smooth muscle and nonmuscle myosin.
Myosin II self-assembles to form thick filaments that are attributed to its long coiled-coil tail domain. The present study has determined a region critical for filament formation of vertebrate smooth muscle and nonmuscle myosin II. A monoclonal antibody recognizing the 28 residues from the C-terminal end of the coiled-coil domain of smooth muscle myosin II completely inhibited filament formation, whereas other antibodies recognizing other parts of the coiled-coil did not. To determine the importance of this region in the filament assembly in vivo, green fluorescent protein (GFP)-tagged smooth muscle myosin was expressed in COS-7 cells, and the filamentous localization of the GFP signal was monitored by fluorescence microscopy. Wild type GFP-tagged smooth muscle myosin colocalized with F-actin during interphase and was also recruited into the contractile ring during cytokinesis. Myosin with the nonhelical tail piece deleted showed similar behavior, whereas deletion of the 28 residues at the C-terminal end of the coiled-coil domain abolished this localization. Deletion of the corresponding region of GFP-tagged nonmuscle myosin IIA also abolished this localization. We conclude that the C-terminal end of the coiled-coil domain, but not the nonhelical tail piece, of myosin II is critical for myosin filament formation both in vitro and in vivo.
Ikebe
M
M
Departments of Physiology and Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. mitsuo.ikebe@umassmed.edu
Komatsu
S
S
Woodhead
J L
JL
Mabuchi
K
K
Ikebe
R
R
Saito
J
J
Craig
R
R
Higashihara
M
M
D000199:Actins / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002455:Cell Division; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins; D007399:Interphase; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009130:Muscle, Smooth / Q000378:metabolism*; D009154:Mutation; D009218:Myosins / Q000737:chemistry* / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014421:Turkey
D000199:Actins; D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009218:Myosins