Abstract
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.
Highlights
Myosin is a molecular motor that interacts with actin filaments and converts chemical energy of ATP to mechanical work
These results suggest that, whereas the critical region for myosin assembly for vertebrate skeletal myosin is near the C terminus, for Dictyostelium myosin it is away from C terminus
The results clearly indicate that the 28 residues at the C-terminal end of the ␣-helical coiled-coil domain, but not the C-terminal nonhelical tail, is the mm19 binding site and suggest that this region is critical for the filament formation of smooth muscle myosin
Summary
We produced a monoclonal antibody that disassembles smooth muscle myosin filaments. The binding site of this antibody is determined to be within the Cterminal 28 residues of the coiled-coil structure of myosin rod. The GFP-tagged truncated myosin mutant lacking these 28 residues in cells failed to show evidence of filamentous structure. The results indicated that the C-terminal 28 residues of the predicted coiled-coil domain of myosin are critical for the thick filament formation of smooth muscle myosin. The corresponding region of human nonmuscle myosin II was shown to be critical for filament formation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
