title: Direct evidence for functional smooth muscle myosin II in the 10S self-inhibited monomeric conformation in airway smooth muscle cells.
abstract: The 10S self-inhibited monomeric conformation of myosin II has been characterized extensively in vitro. Based upon its structural and functional characteristics, it has been proposed to be an assembly-competent myosin pool in equilibrium with filaments in cells. It is known that myosin filaments can assemble and disassemble in nonmuscle cells, and in some smooth muscle cells, but whether or not the disassembled pool contains functional 10S myosin has not been determined. Here we address this question using human airway smooth muscle cells (hASMCs). Using two antibodies against different epitopes on smooth muscle myosin II (SMM), two distinct pools of SMM, diffuse, and stress-fiber-associated, were visualized by immunocytochemical staining. The two SMM pools were functional in that they could be interconverted in two ways: (i) by exposure to 10S- versus filament-promoting buffer conditions, and (ii) by exposure to a peptide that shifts the filament-10S equilibrium toward filaments in vitro by a known mechanism that requires the presence of the 10S conformation. The effect of the peptide was not due to a trivial increase in SMM phosphorylation, and its specificity was demonstrated by use of a scrambled peptide, which had no effect. Based upon these data, we conclude that hASMCs contain a significant pool of functional SMM in the 10S conformation that can assemble into filaments upon changing cellular conditions. This study provides unique direct evidence for the presence of a significant pool of functional myosin in the 10S conformation in cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.
authors.forename: Deanna L
authors.initials: DL
authors.lastname: Milton
authors.forename: Amy N
authors.initials: AN
authors.lastname: Schneck
authors.forename: Dominique A
authors.initials: DA
authors.lastname: Ziech
authors.forename: Mariam
authors.initials: M
authors.lastname: Ba
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Facemyer
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Halayko
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Baker
authors.forename: William T
authors.initials: WT
authors.lastname: Gerthoffer
authors.forename: Christine R
authors.initials: CR
authors.lastname: Cremo
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002461:Cell Line, Transformed; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D024462:Myosin Type II / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation; D012137:Respiratory System / Q000166:cytology; D014118:Toxins, Biological / Q000494:pharmacology
chemical_list: D010455:Peptides; D014118:Toxins, Biological; D024462:Myosin Type II