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.filament assembly | myosin phosphorylation | nuclear myosin II | stress fibers | cytoskeleton

Novel roles for actin in mitochondrial fission

“…There is evidence for such an arrangement in fission yeast cytokinesis, through use of singlemolecule high-resolution colocalization (SHREC) (Laporte et al, 2011). In this study, a distance of 70 nm between the myosin head and its tail at the membrane has been measured, suggesting that the coiled-coil tail of myosin might be folded back on itself, perhaps similar to an inactive compact conformation of many myosin II molecules identified in vitro (Billington et al, 2013;Craig et al, 1983) and in smooth muscle cells (Milton et al, 2011). Recently, activated myosin II dimers have been identified in mammalian culture cells (Shutova et al, 2014).…”

Smooth muscle function and myosin polymerization

“…Milton et al (2011) provided evidence for such a pool of myosin monomers in cultured airway smooth muscle cells. In the present study we provide further evidence suggesting that interconversion of monomeric, oligomeric and filamentous myosin occurs during length adaptation.…”

“…It dissolves filaments and also prevents filament formation when the antibody is added to monomeric myosin (Ikebe et al, 2001). This same antibody was used by Milton et al (2011) and through confocal microscopy they were able to separate filamentous from monomeric myosin, demonstrating relative specificity for myosin monomers; however, weak affinity for myosin filaments cannot be excluded. Signal detection was by a biotin-free, polymeric alkaline phosphatase (AP)-linker antibody conjugate system optimized for the BOND-RX.…”

“…One possibility is that different muscle preparations were used in the studies, i.e. cultured cells (Milton et al, 2011) versus tissue cells (Horowitz et al, 1994). Another possibility is the different cell types used; Horowitz et al used avian gizzard smooth muscle, whereas mammalian airway smooth muscle was used by Milton et al In neither study was the concentration of myosin monomers examined at different cell lengths.…”

“…Length adaptation also leads to shifting of the length-force and force-velocity relationships (Wang et al, 2001;Herrera et al, 2005), indicating changes in the underlying structure of the contractile apparatus. Milton et al (2011) showed that there is a substantial pool of functional myosin monomers that can be converted into filaments in cultured smooth muscle cells, suggesting that normal function of smooth muscle involves myosin polymerization and depolymerization. It is possible that in adaptation to a longer length where more contractile units are needed, myosin monomers are recruited into the actin filament lattice where local conditions favor formation of filaments.…”