Actin dynamics and competition for myosin monomer govern the sequential amplification of myosin filaments

Abstract

Cellular mechanisms governing non-muscle myosin 2 (NM2) filament assembly are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we characterized and quantified the sequential amplification of NM2 filaments within lamella, wherein filaments emanating from single nucleation events continuously partition, forming filament clusters that populate large-scale actomyosin structures deeper in the cell. Individual partitioning events coincide spatially and temporally with the movements of diverging actin fibers, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibers to which they are bound. Finally, we showed that partition frequency and filament growth rate in the lamella depend on MLCK, and that MLCK is competing with centrally-active ROCK for a limiting pool of monomer with which to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and spatio-temporal regulation of NM2 filament assembly in cells.

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

“…These punctates grew brighter as they appeared to be carried away from the cell periphery by retrograde actin flow. At times, the punctates split into two parts, which then each subsequently grew brighter, presumably as more monomeric myosin added to the filaments (32,33).…”

“…A recent study (33) suggests that there is a limited pool of NM2s in cells, that the two major light-chain kinases, MLCK and ROCK, are differentially localized and compete for this pool, and that inhibition of either of these kinases results in overaccumulation of myosin in the vicinity of the other kinase.…”

“…Recent research, using superresolution light-microscopic imaging of living cells by which individual myosin filaments could be discerned, have shown that myosin filaments are very dynamic (32)(33)(34). Myosins tagged with fluorescent fusion proteins were imaged as closely spaced "dumbbells" corresponding to the ∼300-nm-long bipolar filaments assembled in vitro.…”

Nonmuscle myosin IIB regulates epicardial integrity and epicardium-derived mesenchymal cell maturation

“…The dynamics underlying changes in NMII reorganization are just beginning to be understood (Beach et al, 2017;Hu et al, 2017). EMT, the process by which static, well-ordered and apical-basal polarized epithelial cells transform into dynamically migrating mesenchymal cells associated with extensive reorganization of the actomyosin cytoskeleton is of major interest and is important for cardiac development.…”

UNC-45a helps cells manage their stress levels

“…Lappalainen speculates that the small amount of NM-II that folds and forms filaments in UNC-45a knockout cells is sufficient to mediate cytokinesis because there are no other contractile structures in mitotic cells to compete for this limited pool of myosin (4). …”