Ablation of nonmuscle myosin (NM) II-A or NM II-B results in mouse embryonic lethality. Here, we report the results of ablating NM II-C as well as NM II-C/II-B together in mice. NM II-C ablated mice survive to adulthood and show no obvious defects compared with wild-type littermates. However, ablation of NM II-C in mice expressing only 12% of wild-type amounts of NM II-B results in a marked increase in cardiac myocyte hypertrophy compared with the NM II-B hypomorphic mice alone. In addition, these hearts develop interstitial fibrosis associated with diffuse N-cadherin and β-catenin localization at the intercalated discs, where both NM II-B and II-C are normally concentrated. When both NM II-C and II-B are ablated the B-C-/B-C- cardiac myocytes show major defects in karyokinesis. More than 90% of B-C-/B-C- myocytes demonstrate defects in chromatid segregation and mitotic spindle formation accompanied by increased stability of microtubules and abnormal formation of multiple centrosomes. This requirement for NM II in karyokinesis is further demonstrated in the HL-1 cell line derived from mouse atrial myocytes, by using small interfering RNA knockdown of NM II or treatment with the myosin inhibitor blebbistatin. Our study shows that NM II is involved in regulating cardiac myocyte karyokinesis by affecting microtubule dynamics.

Membrane remodeling plays a fundamental role during a variety of biological events. However, the dynamics and the molecular mechanisms regulating this process within cells in mammalian tissues in situ remain largely unknown. In this study, we use intravital subcellular microscopy in live mice to study the role of the actomyosin cytoskeleton in driving the remodeling of membranes of large secretory granules, which are integrated into the plasma membrane during regulated exocytosis. We show that two isoforms of nonmuscle myosin II, NMIIA and NMIIB, control distinct steps of the integration process. Furthermore, we find that F-actin is not essential for the recruitment of NMII to the secretory granules but plays a key role in the assembly and activation of NMII into contractile filaments. Our data support a dual role for the actomyosin cytoskeleton in providing the mechanical forces required to remodel the lipid bilayer and serving as a scaffold to recruit key regulatory molecules.

The cellular mechanisms governing non-muscle myosin II (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 lamellae, 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 fibres, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibres 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.

The integrity and function of the epithelial barrier is dependent on the apical junctional complex (AJC) composed of tight and adherens junctions and regulated by the underlying actin filaments. A major F-actin motor, myosin II, was previously implicated in regulation of the AJC, however direct evidence of the involvement of myosin II in AJC dynamics are lacking and the molecular identity of the myosin II motor that regulates formation and disassembly of apical junctions in mammalian epithelia is unknown. We investigated the role of nonmuscle myosin II (NMMII) heavy chain isoforms, A, B, and C in regulation of epithelial AJC dynamics and function. Expression of the three NMMII isoforms was observed in model intestinal epithelial cell lines, where all isoforms accumulated within the perijunctional F-actin belt. siRNA-mediated downregulation of NMMIIA, but not NMMIIB or NMMIIC expression in SK-CO15 colonic epithelial cells resulted in profound changes of cell morphology and cell-cell adhesions. These changes included acquisition of a fibroblast-like cell shape, defective paracellular barrier, and substantial attenuation of the assembly and disassembly of both adherens and tight junctions. Impaired assembly of the AJC observed after NMMIIA knock-down involved dramatic disorganization of perijunctional actin filaments. These findings provide the first direct non-pharmacological evidence of myosin II-dependent regulation of AJC dynamics in mammalian epithelia and highlight a unique role of NMMIIA in junctional biogenesis.