Highlights d Myosin-IIA templates myosin-II filament stacks for faster cleavage furrow ingression d Myosin-IIA generates cortex tension while myosin-IIB acts as a cortex stabilizer d Cortex tension is tuned by composition of myosin-IIA/B hetero-filaments d MIIB regulates cytokinetic fidelity through two distinct mechanisms
Cardiac muscle cells lack regenerative capacity in postnatal mammals. A concerted effort has been made in the field to determine regulators of cardiomyocyte proliferation and identify therapeutic strategies to induce division, with the ultimate goal of regenerating heart tissue after a myocardial infarct. We sought to optimize a high throughput screening protocol to facilitate this effort. We developed a straight-forward high throughput screen with simple readouts to identify small molecules that modulate cardiomyocyte proliferation. We identify human induced pluripotent stem cell-derived cardiomyocytes (hiCMs) as a model system for such a screen, as a very small subset of hiCMs have the potential to proliferate. The ability of hiCMs to proliferate is density-dependent, and cell density has no effect on the outcome of proliferation: cytokinesis or binucleation. Screening a compound library revealed many regulators of proliferation and cell death. We provide a comprehensive and flexible screening procedure and cellular phenotype information for each compound. We then provide an example of steps to follow after this screen is performed, using three of the identified small molecules at various concentrations, further implicating their target kinases in cardiomyocyte proliferation. This screening platform is flexible and cost-effective, opening the field of cardiovascular cell biology to laboratories without substantial funding or specialized training, thus diversifying this scientific community.
Force generation by the molecular motor myosin II (MII) at the actin cortex is a universal feature of animal cells. Despite its central role in driving cell shape changes, the mechanisms underlying MII regulation at the actin cortex remain incompletely understood. Here we show that Myosin Light Chain Kinase (MLCK) promotes MII turnover at the mitotic cortex. Inhibition of MLCK resulted in an alteration of the relative levels of phosphorylated Regulatory Light Chain (RLC), with MLCK preferentially creating a short-lived pRLC species and Rho associated kinase (ROCK) preferentially creating a stable ppRLC species during metaphase. Slower turnover of MII and altered RLC homeostasis upon MLCK inhibition correlated with increased cortex tension, driving increased membrane bleb initiation and growth, but reduced bleb retraction during mitosis. Taken together, we show that ROCK and MLCK play distinct roles at the actin cortex during mitosis; ROCK activity is required for recruitment of MII to the cortex, while MLCK activity promotes MII turnover. Our findings support the growing evidence that MII turnover is an essential dynamic process influencing the mechanical output of the actin cortex. [Media: see text]
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