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Fission yeast myosin Myo2 is down-regulated in actin affinity by light chain phosphorylation

Significance The separation of daughter cells during cell division, or cytokinesis, is a process that requires contractile rings which develop tension using actin and myosin. Current models of contractile ring dynamics are based on quantitative data from two decades of research using the tractable fission yeast system. However, it is unknown how fission yeast’s essential myosin, Myo2, is regulated in the contractile ring. Here, we find that Myo2 does not assemble into minifilaments, consistent with its role in ring precursor nodes. Unphosphorylated Myo2 exhibits robust enzymatic and motor activity whereas phosphorylation of Myo2’s regulatory light chain reduces its affinity for actin. This reduction likely weakens the tension in the contractile ring, potentially to delay cytokinesis until segregation of chromosomes is complete. Studies in fission yeast Schizosaccharomyces pombe have provided the basis for the most advanced models of the dynamics of the cytokinetic contractile ring. Myo2, a class-II myosin, is the major source of tension in the contractile ring, but how Myo2 is anchored and regulated to produce force is poorly understood. To enable more detailed biochemical/biophysical studies, Myo2 was expressed in the baculovirus/Sf9 insect cell system with its two native light chains, Rlc1 and Cdc4. Milligram yields of soluble, unphosphorylated Myo2 were obtained that exhibited high actin-activated ATPase activity and in vitro actin filament motility. The fission yeast specific chaperone Rng3 was thus not required for expression or activity. In contrast to nonmuscle myosins from animal cells that require phosphorylation of the regulatory light chain for activation, phosphorylation of Rlc1 markedly reduced the affinity of Myo2 for actin. Another unusual feature of Myo2 was that, unlike class-II myosins, which generally form bipolar filamentous structures, Myo2 showed no inclination to self-assemble at approximately physiological salt concentrations, as analyzed by sedimentation velocity ultracentrifugation. This lack of assembly supports the hypothesis that clusters of Myo2 depend on interactions at the cell cortex in structural units called nodes for force production during cytokinesis.