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

Abstract

Studies in fission yeast 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/9 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.

Fission yeast Myo2: Molecular organization and diffusion in the cytoplasm

“…Control preparations of skeletal muscle myosin contained bipolar filaments. These experiments are consistent with independent work by Pollard et al (), who found that Rlc1p phosphorylation does not affect the 7 S sedimentation coefficient or solubility of Myo2 in a range of KCl concentrations.…”

“…Transmission electron micrographs of these specimens contain only single molecules with two heads and a tail (Figure a and b). Pollard et al (Pollard et al, ) published similar micrographs of a few Myo2 molecules under unspecified conditions. The lengths of the Myo2 tails in our specimens varied from about 40 nm to more than 100 nm (Figure c).…”

“…In 200 mM KCl, Myo2 sediments at 6–7 S in sucrose or glycerol gradients (Supporting Information Figure S1a) like similar Type II nonmuscle myosins. Pollard et al () measured a sedimentation coefficient of 6.8 S for purified Myo2 in 50 to 500 mM KCl with or without phosphorylation of the regulatory light chain.…”

“…Fluorescence recovery after photobleaching (FRAP) confirmed the behavior of Myo2 seen in live‐cell FCS experiments. Readers will be interested in a complementary, independent study of the hydrodynamics, solubility and motor activity of Myo2 (Pollard et al, ).…”

Anchoring of actin to the plasma membrane enables tension production in the fission yeast cytokinetic ring

“…Related in vitro measurements in fission yeast were performed in ref. (Pollard et al, 2017), where the ATpase rate of fission yeast myosin Myo2 was measured versus actin concentration at saturating ATP levels.…”