Myo2 regulation in Schizosaccharomyces pombe

Cell division requires complex molecular assemblies, which are regulated by diverse signals. Proper segregation of genetic material during cell division depends on a contractile ring positioned between the separated nuclei; this ring includes as essential components actin and myosin. In the fission yeast Schizosaccharomyces pombe, myosin (Myo2p) localizes to nodes in early mitosis, then condenses into a medial ring which constricts and disappears. Myosin assembly is controlled primarily through its tail domain, which determines if it will form coiled-coil dimers and/or higher order assemblies such as minifilaments. With this in mind, I performed a series of in vivo truncation experiments to determine the minimal portion of the myosin tail necessary for function. Overexpression in live cells causes severe phenotypes, which are proportional to the length of the fragment present; 81 C-terminal amino acids do not localize to the contractile ring, but 165 amino acids do. I subsequently used purified recombinant myosin tail proteins and native Myo2p protein to characterize assembly properties. These proteins all form large aggregates in low salt, instead of filaments or minifilaments. I investigated phosphorylation of the fission yeast Myo2p heavy chain; it is phosphorylated in vivo on serine-1444. Like myosin-II from other species, Myo2 has two heads and a coiled-coiled tail 68 nm long. Phosphorylation does not change Myo2's enzymatic or actin binding activity. A large screen of budding yeast protein kinases found that a type I calmodulin-dependent kinase phosphorylates serine-1444 of Myo2p. Deletion of the fission yeast gene for calmodulin-dependent kinase 1 cm1l + caused dramatic delays in ring assembly and constriction compared to wild-type cells and made more variable the progress of cytokinesis, but the cells are viable, so other kinases may also act on serine-1444, albeit in a less coordinated manner. Our findings indicate that Cmk1p plays a role in coordinating cytokinetic synchronicity and timing, perhaps through effects on interactions of Myo2p with other proteins.