The nuclei of early syncytial Drosophila embryos migrate dramatically toward the poles. The cellular mechanisms driving this process, called axial expansion, are unclear, but myosin II activity is required. By following regulatory myosin light chain (RLC)–green fluorescent protein dynamics in living embryos, we observed cycles of myosin recruitment to the cortex synchronized with mitotic cycles. Cortical myosin is first seen in a patch at the anterocentral part of the embryo at cycle 4. With each succeeding cycle, the patch expands poleward, dispersing at the beginning of each mitosis and reassembling at the end of telophase. Each cycle of actin and myosin recruitment is accompanied by a cortical contraction. The cortical myosin cycle does not require microtubules but correlates inversely with Cdc2/cyclinB (mitosis-promoting factor) activity. A mutant RLC lacking inhibitory phosphorylation sites was fully functional with no effect on the cortical myosin cycle, indicating that Cdc2 must be modulating myosin activity by some other mechanism. An inhibitor of Rho kinase blocks the cortical myosin recruitment cycles and provokes a concomitant failure of axial expansion. These studies suggest a model in which cycles of myosin-mediated contraction and relaxation, tightly linked to Cdc2 and Rho kinase activity, are directly responsible for the axial expansion of the syncytial nuclei.

The Drosophila spaghetti squash ( sqh ) gene encodes the regulatory myosin light chain (RMLC) of nonmuscle myosin II. Biochemical analysis of vertebrate nonmuscle and smooth muscle myosin II has established that phosphorylation of certain amino acids of the RMLC greatly increases the actin-dependent myosin ATPase and motor activity of myosin in vitro. We have assessed the in vivo importance of these sites, which in Drosophila correspond to serine-21 and threonine-20, by creating a series of transgenes in which these specific amino acids were altered. The phenotypes of the transgenes were examined in an otherwise null mutant background during oocyte development in Drosophila females. Germ line cystoblasts entirely lacking a functional sqh gene show severe defects in proliferation and cytokinesis. The ring canals, cytoplasmic bridges linking the oocyte to the nurse cells in the egg chamber, are abnormal, suggesting a role of myosin II in their establishment or maintenance. In addition, numerous aggregates of myosin heavy chain accumulate in the sqh null cells. Mutant sqh transgene sqh -A20, A21 in which both serine-21 and threonine-20 have been replaced by alanines behaves in most respects identically to the null allele in this system, with the exception that no heavy chain aggregates are found. In contrast, expression of sqh -A21, in which only the primary phosphorylation target serine-21 site is altered, partially restores functionality to germ line myosin II, allowing cystoblast division and oocyte development, albeit with some cytokinesis failure, defects in the rapid cytoplasmic transport from nurse cells to cytoplasm characteristic of late stage oogenesis, and some damaged ring canals. Substituting a glutamate for the serine-21 (mutant sqh -E21) allows oogenesis to be completed with minimal defects, producing eggs that can develop normally to produce fertile adults. Flies expressing sqh -A20, in which only the secondary phosphorylation site is absent, appear to be entirely wild type. Taken together, this genetic evidence argues that phosphorylation at serine-21 is critical to RMLC function in activating myosin II in vivo, but that the function can be partially provided by phosphorylation at threonine-20.