MYOSIN LIGHT CHAIN-ACTIVATING PHOSPHORYLATION SITES ARE REQUIRED FOR OOGENESIS IN DROSOPHILA

The Drosophila spaghetti squash (sqh) gene encodes the regulatory myos in 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 t he actin-dependent myosin ATPase and motor activity of myosin in vitro . We have assessed the in vivo importance of these sites, which in Dro sophila correspond to serine-21 and threonine-20, by creating a series of transgenes in which these specific amino acids were altered. The p henotypes 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 bridge s linking the oocyte to the nurse cells in the egg chamber, are abnorm al, suggesting a role of myosin II in their establishment or maintenan ce. 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 Mutant sqh transgene sqh-A20, A21 in which both serine-21 and threonine-20 have been replaced by alanines behaves in most respects identically to the null allele in this system, with the exception that no heavy chain aggregates are found.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, expre ssion of sqh-A21, in which only the primary phosphorylation target ser ine-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 trans port from nurse cells to cytoplasm characteristic of late stage oogene sis, and some damaged ring canals. Substituting a glutamate for the se rine-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 phosphor ylation site is absent, appear to be entirely wild type. Taken togethe r, this genetic evidence argues that phosphorylation at serine-21 is c ritical to RMLC function in activating myosin II in vivo, but that the function can be partially provided by phosphorylation at threonine-20 .