Myosin Light Chain–activating Phosphorylation Sites Are Required for Oogenesis in Drosophila

Abstract

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 Mutant sqh transgene sqh-A20, A21 in which both serine-21 and threonine-20 have been replaced by alanines behaves in most respects identically to the null allele in this system, with the exception that no heavy chain aggregates are found.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.

Distinct pathways control recruitment and maintenance of myosin II at the cleavage furrow during cytokinesis

“…Rho1 activates rho kinase and citron kinase, both of which phosphorylate and activate myosin II RLC (27)(28)(29). However, nonphosphorylatable mutants of the Drosophila myosin II RLC are still correctly localized in egg chambers (30). Thus, it is unclear whether rho1 signaling affects myosin II localization directly or through its effect on the F-actin network.…”

Coronin 1B supports RhoA signaling at cell-cell junctions through Myosin II

“…The phosphomimetic form of the myosin regulatory light chain (MRLC-DD) has been previously shown to restore NMII contractility to cells when its upstream activators are inhibited. 27,28 Indeed, expression of MRLC-DD in Coronin 1B K D MCF-7 cells restored junctional RhoA localization. (Fig.…”

Reassessing the Role and Dynamics of Nonmuscle Myosin II during Furrow Formation in EarlyDrosophilaEmbryos

“…The isolation and cloning of sqh 1 and sqh AX3 are described in Karess et al (1991) and Jordan and Karess (1997)). The FLP-DFS system of Chou and Perrimon (1992) and Chou et al (1993) was used to generate homozygous sqh 1 germline clones as described by Wheatley et al (1995).…”