Excessive Myosin Activity inMbsMutants Causes Photoreceptor Movement Out of theDrosophilaEye Disc Epithelium

Abstract

Neuronal cells must extend a motile growth cone while maintaining the cell body in its original position. In migrating cells, myosin contraction provides the driving force that pulls the rear of the cell toward the leading edge. We have characterized the function of myosin light chain phosphatase, which down-regulates myosin activity, in Drosophila photoreceptor neurons. Mutations in the gene encoding the myosin binding subunit of this enzyme cause photoreceptors to drop out of the eye disc epithelium and move toward and through the optic stalk. We show that this phenotype is due to excessive phosphorylation of the myosin regulatory light chain Spaghetti squash rather than another potential substrate, Moesin, and that it requires the nonmuscle myosin II heavy chain Zipper. Myosin binding subunit mutant cells continue to express apical epithelial markers and do not undergo ectopic apical constriction. In addition, mutant cells in the wing disc remain within the epithelium and differentiate abnormal wing hairs. We suggest that excessive myosin activity in photoreceptor neurons may pull the cell bodies toward the growth cones in a process resembling normal cell migration. INTRODUCTIONThe cytoskeleton plays a variety of roles during development, allowing cells to change their shape, adhesive properties, and motility (Jamora and Fuchs, 2002). Two critical components of the cytoskeleton are actin and nonmuscle myosin II. The contractile activity of actomyosin complexes has been implicated in cell migration, epithelial sheet movements, cytokinesis, axon outgrowth, and cell adhesion (Maciver, 1996;Jacinto et al., 2002;Dent and Gertler, 2003). During migration of several cell types, myosin activity is required to retract the rear of the cell (Ridley et al., 2003).Nonmuscle myosin II consists of a hexamer of two myosin heavy chains (MHC), two myosin light chains (MLC), and two myosin regulatory light chains (MRLC) (Korn and Hammer, 1988). Phosphorylation of key serine and threonine residues on MRLC stimulates the ATPase activity of MHC and promotes its assembly into filaments, leading to stress fiber contraction (Adelstein and Conti, 1975;Craig et al., 1983;Umemoto et al., 1989;Katoh et al., 2001). Mutations in the Drosophila orthologs of these myosin subunits have provided insight into the developmental functions of myosin II. Mutations in zipper (zip), which encodes MHC, cause defects in cytokinesis, closure of the dorsal embryonic epidermis over the amnioserosa, axon patterning, and myofibril formation (Zhao et al., 1988;Young et al., 1993;Bloor and Kiehart, 2001). spaghetti squash (sqh), encoding MRLC, is required for cytokinesis, oogenesis, and imaginal disc eversion (Karess et al., 1991;Wheatley et al., 1995;Edwards and Kiehart, 1996;Jordan and Karess, 1997).Actin-binding proteins of the ezrin, radixin, and moesin (ERM) family are thought to link transmembrane proteins to the actin cytoskeleton (Bretscher, 1999). ERM proteins are activated by phosphorylation of a conserved threonine residue, which inhibits associati...

Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement duringDrosophilametamorphosis

“…Fly stocks used were as follows: Esg-Gal4 (yw; NP5130) (NIG-FLY Stock Center); Esg-Gal4 UAS-GFP (y, w; NP5130, UAS-GFP, UAS-nGFP, UAS-lacZ/CyO) (NIG-FLY Stock Center); Hs-FLP; Ay+Gal4 UAS-GFP (hsp70-FLP; Act FRT y + FRT Gal4, UAS-GFP/CyO; MKRS/TM2) (Ito et al, 1997) (Royou et al, 2004); UAS-Rok CAT (Winter et al, 2001); UAS-P35 [w; ] (Bloomington Stock Center 5072); UASChickadee (L. Cooley, Yale University Medical School, Department of Genetics, New Haven, CT, USA); UAS-MbsN300 (Lee and Treisman, 2004); UAS-Dap (UAS-Dap II.3) (Lane et al, 1996); UAS-EcR-RNAi (UAS-EcR-AB RNAi) (Schubiger et al, 2005) (Oda and Tsukita, 2001); UAS-Tau-GFP (w; UAS-GFP.btau) (Brand, 1995).…”

“…To test whether myosin contraction was required for cell delamination, we overexpressed in LECs a truncated constitutively active form of the Drosophila MBS orthologue (MbsN300) that cannot be phosphorylated by Rok (generalized clonal expression of UAS-MbsN300; see Materials and methods) and inhibits Myosin contractility (Lee and Treisman, 2004). In this condition, a significant proportion of LECs did not extrude and remained within the abdominal epidermis at postmetamorphosis periods (Fig.…”

An in vivo model of apoptosis: linking cell behaviours and caspase substrates in embryos lacking DIAP1

“…Truncated ROCK then activates Myosin II light chain through phosphorylation. To ask whether caspase activation resulted in an increase in MLC phosphorylation in th 5 embryos, we prepared extracts of WT and th 5 homozygous embryos selected 1 hour after the end of cellularisation (C+1 hour), and analysed the extracts in western blots probed with an antibody recognising the phosphorylated form of Drosophila MLC (Lee and Treisman, 2004). We found a significant elevation in the amount of the phosphorylated form of MLC in th 5 embryos versus WT (Mann-Whitney U-test, P<0.001, n=12) (Fig.…”

“…Bound antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch) for 1 hour at room temperature and visualized by ECL chemiluminesce (Amersham). The following antibodies were used: anti-phospho-Myosin light chain 2 [Ser19] (1:200; Cell Signalling Technology) (Lee and Treisman, 2004), Dlg (4F3, 1:200; DSHB), Baz [1:1000 (Wodarz et al, 1999)], tubulin (E7, 1:3000; DSHB) and DE-cadherin [1:100 (Oda et al, 1994)]. Secondary antibodies used were HRP-conjugated anti-rat and antimouse (1:10,000; Jackson ImmunoResearch).…”

The Nonmuscle Myosin Phosphatase PP1β (flapwing) Negatively Regulates Jun N-Terminal Kinase in Wing Imaginal Discs of Drosophila

“…The level of P-Sqh is elevated in Mbs mutant clones (Lee and Treisman 2004). This suggests that Mbs targets PP1c to its substrate Sqh, and therefore flw might not be the only Sqh phosphatase.…”