The leading edge during dorsal closure as a model for epithelial plasticity: Pak is required for recruitment of the Scribble complex and septate junction formation.

Dorsal closure (DC) of the Drosophila embryo is a model for the study of wound healing and developmental epithelial fusions, and involves the sealing of a hole in the epidermis through the migration of the epidermal flanks over the tissue occupying the hole, the amnioserosa. During DC, the cells at the edge of the migrating epidermis extend Rac- and Cdc42-dependent actin-based lamellipodia and filopodia from their leading edge (LE), which exhibits a breakdown in apicobasal polarity as adhesions are severed with the neighbouring amnioserosa cells. Studies using mammalian cells have demonstrated that Scribble (Scrib), an important determinant of apicobasal polarity that functions in a protein complex, controls polarized cell migration through recruitment of Rac, Cdc42 and the serine/threonine kinase Pak, an effector for Rac and Cdc42, to the LE. We have used DC and the follicular epithelium to study the relationship between Pak and the Scrib complex at epithelial membranes undergoing changes in apicobasal polarity and adhesion during development. We propose that, during DC, the LE membrane undergoes an epithelial-to-mesenchymal-like transition to initiate epithelial sheet migration, followed by a mesenchymal-to-epithelial-like transition as the epithelial sheets meet up and restore cell-cell adhesion. This latter event requires integrin-localized Pak, which recruits the Scrib complex in septate junction formation. We conclude that there are bidirectional interactions between Pak and the Scrib complex modulating epithelial plasticity. Scrib can recruit Pak to the LE for polarized cell migration but, as migratory cells meet up, Pak can recruit the Scrib complex to restore apicobasal polarity and cell-cell adhesion.

The Drosophila homologue of Arf-GAP GIT1, dGIT, is required for proper muscle morphogenesis and guidance during embryogenesis.

GIT1-like proteins are GTPase-activating proteins (GAPs) for Arfs and interact with a variety of signaling molecules to function as integrators of pathways controlling cytoskeletal organization and cell motility. In this report, we describe the characterization of a Drosophila homologue of GIT1, dGIT, and show that it is required for proper muscle morphogenesis and myotube guidance in the fly embryo. The dGIT protein is concentrated at the termini of growing myotubes and localizes to muscle attachment sites in late stage embryos. dgit mutant embryos show muscle patterning defects and aberrant targeting in subsets of their muscles. dgit mutant muscles fail to localize the p21-activated kinase, dPak, to their termini. dPak and dGIT form a complex in the presence of dPIX and dpak mutant embryos show similar muscle morphogenesis and targeting phenotypes to that of dgit. We propose that dGIT and dPak are part of a complex that promotes proper muscle morphogenesis and myotube targeting during embryogenesis.

Redundancy and compensation in axon guidance: genetic analysis of the Drosophila Ptp10D/Ptp4E receptor tyrosine phosphatase subfamily.

BACKGROUND: Drosophila has six receptor protein tyrosine phosphatases (RPTPs), five of which are expressed primarily in neurons. Mutations in all five affect axon guidance, either alone or in combination. Highly penetrant central nervous system (CNS) and motor axon guidance alterations are usually observed only when specific combinations of two or more RPTPs are removed. Here, we examine the sixth RPTP, Ptp4E, which is broadly expressed. RESULTS: Ptp4E and Ptp10D are closely related type III RPTPs. Non-drosophilid insect species have only one type III RPTP, which is closest to Ptp10D. We found that Ptp4E mutants are viable and fertile. We then examined Ptp4E Ptp10D double mutants. These die before the larval stage, and have a mild CNS phenotype in which the outer longitudinal 1D4 bundle is frayed. Ptp10D Ptp69D double mutants have a strong CNS phenotype in which 1D4 axons abnormally cross the midline and the outer and middle longitudinal bundles are fused to the inner bundle. To examine if Ptp4E also exhibits synthetic phenotypes in combination with Ptp69D, we made Ptp4E Ptp69D double mutants and Ptp4E Ptp10D Ptp69D triple mutants. No phenotype was observed in the double mutant. The triple mutant phenotype differs from the Ptp10D Ptp69D phenotype in two ways. First, the longitudinal tracts appear more normal than in the double mutant; two or three bundles are observed, although they are disorganized and fused. Second, axons labelled by the SemaIIB-tauMyc marker often cross in the wrong commissure. We also examined motor axon guidance, and found that no phenotypes are observed in any Ptp4E double mutant combination. However, triple mutants in which Ptp4E Ptp10D was combined with Ptp69D or Ptp52F exhibited stronger phenotypes than the corresponding Ptp10D double mutants. CONCLUSION: Type III RPTPs are required for viability in Drosophila, since Ptp4E Ptp10D double mutants die before the larval stage. Unlike Ptp10D, Ptp4E appears to be a relatively minor player in the control of axon guidance. Strong phenotypes are only observed in triple mutants in which both type III RPTPs are eliminated together with Ptp69D or Ptp52F. Our results allow us to construct a complete genetic interaction matrix for all six of the RPTPs.

Bifocal and PP1 interaction regulates targeting of the R-cell growth cone in Drosophila.

Bifocal is a putative cytoskeletal regulator and a Protein phosphatase-1 (PP1) interacting protein that mediates normal photoreceptor morphology in Drosophila. We show here that Bif and PP1-87B as well as their ability to interact with each other are required for photoreceptor growth cone targeting in the larval visual system. Single mutants for bif or PP1-87B show defects in axonal projections in which the axons of the outer photoreceptors bypass the lamina, where they normally terminate. The data show that the functions of bif and PP1-87B in either stabilizing R-cell morphology (for Bif) or regulating the cell cycle (for PP1-87B) can be uncoupled from their function in visual axon targeting. Interestingly, the axon targeting phenotypes are observed in both PP1-87B mutants and PP1-87B overexpression studies, suggesting that an optimal PP1 activity may be required for normal axon targeting. bif mutants also display strong genetic interactions with receptor tyrosine phosphatases, dptp10d and dptp69d, and biochemical studies demonstrate that Bif interacts directly with F-actin in vitro. We propose that, as a downstream component of axon signaling pathways, Bif regulates PP1 activity, and both proteins influence cytoskeleton dynamics in the growth cone of R cells to allow proper axon targeting.

Roles of Bifocal, Homer, and F-actin in anchoring Oskar to the posterior cortex of Drosophila oocytes.

Transport, translation, and anchoring of osk mRNA and proteins are essential for posterior patterning of Drosophila embryos. Here we show that Homer and Bifocal act redundantly to promote posterior anchoring of the osk gene products. Disruption of actin microfilaments, which causes delocalization of Bifocal but not Homer from the oocyte cortex, severely disrupts anchoring of osk gene products only when Homer (not Bifocal) is absent. Our data suggest that two processes, one requiring Bifocal and an intact F-actin cytoskeleton and a second requiring Homer but independent of intact F-actin, may act redundantly to mediate posterior anchoring of the osk gene products.

Subcellular localization of LGN during mitosis: evidence for its cortical localization in mitotic cell culture systems and its requirement for normal cell cycle progression.

Mammalian LGN/AGS3 proteins and their Drosophila Pins orthologue are cytoplasmic regulators of G-protein signaling. In Drosophila, Pins localizes to the lateral cortex of polarized epithelial cells and to the apical cortex of neuroblasts where it plays important roles in their asymmetric division. Using overexpression studies in different cell line systems, we demonstrate here that, like Drosophila Pins, LGN can exhibit enriched localization at the cell cortex, depending on the cell cycle and the culture system used. We find that in WISH, PC12, and NRK but not COS cells, LGN is largely directed to the cell cortex during mitosis. Overexpression of truncated protein domains further identified the Galpha-binding C-terminal portion of LGN as a sufficient domain for cortical localization in cell culture. In mitotic COS cells that normally do not exhibit cortical LGN localization, LGN is redirected to the cell cortex upon overexpression of Galpha subunits of heterotrimeric G-proteins. The results also show that the cortical localization of LGN is dependent on microfilaments and that interfering with LGN function in cultured cell lines causes early disruption to cell cycle progression.

A mouse homologue of Drosophila pins can asymmetrically localize and substitute for pins function in Drosophila neuroblasts.

Asymmetric cell division is a fundamental mechanism used to generate cellular diversity in invertebrates and vertebrates. In Drosophila, asymmetric division of neuroblasts is achieved by the asymmetric segregation of cell fate determinants Prospero and Numb into the basal daughter cell. Asymmetric segregation of cell fate determinants requires an apically localized protein complex that includes Inscuteable, Pins, Bazooka, DmPar-6, DaPKC and Galphai. Pins acts to stabilize the apical complex during neuroblast divisions. Pins interacts and colocalizes with Inscuteable, as well as maintaining its apical localization. We have isolated a mouse homologue of pins (Pins) and characterized its expression profile. Mouse PINS shares high similarity in sequence and structure with Pins and other Pins-like proteins from mammals. Pins is expressed in many mouse tissues but its expression is enriched in the ventricular zone of the developing central nervous systems. PINS localizes asymmetrically to the apical cortex of mitotic neuroblasts when ectopically expressed in Drosophila embryos. Like Pins, its N-terminal tetratricopeptide repeats can directly interact with the asymmetric localization domain of Insc, and its C-terminal GoLoco-containing region can direct localization to the neuroblast cortex. We further show that Pins can fulfill all aspects of pins function in Drosophila neuroblast asymmetric cell divisions. Our results suggest a conservation of function between the fly and mammalian Pins homologues.