CalpB modulates border cell migration in Drosophila egg chambers.

BACKGROUND: Calpains are calcium regulated intracellular cysteine proteases implicated in a variety of physiological functions and pathological conditions. The Drosophila melanogaster genome contains only two genes, CalpA and CalpB coding for canonical, active calpain enzymes. The movement of the border cells in Drosophila egg chambers is a well characterized model of the eukaryotic cell migration. Using this genetically pliable model we can investigate the physiological role of calpains in cell motility. RESULTS: We demonstrate at the whole organism level that CalpB is implicated in cell migration, while the structurally related CalpA paralog can not fulfill the same function. The downregulation of the CalpB gene by mutations or RNA interference results in a delayed migration of the border cells in Drosophila egg chambers. This phenotype is significantly enhanced when the focal adhesion complex genes encoding for α-PS2 integrin ( if), ß-PS integrin (mys) and talin (rhea) are silenced. The reduction of CalpB activity diminishes the release of integrins from the rear end of the border cells. The delayed migration and the reduced integrin release phenotypes can be suppressed by expressing wild-type talin-head in the border cells but not talin-head(R367A), a mutant form which is not able to bind ß-PS integrin. CalpB can cleave talin in vitro, and the two proteins coimmunoprecipitate from Drosophila extracts. CONCLUSIONS: The physiological function of CalpB in border cell motility has been demonstrated in vivo. The genetic interaction between the CalpB and the if, mys, as well as rhea genes, the involvement of active talin head-domains in the process, and the fact that CalpB and talin interact with each other collectively suggest that the limited proteolytic cleavage of talin is one of the possible mechanisms through which CalpB regulates cell migration.

Type ID unconventional myosin controls left-right asymmetry in Drosophila.

Breaking left-right symmetry in Bilateria embryos is a major event in body plan organization that leads to polarized adult morphology, directional organ looping, and heart and brain function. However, the molecular nature of the determinant(s) responsible for the invariant orientation of the left-right axis (situs choice) remains largely unknown. Mutations producing a complete reversal of left-right asymmetry (situs inversus) are instrumental for identifying mechanisms controlling handedness, yet only one such mutation has been found in mice (inversin) and snails. Here we identify the conserved type ID unconventional myosin 31DF gene (Myo31DF) as a unique situs inversus locus in Drosophila. Myo31DF mutations reverse the dextral looping of genitalia, a prominent left-right marker in adult flies. Genetic mosaic analysis pinpoints the A8 segment of the genital disc as a left-right organizer and reveals an anterior-posterior compartmentalization of Myo31DF function that directs dextral development and represses a sinistral default state. As expected of a determinant, Myo31DF has a trigger-like function and is expressed symmetrically in the organizer, and its symmetrical overexpression does not impair left-right asymmetry. Thus Myo31DF is a dextral gene with actin-based motor activity controlling situs choice. Like mouse inversin, Myo31DF interacts and colocalizes with beta-catenin, suggesting that situs inversus genes can direct left-right development through the adherens junction.

Germ line specific expression of a protein phosphatase Y interacting protein (PPYR1) in Drosophila.

PPYR1, the product of the CG15031 gene, was identified as a protein phosphatase Y (PPY) interacting protein in Drosophila melanogaster using a yeast two-hybrid screen. PPYR1 displays a biphasic expression pattern: the maternal protein is abundant in the developing egg chambers and in the early embryos, while the zygotic protein appears later in development and is localized specifically in the testes of the males. The maternal and zygotic gene products differ from each other in their size having apparent molecular masses of 47 and 66 kDa, respectively. The maternal PPYR1 is localized in the cytoplasm of the follicular and nurse cells and is deposited as a ribonucleoprotein complex in the oocyte. In the early embryos, the PPYR1 is distributed evenly, and it gradually diminishes during embryonic development. Zygotic PPYR1 is expressed exclusively in the testes, predominantly in the cytoplasm of the spermatocytes. PPY is localized in the nuclei of the same cells. Our results suggest that PPYR1 has two distinct developmental isoforms: a maternal protein the expression of which is independent of PPY and a zygotic protein which is co-expressed with PPY.

Tissue- and developmental stage-specific changes in the subcellular localization of the 26S proteasome in the ovary of Drosophila melanogaster.

The intracellular localization of the 26S proteasome in the different ovarian cell types of Drosophila melanogaster was studied by means of immunofluorescence staining and laser scanning microscopy, with the use of antibodies specific for regulatory complex subunits or the catalytic core of the 26S proteasome. During the previtellogenic phase of oogenesis (stages 1-6), strong cytoplasmic staining was observed in the nurse cells and follicular epithelial cells, but the proteasome was not detected in the nuclei of these cell types. The subcellular distribution of the 26S proteasome was completely different in the oocyte. Besides a constant, very faint cytoplasmic staining, there was a gradual nuclear accumulation of proteasomes during the previtellogenic phase of oogenesis. A characteristic subcellular redistribution of the 26S proteasome occurred in the ovarian cells during the vitellogenic phase of oogenesis. There was a gradual decline in the concentration of the 26S proteasome in the nucleus of the oocyte, and in the stage 10 oocyte the proteasome could barely be detected in the nucleus. This was accompanied by a massive nuclear accumulation of proteasomes in the follicular epithelial cells. These results demonstrate that the subcellular distribution of the 26S proteasome in higher eukaryotes is strictly tissue- and developmental stage-specific.

The retinoic-like juvenile hormone controls the looping of left-right asymmetric organs in Drosophila.

In vertebrate development, the establishment of left-right asymmetry is essential for sidedness and the directional looping of organs like the heart. Both the nodal pathway and retinoic acid play major and conserved regulatory roles in these processes. We carried out a novel screen in Drosophila to identify mutants that specifically affect the looping of left-right asymmetric organs. We report the isolation of spin, a novel mutant in which the looping of the genitalia and spermiduct are incomplete; under-rotation of the genitalia indicates that spin controls looping morphogenesis but not direction, thus uncoupling left-right asymmetry and looping morphogenesis. spin is a novel, rotation-specific allele of the fasciclin2 (Fas2) gene, which encodes a cell-adhesion protein involved in several aspects of neurogenesis. In spin mutants, the synapses connecting specific neurosecretory cells to the corpora allata are affected. The corpus allatum is part of the ring gland and is involved in the control of juvenile hormone titers during development. Our genetic and pharmacological results indicate that Fas2(spin) rotation defects are linked to an abnormal endocrine function and an elevated level of juvenile hormone. As juvenile hormone is an insect sesquiterpenoid related to retinoic acid, these results establish a new genetic model for studying organ looping and demonstrate an evolutionarily conserved role for terpenoids in this process.

Importin-alpha 2 is critically required for the assembly of ring canals during Drosophila oogenesis.

The interstitial deletion D14 affecting the importin-alpha 2 gene of Drosophila, or imp-alpha 2(D14), causes recessive female sterility characterized by a block of nurse cell-oocyte transport during oogenesis. In wild-type egg chambers, the Imp-alpha 2 protein is uniformly distributed in the nurse cell cytoplasm with a moderate accumulation along the oocyte cortex. Cytochalasin D treatment of wild-type egg chambers disrupts the in vivo association of Imp-alpha 2 with F-actin and results in its release from the oocyte cortex and its transfer into nurse cell nuclei. Binding assay shows that the interaction of Imp-alpha 2 with F-actin, albeit not monomeric actin, requires the occurrence of NLS peptides. Phenotypic analysis of imp-alpha 2(D14) ovaries reveals that the block of nurse cell-oocyte transport results from the occlusion of the ring canals that constitute cytoplasmic bridges between the nurse cells and the oocyte. Immunohistochemistry shows that, although the Imp-alpha2 protein cannot be detected on the ring canals, the Kelch protein, a known ring canal component, fails to bind to ring canals in imp-alpha 2(D14) egg chambers. Since loss-of-function mutations of kelch results in a similar dumpless phenotype, we propose that the Imp-alpha 2 protein plays a critical role in Kelch function by regulating its deposition on ring canals during their assembly.

Structural and enzymatic characterization of Drosophila Dm2-MMP, a membrane-bound matrix metalloproteinase with tissue-specific expression.

We report the isolation and characterization of a cDNA encoding Dm2-MMP, the second matrix metalloproteinase (MMP) identified in the Drosophila melanogaster genome. The cloned cDNA codes for a polypeptide of 758 residues that displays a domain organization similar to that of other MMPs, including signal peptide, propeptide, catalytic, and hemopexin domains. However, the structure of Dm2-MMP is unique because of the presence of an insertion of 214 amino acids between the catalytic and hemopexin domains that is not present in any of the previously described MMPs. Dm2-MMP also contains a C-terminal extension predicted to form a cleavable glycosylphosphatidylinositol anchor site. Western blot and immunofluorescence analysis of S2 cells transfected with the isolated cDNA confirmed that Dm2-MMP is localized at the cell surface. Production of the catalytic domain of Dm2-MMP in Escherichia coli and analysis of its enzymatic activity revealed that this proteinase cleaves several synthetic peptides used for analysis of vertebrate MMPs. This proteolytic activity was abolished by MMP inhibitors such as BB-94, confirming that the isolated cDNA codes for an enzymatically active metalloproteinase. Reverse transcription-PCR analysis showed that Dm2-MMP is expressed at low levels in all of the developmental stages of Drosophila as well as in adult flies. However, detailed in situ hybridization at the larval stage revealed a strong tissue-specific expression in discrete regions of the brain and eye imaginal discs. According to these results, we propose that Dm2-MMP plays both general proteolytic functions during Drosophila development and in adult tissues and specific roles in eye development and neural tissues through the degradation and remodeling of the extracellular matrix.