DJ-1, a novel regulator of the tumor suppressor PTEN.

The phosphatidylinositol 3' kinase (PI3'K) pathway, which regulates cell survival, is antagonized by the PTEN tumor suppressor. The regulation of PTEN is unclear. A genetic screen of Drosophila gain-of-function mutants identified DJ-1 as a suppressor of PTEN function. In mammalian cells, DJ-1 underexpression results in decreased phosphorylation of PKB/Akt, while DJ-1 overexpression leads to hyperphosphorylation of PKB/Akt and increased cell survival. In primary breast cancer samples, DJ-1 expression correlates negatively with PTEN immunoreactivity and positively with PKB/Akt hyperphosphorylation. In 19/23 primary non-small cell lung carcinoma samples, DJ-1 expression was increased compared to paired nonneoplastic lung tissue, and correlated positively with relapse incidence. DJ-1 is thus a key negative regulator of PTEN that may be a useful prognostic marker for cancer.

Nonautonomous planar polarity patterning in Drosophila: dishevelled-independent functions of frizzled.

The frizzled (fz) gene of Drosophila is required for planar polarity establishment in the adult cuticle, acting both cell autonomously and nonautonomously. We demonstrate that these two activities of fz in planar polarity are temporally separable in both the eye and wing. The nonautonomous function is dishevelled (dsh) independent, and its loss results in polarity phenotypes that resemble those seen for mutations in dachsous (ds). Genetic interactions and epistasis analysis suggest that fz, ds, and fat (ft) act together in the long-range propagation of polarity signals in the eye and wing. We also find evidence that polarity information may be propagated by modulation of the binding affinities of the cadherins encoded by the ds and ft loci.

WASp is required for the correct temporal morphogenesis of rhabdomere microvilli.

Microvilli are actin-based fingerlike membrane projections that form the basis of the brush border of enterocytes and the Drosophila melanogaster photoreceptor rhabdomere. Although many microvillar cytoskeletal components have been identified, the molecular basis of microvillus formation is largely undefined. Here, we report that the Wiskott-Aldrich syndrome protein (WASp) is necessary for rhabdomere microvillus morphogenesis. We show that WASp accumulates on the photoreceptor apical surface before microvillus formation, and at the time of microvillus initiation WASp colocalizes with amphiphysin and moesin. The loss of WASp delays the enrichment of F-actin on the apical photoreceptor surface, delays the appearance of the primordial microvillar projections, and subsequently leads to malformed rhabdomeres.

Axonal transport of mitochondria to synapses depends on milton, a novel Drosophila protein.

A protein required to localize mitochondria to Drosophila nerve terminals has been identified genetically. Photoreceptors mutant for milton show aberrant synaptic transmission despite normal phototransduction. Without Milton, synaptic terminals and axons lack mitochondria, although mitochondria are numerous in neuronal cell bodies. In contrast, synaptic vesicles continue to be transported to and concentrated at synapses. Milton protein is associated with mitochondria and is present primarily in axons and synapses. A likely explanation of the apparent trafficking defect is offered by the coimmunoprecipitation of Milton and kinesin heavy chain. Transfected into HEK293T cells, Milton induces a redistribution of mitochondria within the cell. We propose that Milton is a mitochondria-associated protein required for kinesin-mediated transport of mitochondria to nerve terminals.

Human wild-type tau interacts with wingless pathway components and produces neurofibrillary pathology in Drosophila.

Pathologic alterations in the microtubule-associated protein tau have been implicated in a number of neurodegenerative disorders, including Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and frontotemporal dementia (FTD). Here, we show that tau overexpression, in combination with phosphorylation by the Drosophila glycogen synthase kinase-3 (GSK-3) homolog and wingless pathway component (Shaggy), exacerbated neurodegeneration induced by tau overexpression alone, leading to neurofibrillary pathology in the fly. Furthermore, manipulation of other wingless signaling molecules downstream from shaggy demonstrated that components of the Wnt signaling pathway modulate neurodegeneration induced by tau pathology in vivo but suggested that tau phosphorylation by GSK-3beta differs from canonical Wnt effects on beta-catenin stability and TCF activity. The genetic system we have established provides a powerful reagent for identification of novel modifiers of tau-induced neurodegeneration that may serve as future therapeutic targets.

Mispatterning in the ommatidia of Apis mellifera pupae treated with a juvenile hormone analogue.

To further understand the function of morphogenetic hormones in honeybee eye differentiation, the alterations in ommatidial patterning induced by pyriproxyfen, a juvenile hormone (JH) analogue, were studied by scanning and transmission electron microscopy. Prepupae of prospective honeybee workers were treated with pyriproxyfen and the effects on ommatidial differentiation were described at the end of the pupal development. The results show that the entire ommatidia, i.e., the dioptric as well as the receptor systems, were affected by the JH analogue. The wave of ommatidial differentiation, which progresses from the posterior to the anterior region of the pupal eyes, was arrested. In treated pupae, the rhabdomeres only differentiated at the apical axis of the retinula, the secondary and tertiary pigment cells did not develop their cytoplasm protrusions, and the cone cell quartet did not pattern correctly. Simultaneously, an intense vacuolization was observed in cells forming ommatidia. In a previous study we showed that pyriproxyfen exerts an inhibition on pupal ecdysteroid secretion. In this sense, the arrested ommatidial differentiation in pyriproxyfen-treated pupae could be due to a secondary effect resulting from an alteration in pupal ecdysteroid titers.

A Drosophila ortholog of the human MRJ modulates polyglutamine toxicity and aggregation.

In the Drosophila eye, proteins with an expanded polyglutamine (polyQ) tract form nuclear and cytoplasmic inclusions and produce cytotoxicity, demonstrated as loss of eye pigmentation and structural integrity. An EP P-element that suppressed the loss of eye pigmentation was inserted 9.7 kb upstream of dmrj, a gene that encodes an ortholog of a brain-enriched cochaperone, the human MRJ (mammalian relative of DnaJ). Despite the large distance between them, quantitative polymerase chain reaction indicated that the EP could overexpress dmrj. In the retina and other neurons, transgenic dMRJ suppressed polyQ toxicity and colocalized with its inclusions. In the photoreceptors, expression of another suppressor with a J domain, dHDJ1, but not dMRJ, prior to expression of expanded polyQs dramatically promoted cytoplasmic aggregation. However, both proteins increased the level of detergent-soluble, monomeric polyQ-expanded proteins. These findings exemplify the functional similarities and differences between J domain proteins in suppressing polyQ toxicity.

The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila Homolog of YAP.

Coordination between cell proliferation and cell death is essential to maintain homeostasis in multicellular organisms. In Drosophila, these two processes are regulated by a pathway involving the Ste20-like kinase Hippo (Hpo) and the NDR family kinase Warts (Wts; also called Lats). Hpo phosphorylates and activates Wts, which in turn, through unknown mechanisms, negatively regulates the transcription of cell-cycle and cell-death regulators such as cycE and diap1. Here we identify Yorkie (Yki), the Drosophila ortholog of the mammalian transcriptional coactivator yes-associated protein (YAP), as a missing link between Wts and transcriptional regulation. Yki is required for normal tissue growth and diap1 transcription and is phosphorylated and inactivated by Wts. Overexpression of yki phenocopies loss-of-function mutations of hpo or wts, including elevated transcription of cycE and diap1, increased proliferation, defective apoptosis, and tissue overgrowth. Thus, Yki is a critical target of the Wts/Lats protein kinase and a potential oncogene.

The v-ATPase V0 subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila.

The V(0) complex forms the proteolipid pore of an ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been proposed largely based on yeast vacuolar fusion experiments. We have isolated mutations in the largest V(0) component vha100-1 in flies in an unbiased genetic screen for synaptic malfunction. The protein is only required in neurons, colocalizes with markers for synaptic vesicles as well as active zones, and interacts with t-SNAREs. Loss of vha100-1 leads to vesicle accumulation in synaptic terminals, suggesting a deficit in release. The amplitude of spontaneous release events and release with hypertonic stimulation indicate normal levels of neurotransmitter loading, yet mutant embryos display severe defects in evoked synaptic transmission and FM1-43 uptake. Our data suggest that Vha100-1 functions downstream of SNAREs in synaptic vesicle fusion.

Genetic mosaic analysis of the equatorial-less mutation in Drosophila melanogaster.

eql (equatorial-less) is a recessive lethal mutation on the second chromosome of Drosophila melanogaster. J. Campos-Ortega found that eql clones in somatic mosaic flies have reduced numbers of photoreceptor cells, and he suggested that only the R1, R6, and R7 photoreceptor cells were missing in this mutant. These photoreceptor cells help to define the inverted orientation of ommatidial facets along the equatorial midline of the fly eye, hence the mutation was named "equatorial-less." We have conducted a detailed analysis of the eql mutation, by serial section reconstruction of eql clones marked with bw- or w- in somatic mosaic flies. We found that all photoreceptor cell types (R1-R8) could be deleted by the eql mutation, and in rare cases the number of photoreceptor cells was increased. The apparent lack of photoreceptor cell type specificity was confirmed by our analysis of genetically mosaic facets, which indicated that no single photoreceptor cell, or subset of photoreceptor cells, was uniquely required to express eql+. Rather, eql appears to function in all photoreceptor cells, and possibly in all eye precursor cells. The distribution of photoreceptor cell numbers in w eql facets was consistent with the hypothesis that each photoreceptor cell was deleted independently of the others. The eql gene is located on the right arm of chromosome 2 at map location 2-104.5 +/- 0.7 and lies between the polytene chromosome bands 59D8 and 60A7.

Drosophila betaHeavy-spectrin is essential for development and contributes to specific cell fates in the eye.

The spectrin membrane skeleton is a ubiquitous cytoskeletal structure with several cellular roles, including the maintenance of cell integrity, determination of cell shape and as a contributor to cell polarity. We have isolated mutations in the gene encoding &bgr ;Heavy-spectrin in Drosophila, and have named this essential locus karst. karst mutant individuals have a pleiotropic phenotype characterized by extensive larval lethality and, in adult escapers, rough eyes, bent wings, tracheal defects and infertility. Within karst mutant eyes, a significant number of ommatidia specifically lack photoreceptor R7 alongside more complex morphological defects. Immunolocalization of betaHeavy-spectrin in wild-type eye-antennal and wing imaginal discs reveals that betaHeavy-spectrin is present in a restricted subdomain of the membrane skeleton that colocalizes with DE-cadherin. We propose a model where normal levels of Sevenless signaling are dependent on tight cell-cell adhesion facilitated by the betaHeavy-spectrin membrane skeleton. Immunolocalization of betaHeavy-spectrin in the adult and larval midgut indicates that it is a terminal web protein, but we see no gross morphological defects in the adult apical brush border in karst mutant flies. Rhodamine phalloidin staining of karst mutant ovaries similarly reveals no conspicuous defect in the actin cytoskeleton or cellular morphology in egg chambers. This is in contrast to mutations in alpha-spectrin, the molecular partner of betaHeavy-spectrin, which affect cellular structure in both the larval gut and adult ovaries. Our results emphasize the fundamental contribution of the spectrin membrane skeleton to normal development and reveals a critical interplay between the integrity of a cell's membrane skeleton, the structure of cell-cell contacts and cell signaling.

Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons.

Behavior is a manifestation of temporally and spatially defined neuronal activities. To understand how behavior is controlled by the nervous system, it is important to identify the neuronal substrates responsible for these activities, and to elucidate how they are integrated into a functional circuit. I introduce a novel and general method to conditionally perturb anatomically defined neurons in intact Drosophila. In this method, a temperature-sensitive allele of shibire (shi(ts1)) is overexpressed in neuronal subsets using the GAL4/UAS system. Because the shi gene product is essential for synaptic vesicle recycling, and shi(ts1) is semidominant, a simple temperature shift should lead to fast and reversible effects on synaptic transmission of shi(ts1) expressing neurons. When shi(ts1) expression was directed to cholinergic neurons, adult flies showed a dramatic response to the restrictive temperature, becoming motionless within 2 min at 30 degrees C. This temperature-induced paralysis was reversible. After being shifted back to the permissive temperature, they readily regained their activity and started to walk in 1 min. When shi(ts1) was expressed in photoreceptor cells, adults and larvae exhibited temperature-dependent blindness. These observations show that the GAL4/UAS system can be used to express shi(ts1) in a specific subset of neurons to cause temperature-dependent changes in behavior. Because this method allows perturbation of the neuronal activities rapidly and reversibly in a spatially and temporally restricted manner, it will be useful to study the functional significance of particular neuronal subsets in the behavior of intact animals.

The Drosophila RCC1 homolog, Bj1, regulates nucleocytoplasmic transport and neural differentiation during Drosophila development.

The Bj1 gene encodes the Drosophila homolog of RCC1, the guanine-nucleotide exchange factor for RanGTPase. Here, we provide the first phenotypic characterization of a RCC1 homolog in a developmental model system. We identified Bj1 (dRCC1) in a genetic screen to identify mutations that alter central nervous system development. We find that zygotic dRCC1 mutant embryos exhibit specific defects in the development and differentiation of lateral CNS neurons although cell division and the cell cycle appear grossly normal. dRCC1 mutant nerve cords contain abnormally large cells with compartmentalized nuclei and exhibit increased transcription in the lateral CNS. As RCC1 is an important component of the nucleocytoplasmic transport machinery, we find that dRCC1 function is required for nuclear import of nuclear localization signal sequence (NLS)-carrying cargo molecules. Finally, we show that dRCC1 is required for cell proliferation and/or survival during germline, eye and wing development and that dRCC1 appears to facilitate apoptosis.

salvador Promotes both cell cycle exit and apoptosis in Drosophila and is mutated in human cancer cell lines.

The number of cells in an organism is determined by regulating both cell proliferation and cell death. Relatively few mechanisms have been identified that can modulate both of these processes. In a screen for Drosophila mutations that result in tissue overgrowth, we identified salvador (sav), a gene that promotes both cell cycle exit and cell death. Elevated Cyclin E and DIAP1 levels are found in mutant cells, resulting in delayed cell cycle exit and impaired apoptosis. Salvador contains two WW domains and binds to the Warts (or LATS) protein kinase. The human ortholog of salvador (hWW45) is mutated in three cancer cell lines. Thus, salvador restricts cell numbers in vivo by functioning as a dual regulator of cell proliferation and apoptosis.