What's new in edition 64 April 2012 Gene sites new with this edition

The Interactive Fly was first released July/August 1996, with updates provided at approximately one month intervals, through September 1997 (edition 13). Updating quarterly started with edition 14. With edition 40, the Interactive Fly began to schedule updates three times a year: fall, winter and spring.

Gene sites new with this edition of the Interactive Fly:

BM-40-SPARC

SPARC is an evolutionarily conserved collagen-binding extracellular matrix (ECM) glycoprotein whose morphogenetic contribution(s) to embryonic development remain elusive despite decades of research. This study used Drosophila genetics to gain insight into the role of SPARC during embryogenesis. In Drosophila embryos, high levels of SPARC and other basal lamina components (such as network-forming collagen IV, laminin (see Laminin A and Laminin B1) and perlecan) are synthesized and secreted by haemocytes, and assembled into basal laminae. A SPARC mutant was generated by P-element mutagenesis that is embryonic lethal because of multiple developmental defects. Whereas no differences in collagen IV immunostaining were observed in haemocytes between wild-type and SPARC-mutant embryos, collagen IV was not visible in basal laminae of SPARC-mutant embryos. In addition, the laminin network of SPARC-mutant embryos appeared fragmented and discontinuous by late embryogenesis. Transgenic expression of SPARC protein by haemocytes in SPARC-mutant embryos restored collagen IV and laminin continuity in basal laminae. However, transgenic expression of SPARC by neural cells failed to rescue collagen IV in basal laminae, indicating that the presence of collagen IV deposition requires SPARC expression by haemocytes. A previous finding that haemocyte-derived SPARC protein levels are reduced in collagen-IV-mutant embryos and the observation that collagen-IV-mutant embryos showed a striking phenotypic similarity to SPARC-mutant embryos suggests a mutual dependence between these major basal laminae components during embryogenesis. Patterning defects and impaired condensation of the ventral nerve cord also resulted from the loss of SPARC expression prior to haemocyte migration. Hence, SPARC is required for basal lamina maturation and condensation of the ventral nerve cord during Drosophila embryogenesis (Martinek, 2008).

Benign gonial cell neoplasm

The fate of stem cells is intricately regulated by numerous extrinsic and intrinsic factors that promote maintenance or differentiation. The RNA-binding translational repressor Pumilio (Pum) in conjunction with Nanos (Nos) is required for self-renewal, whereas Bam (bag-of-marbles) and Bgcn (benign gonial cell neoplasm) promote differentiation of germ line stem cells in the Drosophila ovary. Genetic analysis suggests that Bam and Bgcn antagonize Pum/Nos function to promote differentiation; however, the molecular basis of this epistatic relationship is currently unknown. This study shows that Bam and Bgcn inhibit Pum function through direct binding. A ternary complex involving Bam, Bgcn, and Pum has been identified in which Bam, but not Bgcn, directly interacts with Pum, and this interaction is greatly increased by the presence of Bgcn. In a heterologous reporter assay to monitor Pum activity, Bam, but not Bgcn, inhibits Pum activity. Notably, the N-terminal region of Pum, which lacks the C-terminal RNA-binding Puf domain, mediates both the ternary protein interaction and the Bam inhibition of Pum function. These studies suggest that, in cystoblasts, Bam and Bgcn may directly inhibit Pum/Nos activity to promote differentiation of germ line stem cells (Kim, 2010).

Eukaryotic initiation factor 4E

In female fruit flies, Sex-lethal (Sxl) turns off the X chromosome dosage compensation system by a mechanism involving a combination of alternative splicing and translational repression of the male specific lethal-2 (msl-2) mRNA. A genetic screen identified the translation initiation factor eif4e as a gene that acts together with Sxl to repress expression of the Msl-2 protein. However, eif4e is not required for Sxl mediated repression of msl-2 mRNA translation. Instead, eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs. Like other factors required for Sxl regulation of splicing, eif4e shows maternal-effect female-lethal interactions with Sxl. This female lethality can be enhanced by mutations in other co-factors that promote female-specific splicing and is caused by a failure to properly activate the Sxl-positive autoregulatory feedback loop in early embryos. In this feedback loop Sxl proteins promote their own synthesis by directing the female-specific alternative splicing of Sxl-Pm pre-mRNAs. Analysis of pre-mRNA splicing when eif4e activity is compromised demonstrates that Sxl-dependent female-specific splicing of both Sxl-Pm and msl-2 pre-mRNAs requires eif4e activity. Consistent with a direct involvement in Sxl-dependent alternative splicing, eIF4E is associated with unspliced Sxl-Pm pre-mRNAs and is found in complexes that contain early acting splicing factors -- the U1/U2 snRNP protein Sans-fils (Snf), the U1 snRNP protein U1-70k, U2AF38, U2AF50, and the Wilms' Tumor 1 Associated Protein Fl(2)d--that have been directly implicated in Sxl splicing regulation (Graham, 2011).

Pten-induced putative kinase 1

Mutations in PTEN-induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson's disease (PD), PARK6. To investigate the mechanism of PINK1 pathogenesis, the Drosophila Pink1 knockout (KO) model was used. In mitochondria isolated from Pink1-KO flies, mitochondrial respiration driven by the electron transport chain (ETC) is significantly reduced. This reduction is the result of a decrease in ETC complex I and IV enzymatic activity. As a consequence, Pink1-KO flies also display a reduced mitochondrial ATP synthesis. Because mitochondrial dynamics is important for mitochondrial function and Pink1-KO flies have defects in mitochondrial fission, whether fission machinery deficits underlie the bioenergetic defect in Pink1- KO flies was investigated. It was found that the bioenergetic defects in the Pink1-KO can be ameliorated by expression of Drp1, a key molecule in mitochondrial fission. Further investigation of the ETC complex integrity in wild type, Pink1-KO, PInk1-KO/Drp1 transgenic, or Drp1 transgenic flies indicates that the reduced ETC complex activity is likely derived from a defect in the ETC complex assembly, which can be partially rescued by increasing mitochondrial fission. Taken together, these results suggest a unique pathogenic mechanism of PINK1 PD: The loss of PINK1 impairs mitochondrial fission, which causes defective assembly of the ETC complexes, leading to abnormal bioenergetics (Liu, 2011).

Quiver

Sleep is an essential process conserved from flies to humans. The importance of sleep is underscored by its tight homeostatic control. In this study, through a forward-genetic screen, a novel gene, sleepless (quiver), was identified that is required for sleep in Drosophila. sleepless encodes a brain-enriched, glycosyl-phosphatidylinositol-anchored protein. Loss of Sleepless protein causes an extreme (>80%) reduction in sleep. Furthermore, a moderate reduction in Sleepless protein has minimal effects on baseline sleep, but markedly reduces recovery sleep following sleep deprivation. Genetic and molecular analyses reveal that quiver, a mutation that impairs Shaker-dependent K+ current, is an allele of sleepless. Consistent with this finding, Shaker protein level is reduced in sleepless mutants. It is proposed that Sleepless is a signaling molecule that connects sleep drive to lowered membrane excitability (Koh, 2008).

N-synaptobrevin

Soluble NSF attachment protein receptors (SNAREs) are the core proteins in membrane fusion. The neuron-specific synaptic v-SNARE n-Syb (neuronal Synaptobrevin) is a vesicular protein that plays a key role during synaptic vesicle exocytosis. This paper reports that loss of n-syb caused slow neurodegeneration independent of its well documented role in neurotransmitter release in adult Drosophila photoreceptor neurons. In addition to synaptic vesicles, n-Syb localizes to endosomal vesicles. Loss of n-syb lead to endosomal accumulations, transmembrane protein degradation defects, and a secondary increase in autophagy. The evidence suggests a primary defect of impaired delivery of vesicles that contain degradation proteins, including the acidification-activated Cathepsin proteases and the neuron-specific proton pump and V0 adenosine triphosphatase component V100. Overexpressing V100 partially rescues n-syb-dependent degeneration through an acidification-independent endosomal sorting mechanism. Collectively, these findings reveal a role for n-Syb in a neuron-specific sort-and-degrade mechanism that protects neurons from degeneration. These findings further shed light on which intraneuronal compartments exhibit increased or decreased neurotoxicity (Haberman, 2012).

Serotonin receptor 7

The 5-HT₇ receptor remains one of the less well characterized serotonin receptors. Although it has been demonstrated to be involved in the regulation of mood, sleep, and circadian rhythms, as well as relaxation of vascular smooth muscles in mammals, the precise mechanisms underlying these functions remain largely unknown. The fruit fly is an attractive model organism to study neuropharmacological, molecular, and behavioral processes that are largely conserved with mammals. Drosophila express a homolog of the mammalian 5-HT₇ receptor, as well as homologs for the mammalian 5-HT_1A, and 5-HT₂, receptors. Each fly receptor couples to the same effector pathway as their mammalian counterpart and has been demonstrated to mediate similar behavioral responses. This study reports on the expression and function of the 5-HT₇ receptor in Drosophila. In the larval central nervous system, expression is detected postsynaptically in discreet cells and neuronal circuits. In the adult brain there is strong expression in all large-field R neurons that innervate the ellipsoid body, as well as in a small group of cells that cluster with the PDF-positive LNvs neurons that mediate circadian activity. Following both pharmacological and genetic approaches, 5-HT₇ activity was found to be essential for normal courtship and mating behaviors in the fly, where it appears to mediate levels of interest in both males and females. This is the first reported evidence of direct involvement of a particular serotonin receptor subtype in courtship and mating in the fly (Becnel, 2011).

Set1

Histone H3 lysine 4 trimethylation (H3K4me3) is a major hallmark of promoter-proximal histones at transcribed genes. This study reports that a previously uncharacterized Drosophila H3K4 methyltransferase, dSet1, and not the other putative histone H3K4 methyltransferases (Trithorax; Trithorax-related protein), is predominantly responsible for histone H3K4 trimethylation. Functional and proteomics studies reveal that dSet1 is a component of a conserved H3K4 trimethyltransferase complex and polytene staining and live cell imaging assays show widespread association of dSet1 with transcriptionally active genes. dSet1 is present at the promoter region of all tested genes, including activated Hsp70 and Hsp26 heat shock genes and is required for optimal mRNA accumulation from the tested genes. In the case of Hsp70, the mRNA production defect in dSet1 RNAi-treated cells is accompanied by retention of Pol II at promoters. These data suggest that dSet1-dependent H3K4me3 is responsible for the generation of a chromatin structure at active promoters that ensures optimal Pol II release into productive elongation (Ardehali, 2011).

Synapse protein 25

Current models of synaptic vesicle trafficking implicate a core complex of proteins comprised of N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAREs (an acronym derived from 'SNAP (Soluble NSF Attachment Protein) REceptor', consisting of proteins that mediated vesicular fusion with the target membrane) in synaptic vesicle fusion and neurotransmitter release. Despite this progress, major challenges remain in establishing the in vivo functions of these proteins and their roles in determining the physiological properties of synapses. The present study employs glutamatergic adult neuromuscular synapses of Drosophila, which exhibit conserved properties of short-term synaptic plasticity with respect to mammalian glutamatergic synapses, to address these issues through genetic analysis. The findings establish an in vivo role for the target-Snare (t-Snare) protein SNAP-25, one part of the four helix bundle of proteins referred to as SNAPs, in synaptic vesicle priming, and support a zippering model of SNARE function in this process. Moreover, these studies define the contribution of SNAP-25-dependent vesicle priming to the detailed properties of short-term depression elicited by paired-pulse (PP) and train stimulation. In contrast, NSF is shown here not to be required for wild-type PP depression, but to be critical for maintaining neurotransmitter release during sustained stimulation. In keeping with this role, disruption of NSF function results in activity-dependent redistribution of the t-SNARE proteins, Syntaxin and SNAP-25, away from neurotransmitter release sites (active zones). These findings support a role for NSF in replenishing active zone t-SNAREs for subsequent vesicle priming, and provide new insight into the spatial organization of SNARE protein cycling during synaptic activity. Together, the results reported in this study establish in vivo contributions of SNAP-25 and NSF to synaptic vesicle trafficking and define molecular mechanisms determining conserved functional properties of short-term depression (Kawasaki, 2009).

Synapsin

Synapsin is a phosphoprotein reversibly associated with synaptic vesicles. Synapsin function in mediating synaptic activity was investigated during intense stimulation at Drosophila motor boutons. Electron microscopy analysis of synapsin⁻ boutons demonstrated that synapsin maintains vesicle clustering over the periphery of the bouton. Cyclosporin A pretreatment disrupted peripheral vesicle clustering, presumably due to increasing synapsin phosphorylated state. Labeling recycling vesicles with a fluorescent dye FM1-43 followed by photoconversion of the dye into electron dense product demonstrated that synapsin deficiency does not affect mixing of the reserve and recycling vesicle pools but selectively reduces the size of the reserve pool. Intense stimulation produced a significant increase in vesicle abundance and vesicle redistribution toward the central core of synapsin ⁺ boutons, while in synapsin⁻ boutons the area occupied by vesicles did not change and the increase in vesicle numbers was not as prominent. However, intense stimulation produced an increase in basal release at synapsin⁻ but not in synapsin⁺ boutons, suggesting that synapsin may direct vesicles to the reserve pool. Finally, synapsin deficiency inhibited an increase in quantal size and formation of endosome-like cisternae, which was activated either by intense electrical stimulation or by high K⁺ application. Taken together, these results elucidate a novel synapsin function, specifically, promoting vesicle reuptake and reserve pool formation upon intense stimulation (Akbergenova, 2010).

Viking and Collagen type IV

Basement membranes (BMs) are resilient polymer structures that surround organs in all animals. Tissues, however, undergo extensive morphological changes during development. It is not known whether the assembly of BM components plays an active morphogenetic role. To study in vivo the biogenesis and assembly of Collagen IV, the main constituent of BMs, a GFP-based RNAi method (iGFPi) was used that was designed to knock down any GFP trapped protein in Drosophila. With this method it was found that Collagen IV is synthesized by the fat body, secreted to the hemolymph (insect blood), and continuously incorporated into the BMs of the larva. Incorporation of Collagen IV determines organ shape, first by mechanically constricting cells and second through recruitment of trol, the Drosophila Perlecan, which counters constriction by Collagen IV. These results uncover incorporation of Collagen IV and Perlecan into BMs as a major determinant of organ shape and animal form (Pastor-Pareja, 2011).

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