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Sample records for drosophila sex-lethal protein

  1. PPS, a large multidomain protein, functions with sex-lethal to regulate alternative splicing in Drosophila.

    PubMed

    Johnson, Matthew L; Nagengast, Alexis A; Salz, Helen K

    2010-03-05

    Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal (Sxl). Sxl expression is controlled via a negative regulatory mechanism where inclusion of the translation-terminating male exon is blocked in females. Previous studies have shown that the mechanism leading to exon skipping is autoregulatory and requires the SXL protein to antagonize exon inclusion by interacting with core spliceosomal proteins, including the U1 snRNP protein Sans-fille (SNF). In studies begun by screening for proteins that interact with SNF, we identified PPS, a previously uncharacterized protein, as a novel component of the machinery required for Sxl male exon skipping. PPS encodes a large protein with four signature motifs, PHD, BRK, TFS2M, and SPOC, typically found in proteins involved in transcription. We demonstrate that PPS has a direct role in Sxl male exon skipping by showing first that loss of function mutations have phenotypes indicative of Sxl misregulation and second that the PPS protein forms a complex with SXL and the unspliced Sxl RNA. In addition, we mapped the recruitment of PPS, SXL, and SNF along the Sxl gene using chromatin immunoprecipitation (ChIP), which revealed that, like many other splicing factors, these proteins bind their RNA targets while in close proximity to the DNA. Interestingly, while SNF and SXL are specifically recruited to their predicted binding sites, PPS has a distinct pattern of accumulation along the Sxl gene, associating with a region that includes, but is not limited to, the SxlPm promoter. Together, these data indicate that PPS is different from other splicing factors involved in male-exon skipping and suggest, for the first time, a functional link between transcription and SXL-mediated alternative splicing. Loss of zygotic PPS function, however, is lethal to both sexes, indicating that its role may be of broad significance.

  2. Biochemical and Functional Analysis of Drosophila-Sciara Chimeric Sex-Lethal Proteins

    PubMed Central

    Ruiz, María Fernanda; Sarno, Francesca; Zorrilla, Silvia; Rivas, Germán; Sánchez, Lucas

    2013-01-01

    Background The Drosophila SXL protein controls sex determination and dosage compensation. It is a sex-specific factor controlling splicing of its own Sxl pre-mRNA (auto-regulation), tra pre-mRNA (sex determination) and msl-2 pre-mRNA plus translation of msl-2 mRNA (dosage compensation). Outside the drosophilids, the same SXL protein has been found in both sexes so that, in the non-drosophilids, SXL does not appear to play the key discriminating role in sex determination and dosage compensation that it plays in Drosophila. Comparison of SXL proteins revealed that its spatial organisation is conserved, with the RNA-binding domains being highly conserved, whereas the N- and C-terminal domains showing significant variation. This manuscript focuses on the evolution of the SXL protein itself and not on regulation of its expression. Methodology Drosophila-Sciara chimeric SXL proteins were produced. Sciara SXL represents the non-sex-specific function of ancient SXL in the non-drosophilids from which presumably Drosophila SXL evolved. Two questions were addressed. Did the Drosophila SXL protein have affected their functions when their N- and C-terminal domains were replaced by the corresponding ones of Sciara? Did the Sciara SXL protein acquire Drosophila sex-specific functions when the Drosophila N- and C-terminal domains replaced those of Sciara? The chimeric SXL proteins were analysed in vitro to study their binding affinity and cooperative properties, and in vivo to analyse their effect on sex determination and dosage compensation by producing Drosophila flies that were transgenic for the chimeric SXL proteins. Conclusions The sex-specific properties of extant Drosophila SXL protein depend on its global structure rather than on a specific domain. This implies that the modifications, mainly in the N- and C-terminal domains, that occurred in the SXL protein during its evolution within the drosophilid lineage represent co-evolutionary changes that determine the appropriate

  3. Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis.

    PubMed

    Chau, Johnnie; Kulnane, Laura Shapiro; Salz, Helen K

    2012-06-12

    Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. We address the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. We find that this dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3' untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior.

  4. Effects of Wolbachia Infection and ovarian tumor Mutations on Sex-lethal Germline Functioning in Drosophila

    PubMed Central

    Sun, Sha; Cline, Thomas W.

    2009-01-01

    Wolbachia is a ubiquitous intracellular endosymbiont of invertebrates. Surprisingly, infection of Drosophila melanogaster by this maternally inherited bacterium restores fertility to females carrying ovarian tumor (cystocyte overproliferation) mutant alleles of the Drosophila master sex-determination gene, Sex-lethal (Sxl). We scanned the Drosophila genome for effects of infection on transcript levels in wild-type previtellogenic ovaries that might be relevant to this suppression of female-sterile Sxl mutants by Wolbachia. Yolk protein gene transcript levels were most affected, being reduced by infection, but no genes showed significantly more than a twofold difference. The yolk gene effect likely signals a small, infection-induced delay in egg chamber maturation unrelated to suppression. In a genetic study of the Wolbachia–Sxl interaction, we established that germline Sxl controls meiotic recombination as well as cystocyte proliferation, but Wolbachia only influences the cystocyte function. In contrast, we found that mutations in ovarian tumor (otu) interfere with both Sxl germline functions. We were led to otu through characterization of a spontaneous dominant suppressor of the Wolbachia–Sxl interaction, which proved to be an otu mutation. Clearly Sxl and otu work together in the female germline. These studies of meiosis in Sxl mutant females revealed that X chromosome recombination is considerably more sensitive than autosomal recombination to reduced Sxl activity. PMID:19171941

  5. Functional conservation of the sex-lethal sex determining promoter, Sxl-Pe, in Drosophila virilis.

    PubMed

    Jinks, Timothy Morgan; Calhoun, Gretchen; Schedl, Paul

    2003-05-01

    The primary sex determination signal in Drosophila melanogaster, the ratio of X chromosomes to autosomes, sets the activity state of the switch gene, Sex-lethal ( Sxl), by regulating the establishment promoter, m-Sxl-Pe. We have identified and characterized the establishment promoter, v-Sxl-Pe, of the distantly related species Drosophila virilis. Like melanogaster, the virilis Sxl-Pe is organized into four sub-domains: the Sxl-Pe mRNA leader and exon E1 of Sxl protein, the core promoter, the sex-specific element and the augmentation element. The core promoter and sex-specific element of v-Sxl-Pe show considerable sequence similarity to m-Sxl-Pe and contain target sites for components of the X/A signaling system. While the augmentation element of v-Sxl-Pe also has sequence motifs that could function as target sites for the X/A signaling system, it shows little similarity to the melanogaster augmentation element. Functional studies reveal that v-Sxl-Pe drives sex-specific expression in D. melanogaster embryos and that the activity of the virilis promoter is controlled by known components of the melanogaster X/A counting system. Although v-Sxl-Pe responds appropriately to the melanogaster sex determination signal, it is less active than Sxl-Pe from melanogaster. Unexpectedly, the reduced activity is due to differences in the activity of the conserved core promoter, while the non-conserved augmentation element functions effectively. These findings suggest that low-affinity target sites for the X/A counting system are critical for the functioning of Sxl-Pe.

  6. Transcriptional regulation of the Sex-lethal gene by helix-loop-helix proteins.

    PubMed

    Hoshijima, K; Kohyama, A; Watakabe, I; Inoue, K; Sakamoto, H; Shimura, Y

    1995-09-11

    Somatic sex determination in Drosophila depends on the expression of Sex-lethal (Sxl), whose level is determined by the relative number of X chromosomes and sets of autosomes (X:A ratio). The first step in regulation of Sxl expression is transcriptional control from its early promoter and several genes encoding transcription factors of the helix-loop-helix (HLH) family such as daughterless (da), sisterless-b (sis-b), deadpan (dpn) and extramacrochaetae (emc) have been implicated. By the use of transfection assays and in vitro binding experiments, here we show that da/sis-b heterodimers bind several sites on the Sxl early promoter with different affinities and consequently tune the level of active transcription from this promoter. Interestingly, our data indicate that repression by the dpn product of da/sis-b dependent activation results from specific binding of dpn protein to a unique site within the promoter. This contrasts with the mode of emc repression, which inhibits the formation of the da/sis-b heterodimers. These results reveal the molecular mechanisms by which Sxl gene transcription is positively or negatively regulated to control somatic sex determination.

  7. CRISPR/Cas9-mediated mutagenesis of the white and Sex lethal loci in the invasive pest, Drosophila suzukii.

    PubMed

    Li, Fang; Scott, Maxwell J

    2016-01-22

    Drosophila suzukii (commonly called spotted wing Drosophila) is an invasive pest of soft-skinned fruit (e.g. blueberries, strawberries). A high quality reference genome sequence is available but functional genomic tools, such as used in Drosophila melanogaster, remain to be developed. In this study we have used the CRISPR/Cas9 system to introduce site-specific mutations in the D. suzukii white (w) and Sex lethal (Sxl) genes. Hemizygous males with w mutations develop white eyes and the mutant genes are transmissible to the next generation. Somatic mosaic females that carry mutations in the Sxl gene develop abnormal genitalia and reproductive tissue. The D. suzukii Sxl gene could be an excellent target for a Cas9-mediated gene drive to suppress populations of this highly destructive pest. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Transposon insertions causing constitutive sex-lethal activity in Drosophila melanogaster affect Sxl sex-specific transcript splicing

    SciTech Connect

    Berstein, M.; Cline, T.W. |; Lersch, R.A.; Subrahmanyan, L.

    1995-02-01

    Sex-lethal (Sxl) gene products induce female development in Drosophila melanogaster and suppress the transcriptional hyperactivation of X-linked genes responsible for male X-chromosome dosage compensation. Control of Sxl functioning by the dose of X-chromosomes normally ensures that the female-specific functions of this developmental switch gene are only expressed in diplo-X individuals. Although the immediate effect of X-chromosome dose is on Sxl transcription, during most of the life cycle {open_quotes}on{close_quotes} vs. {open_quotes}off{close_quotes} reflects alternative Sxl RNA splicing, with the female (productive) splicing mode maintained by a positive feedback activity of SXL protein on Sxl pre-mRNA splicing. {open_quotes}Male-lethal{close_quotes} (Sxl{sup M}) gain-of-function alleles subvert Sxl control by X-chromosome dose, allowing female Sxl functions to be expressed independent of the positive regulators upstream of Sxl. As a consequence, Sxl{sup M} haplo-X animals (chromosomal males) die because of improper dosage compensation, and Sxl{sup m} chromosomal females survive the otherwise lethal effects of mutations in upstream positive regulators. Transcript analysis of double-mutant male-viable Sxl{sup M} derivatives in which the Sxl{sup M} insertion is cis to loss-of-function mutations, combined with other results reported here, indicates that the constitutive character of these Sxl{sup M} alleles is a consequence of an alteration of the structure of the pre-mRNA that allow some level of female splicing to occur even in the absence of functional SXL protein. Surprisingly, however, most of the constitutive character of Sxl{sup M} alleles appears to depend on the mutant alleles` responsiveness, perhaps greater than wild-type, to the autoregulatory splicing activity of the wild-type SXL proteins they produce. 47 refs., 10 figs., 4 tabs.

  9. The complex set of late transcripts from the Drosophila sex determination gene sex-lethal encodes multiple related polypeptides.

    PubMed Central

    Samuels, M E; Schedl, P; Cline, T W

    1991-01-01

    Sex-lethal (Sxl), a key sex determination gene in Drosophila melanogaster, is known to express a set of three early transcripts arising during early embryogenesis and a set of seven late transcripts occurring from midembryogenesis through adulthood. Among the late transcripts, male-specific mRNAs were distinguished from their female counterparts by the presence of an extra exon interrupting an otherwise long open reading frame (ORF). We have now analyzed the structures of the late Sxl transcripts by cDNA sequencing, Northern (RNA) blotting, primer extension, and RNase protection. The late transcripts appear to use a common 5' end but differ at their 3' ends by the use of alternative polyadenylation sites. Two of these sites lack canonical AATAAA sequences, and their use correlates in females with the presence of a functional germ line, suggesting possible tissue-specific polyadenylation. Besides the presence of the male-specific exon, no additional sex-specific splicing events were detected, although a number of non-sex-specific splicing variants were observed. In females, the various forms of late Sxl transcript potentially encode up to six slightly different polypeptides. All of the protein-coding differences occur outside the previously defined ribonucleoprotein motifs. One class of Sxl mRNAs also includes a second long ORF in the same frame as the first ORF but separated from it by a single ochre codon. The function of this second ORF is unknown. Significant amounts of apparently partially processed Sxl RNAs were observed, consistent with the hypothesis that the regulated Sxl splices occur relatively slowly. Images PMID:1710769

  10. Misregulation of Sex-Lethal and Disruption of Male-Specific Lethal Complex Localization in Drosophila Species Hybrids

    PubMed Central

    Pal Bhadra, Manika; Bhadra, Utpal; Birchler, James A.

    2006-01-01

    A major model system for the study of evolutionary divergence between closely related species has been the unisexual lethality resulting from reciprocal crosses of Drosophila melanogaster and D. simulans. Sex-lethal (Sxl), a critical gene for sex determination, is misregulated in these hybrids. In hybrid males from D. melanogaster mothers, there is an abnormal expression of Sxl and a failure of localization of the male-specific lethal (MSL) complex to the X chromosome, which causes changes in gene expression. Introduction of a Sxl mutation into this hybrid genotype will allow expression of the MSL complex but there is no sequestration to the X chromosome. Lethal hybrid rescue (Lhr), which allows hybrid males from this cross to survive, corrects the SXL and MSL defects. The reciprocal cross of D. simulans mothers by D. melanogaster males exhibits underexpression of Sxl in embryos. PMID:16951071

  11. Control of Drosophila Sex-lethal pre-mRNA splicing by its own female-specific product.

    PubMed Central

    Sakamoto, H; Inoue, K; Higuchi, I; Ono, Y; Shimura, Y

    1992-01-01

    Drosophila melanogaster somatic sexual differentiation is accomplished by serial function of the products of sex-determination genes. Sex-lethal (Sxl), is one such gene. It is functionally expressed only in female flies. The sex-specific expression of this gene is regulated by alternative mRNA splicing which results in either the inclusion or exclusion of the translation stop codon containing third exon. Although previous genetic and molecular analyses suggest that functional Sxl expression is maintained by a positive feedback loop, where the female-specific Sxl product promotes the synthesis of its own female-specific mRNA, the mechanistic details of such regulation have remained unclear. We have developed a cotransfection system using Drosophila cultured (Kc) cells in which Sxl primary transcripts are expressed with or without the female specific Sxl product. Here we show that the female-specific Sxl product induces the synthesis of its own female-specific mRNA by negative control of male-specific splicing. Deletion, substitution, and binding experiments have demonstrated that multiple uridine-rich sequences in the introns around the male-specific third exon are involved in the splicing regulation of Sxl pre-mRNA. Images PMID:1454517

  12. Drosophila switch gene Sex-lethal can bypass its switch-gene target transformer to regulate aspects of female behavior.

    PubMed

    Evans, Daniel S; Cline, Thomas W

    2013-11-19

    The switch gene Sex-lethal (Sxl) was thought to elicit all aspects of Drosophila female somatic differentiation other than size dimorphism by controlling only the switch gene transformer (tra). Here we show instead that Sxl controls an aspect of female sexual behavior by acting on a target other than or in addition to tra. We inferred the existence of this unknown Sxl target from the observation that a constitutively feminizing tra transgene that restores fertility to tra(-) females failed to restore fertility to Sxl-mutant females that were adult viable but functionally tra(-). The sterility of these mutant females was caused by an ovulation failure. Because tra expression is not sufficient to render these Sxl-mutant females fertile, we refer to this pathway as the tra-insufficient feminization (TIF) branch of the sex-determination regulatory pathway. Using a transgene that conditionally expresses two Sxl feminizing isoforms, we find that the TIF branch is required developmentally for neurons that also sex-specifically express fruitless, a tra gene target controlling sexual behavior. Thus, in a subset of fruitless neurons, targets of the TIF and tra pathways appear to collaborate to control ovulation. In most insects, Sxl has no sex-specific functions, and tra, rather than Sxl, is both the target of the primary sex signal and the gene that maintains the female developmental commitment via positive autoregulation. The TIF pathway may represent an ancestral female-specific function acquired by Sxl in an early evolutionary step toward its becoming the regulator of tra in Drosophila.

  13. Drosophila switch gene Sex-lethal can bypass its switch-gene target transformer to regulate aspects of female behavior

    PubMed Central

    Evans, Daniel S.; Cline, Thomas W.

    2013-01-01

    The switch gene Sex-lethal (Sxl) was thought to elicit all aspects of Drosophila female somatic differentiation other than size dimorphism by controlling only the switch gene transformer (tra). Here we show instead that Sxl controls an aspect of female sexual behavior by acting on a target other than or in addition to tra. We inferred the existence of this unknown Sxl target from the observation that a constitutively feminizing tra transgene that restores fertility to tra− females failed to restore fertility to Sxl-mutant females that were adult viable but functionally tra−. The sterility of these mutant females was caused by an ovulation failure. Because tra expression is not sufficient to render these Sxl-mutant females fertile, we refer to this pathway as the tra-insufficient feminization (TIF) branch of the sex-determination regulatory pathway. Using a transgene that conditionally expresses two Sxl feminizing isoforms, we find that the TIF branch is required developmentally for neurons that also sex-specifically express fruitless, a tra gene target controlling sexual behavior. Thus, in a subset of fruitless neurons, targets of the TIF and tra pathways appear to collaborate to control ovulation. In most insects, Sxl has no sex-specific functions, and tra, rather than Sxl, is both the target of the primary sex signal and the gene that maintains the female developmental commitment via positive autoregulation. The TIF pathway may represent an ancestral female-specific function acquired by Sxl in an early evolutionary step toward its becoming the regulator of tra in Drosophila. PMID:24191002

  14. A theoretical model for the regulation of Sex-lethal, a gene that controls sex determination and dosage compensation in Drosophila melanogaster.

    PubMed Central

    Louis, Matthieu; Holm, Liisa; Sánchez, Lucas; Kaufman, Marcelle

    2003-01-01

    Cell fate commitment relies upon making a choice between different developmental pathways and subsequently remembering that choice. Experimental studies have thoroughly investigated this central theme in biology for sex determination. In the somatic cells of Drosophila melanogaster, Sex-lethal (Sxl) is the master regulatory gene that specifies sexual identity. We have developed a theoretical model for the initial sex-specific regulation of Sxl expression. The model is based on the well-documented molecular details of the system and uses a stochastic formulation of transcription. Numerical simulations allow quantitative assessment of the role of different regulatory mechanisms in achieving a robust switch. We establish on a formal basis that the autoregulatory loop involved in the alternative splicing of Sxl primary transcripts generates an all-or-none bistable behavior and constitutes an efficient stabilization and memorization device. The model indicates that production of a small amount of early Sxl proteins leaves the autoregulatory loop in its off state. Numerical simulations of mutant genotypes enable us to reproduce and explain the phenotypic effects of perturbations induced in the dosage of genes whose products participate in the early Sxl promoter activation. PMID:14668388

  15. Behavioral and pheromonal phenotypes associated with expression of loss-of-function mutations in the sex-lethal gene of Drosophila melanogaster.

    PubMed

    Tompkins, L; McRobert, S P

    1995-02-01

    We have shown that female-specific functions of the sex determination gene Sex-lethal (Sxl) regulate sexual behavior and synthesis of the three major sex pheromones that have been identified in normal, sexually mature Drosophilia melanogaster males and virgin females. Diplo-X flies, heterozygous in trans for two partial loss-of-function Sxl mutations, elicit less courtship than normal females and produce large quantities of the inhibitory pheromones that normal males synthesize. In addition, the mutant flies fail to synthesize the female-predominant aphrodisiac pheromone or make very small quantities of this compound.

  16. Molecular analysis and developmental expression of the Sex-lethal gene of Sciara ocellaris (Diptera order, Nematocera suborder).

    PubMed

    Ruiz, M F; Goday, C; González, P; Sánchez, L

    2003-06-01

    This paper reports the cloning and characterization in Sciara ocellaris of the gene homologous to Sex-lethal (Sxl) of Drosophila melanogaster. This gene plays the key role controlling sex determination and dosage compensation in the latter species. The Sciara Sxl gene produces a single transcript encoding a single protein in both males and females. This protein, found inside the nucleus, is expressed in all tissues. Both Sciara and Drosophila Sxl proteins are highly conserved at their two RNA-binding domains. In both Sciara sexes, the Sxl protein co-localizes with transcription-active regions dependent on RNA polymerase II but not on RNA polymerase I. It would appear that in Sciara, Sxl does not appear to play the key discriminative role in controlling sex determination and dosage compensation that it plays in Drosophila.

  17. The master switch gene Sex-lethal promotes female development by negatively regulating the N signaling pathway

    PubMed Central

    Penn, Jill K M; Schedl, Paul

    2011-01-01

    Summary Notch (N) signaling is used for cell fate determination in many different developmental contexts. Here we show that the master control gene for sex determination in Drosophila melanogaster, Sex-lethal (Sxl), negatively regulates the N signaling pathway in females. In genetic assays, reducing Sxl activity suppresses the phenotypic effects of N mutations while increasing Sxl activity enhances the effects. Sxl appears to negatively regulate the pathway by reducing N protein accumulation and higher levels of N are found in Sxl−clones than in adjacent wild type cells. The inhibition of N expression does not depend on the known downstream targets of Sxl; however we find that Sxl protein can bind to N mRNAs. Finally our results indicate that downregulation of the N pathway by Sxl contributes to sex specific differences in morphology and suggest that it may also play an important role in follicle cell specification during oogenesis. PMID:17276344

  18. Interaction of the sex-lethal RNA binding domains with RNA.

    PubMed Central

    Kanaar, R; Lee, A L; Rudner, D Z; Wemmer, D E; Rio, D C

    1995-01-01

    Sex determination and X chromosome dosage compensation in Drosophila melanogaster are directed by the Sex-lethal (Sxl) protein. In part, Sxl functions by regulating the splicing of the transformer pre-mRNA by binding to a 3' splice site polypyrimidine tract. Polypyrimidine tracts are essential for splicing of metazoan pre-mRNAs. To unravel the mechanism of splicing regulation at polypyrimidine tracts we analyzed the interaction of Sxl with RNA. The RNA binding activity of Sxl was mapped to the two ribonucleoprotein consensus sequence domains of the protein. Quantitation of binding showed that both RNA binding domains (RBDs) were required in cis for site-specific RNA binding. Individual RBDs interacted with RNA more weakly and had lost the ability to discriminate between wild-type and mutant transformer polypyrimidine tracts. Structural elements in one of the RBDs that are likely to interact with a polypyrimidine tract were identified using nuclear magnetic resonance techniques. In addition, our data suggest that multiple imino protons of the transformer polypyrimidine tract were involved in hydrogen bonding. Interestingly, in vitro Sxl bound with equal affinity to polypyrimidine tracts of pre-mRNAs that it does not regulate in vivo. We discuss the implications of this finding for the mechanism through which Sxl may gain selectivity for particular polypyrimidine tracts in vivo. Images PMID:7556096

  19. The gene Sex-lethal of the Sciaridae family (order Diptera, suborder Nematocera) and its phylogeny in dipteran insects.

    PubMed

    Serna, Esther; Gorab, Eduardo; Ruiz, M Fernanda; Goday, Clara; Eirín-López, José M; Sánchez, Lucas

    2004-10-01

    This article reports the cloning and characterization of the gene homologous to Sex-lethal (Sxl) of Drosophila melanogaster from Sciara coprophila, Rhynchosciara americana, and Trichosia pubescens. This gene plays the key role in controlling sex determination and dosage compensation in D. melanogaster. The Sxl gene of the three species studied produces a single transcript encoding a single protein in both males and females. Comparison of the Sxl proteins of these Nematocera insects with those of the Brachycera showed their two RNA-binding domains (RBD) to be highly conserved, whereas significant variation was observed in both the N- and C-terminal domains. The great majority of nucleotide changes in the RBDs were synonymous, indicating that purifying selection is acting on them. In both sexes of the three Nematocera insects, the Sxl protein colocalized with transcription-active regions dependent on RNA polymerase II but not on RNA polymerase I. Together, these results indicate that Sxl does not appear to play a discriminatory role in the control of sex determination and dosage compensation in nematocerans. Thus, in the phylogenetic lineage that gave rise to the drosophilids, evolution coopted for the Sxl gene, modified it, and converted it into the key gene controlling sex determination and dosage compensation. At the same time, however, certain properties of the recruited ancestral Sxl gene were beneficial, and these are maintained in the evolved Sxl gene, allowing it to exert its sex-determining and dose compensation functions in Drosophila.

  20. In vivo interactions of the Drosophila Hairy and Runt transcriptional repressors with target promoters.

    PubMed

    Jiménez, G; Pinchin, S M; Ish-Horowicz, D

    1996-12-16

    The Hairy and Runt pair-rule proteins regulate Drosophila segmentation by repressing transcription. To explore the ability of these proteins to function as promoter-bound regulators in vivo, we examined the effects of Hairy and Runt derivatives containing heterologous transcriptional activation domains (HairyAct and RunAct). Using this approach, we find that Hairy and Runt efficiently target such activation domains to specific segmentation gene promoters, leading to rapid induction of transcription. Our results strongly suggest that Hairy normally acts as a promoter-bound repressor of fushi tarazu, runt and odd-skipped, and that Runt directly represses even-skipped. We also show that expressing HairyAct in early blastoderm embryos causes ectopic Sex-lethal expression and male-specific lethality, implying that the Hairy-related denominator element Deadpan represses Sex-lethal during sex determination by directly recognizing the early Sex-lethal promoter.

  1. Protein-protein interactions among components of the Drosophila primary sex determination signal.

    PubMed

    Liu, Y; Belote, J M

    1995-07-28

    Sex determination in Drosophila melanogaster is initiated in the early embryo by a signal provided by three types of genes: (1) X-linked numerator elements [e.g., sisterless-a (sis-a) and sisterless-b (sis-b)], (2) autosomally linked denominator elements [e.g., deadpan (dpn)], and (3) maternal factors [e.g., daughterless (da)]. This signal acts to stimulate transcription from an embryo-specific promoter of the master regulatory gene Sex-lethal (Sxl) in embryos that have two X chromosomes (females), while it fails to activate Sxl in those with only one X (males). It has been previously proposed that competitive dimerizations among the components of this signal might provide the molecular basis for this sex specificity. Here, we use the yeast two-hybrid system to demonstrate specific protein-protein interactions among the above-mentioned factors, and to delimit their interacting domains. These results support and extend the model of the molecular basis of the X/A ratio signal.

  2. 31 Flavors of Drosophila Rab proteins

    SciTech Connect

    Zhang, Jun; Schulze, Karen L.; Hiesinger, P. Robin; Suyama, Kaye; Wang, Stream; Fish, Matthew; Acar, Melih; Hoskins, Roger A.; Bellen, HugoJ.; Scott, Matthew P.

    2007-04-03

    Rab proteins are small GTPases that play important roles intransport of vesicle cargo and recruitment, association of motor andother proteins with vesicles, and docking and fusion of vesicles atdefined locations. In vertebrates, more than 75 Rab genes have beenidentified, some of which have been intensively studied for their rolesin endosome and synaptic vesicle trafficking. Recent studies of thefunctions of certain Rab proteins have revealed specific roles inmediating developmental signal transduction. We have begun a systematicgenetic study of the 33 Rab genes in Drosophila. Most of the fly proteinsare clearly related to specific vertebrate proteins. We report here thecreation of a set of transgenic fly lines that allow spatially andtemporally regulated expression of Drosophila Rab proteins. We generatedfluorescent protein-tagged wild-type, dominant-negative, andconstitutively active forms of 31 Drosophila Rab proteins. We describeDrosophila Rab expression patterns during embryogenesis, the subcellularlocalization of some Rab proteins, and comparisons of the localization ofwild-type, dominant-negative, and constitutively active forms of selectedRab proteins. The high evolutionary conservation and low redundancy ofDrosophila Rab proteins make these transgenic lines a useful toolkit forinvestigating Rab functions in vivo.

  3. Heat shock proteins and Drosophila aging.

    PubMed

    Tower, John

    2011-05-01

    Since their discovery in Drosophila, the heat shock proteins (Hsps) have been shown to regulate both stress resistance and life-span. Aging is characterized by increased oxidative stress and the accumulation of abnormal (malfolded) proteins, and these stresses induce Hsp gene expression through the transcription factor HSF. In addition, a subset of Hsps is induced by oxidative stress through the JNK signaling pathway and the transcription factor Foxo. The Hsps counteract the toxicity of abnormal proteins by facilitating protein refolding and turnover, and through other mechanisms including inhibition of apoptosis. The Hsps are up-regulated in tissue-specific patterns during aging, and their expression correlates with, and sometimes predicts, life span, making them ideal biomarkers of aging. The tools available for experimentally manipulating gene function and assaying healthspan in Drosophila provides an unparalleled opportunity to further study the role of Hsps in aging.

  4. Heat shock proteins and Drosophila aging

    PubMed Central

    Tower, John

    2010-01-01

    Since their discovery in Drosophila, the heat shock proteins (Hsps) have been shown to regulate both stress resistance and life span. Aging is characterized by increased oxidative stress and the accumulation of abnormal (malfolded) proteins, and these stresses induce Hsp gene expression through the transcription factor HSF. In addition, a subset of Hsps is induced by oxidative stress through the JNK signaling pathway and the transcription factor Foxo. The Hsps counteract the toxicity of abnormal proteins by facilitating protein refolding and turnover, and through other mechanisms including inhibition of apoptosis. The Hsps are up-regulated in tissue-specific patterns during aging, and their expression correlates with, and sometimes predicts, life span, making them ideal biomarkers of aging. The tools available for experimentally manipulating gene function and assaying healthspan in Drosophila provides an unparalleled opportunity to further study the role of Hsps in aging. PMID:20840862

  5. Biases in Drosophila melanogaster protein trap screens

    PubMed Central

    Aleksic, Jelena; Lazic, Ranko; Müller, Ilka; Russell, Steven R; Adryan, Boris

    2009-01-01

    Background The ability to localise or follow endogenous proteins in real time in vivo is of tremendous utility for cell biology or systems biology studies. Protein trap screens utilise the random genomic insertion of a transposon-borne artificial reporter exon (e.g. encoding the green fluorescent protein, GFP) into an intron of an endogenous gene to generate a fluorescent fusion protein. Despite recent efforts aimed at achieving comprehensive coverage of the genes encoded in the Drosophila genome, the repertoire of genes that yield protein traps is still small. Results We analysed the collection of available protein trap lines in Drosophila melanogaster and identified potential biases that are likely to restrict genome coverage in protein trap screens. The protein trap screens investigated here primarily used P-element vectors and thus exhibit some of the same positional biases associated with this transposon that are evident from the comprehensive Drosophila Gene Disruption Project. We further found that protein trap target genes usually exhibit broad and persistent expression during embryonic development, which is likely to facilitate better detection. In addition, we investigated the likely influence of the GFP exon on host protein structure and found that protein trap insertions have a significant bias for exon-exon boundaries that encode disordered protein regions. 38.8% of GFP insertions land in disordered protein regions compared with only 23.4% in the case of non-trapping P-element insertions landing in coding sequence introns (p < 10-4). Interestingly, even in cases where protein domains are predicted, protein trap insertions frequently occur in regions encoding surface exposed areas that are likely to be functionally neutral. Considering the various biases observed, we predict that less than one third of intron-containing genes are likely to be amenable to trapping by the existing methods. Conclusion Our analyses suggest that the utility of P

  6. Biases in Drosophila melanogaster protein trap screens.

    PubMed

    Aleksic, Jelena; Lazic, Ranko; Müller, Ilka; Russell, Steven R; Adryan, Boris

    2009-05-28

    The ability to localise or follow endogenous proteins in real time in vivo is of tremendous utility for cell biology or systems biology studies. Protein trap screens utilise the random genomic insertion of a transposon-borne artificial reporter exon (e.g. encoding the green fluorescent protein, GFP) into an intron of an endogenous gene to generate a fluorescent fusion protein. Despite recent efforts aimed at achieving comprehensive coverage of the genes encoded in the Drosophila genome, the repertoire of genes that yield protein traps is still small. We analysed the collection of available protein trap lines in Drosophila melanogaster and identified potential biases that are likely to restrict genome coverage in protein trap screens. The protein trap screens investigated here primarily used P-element vectors and thus exhibit some of the same positional biases associated with this transposon that are evident from the comprehensive Drosophila Gene Disruption Project. We further found that protein trap target genes usually exhibit broad and persistent expression during embryonic development, which is likely to facilitate better detection. In addition, we investigated the likely influence of the GFP exon on host protein structure and found that protein trap insertions have a significant bias for exon-exon boundaries that encode disordered protein regions. 38.8% of GFP insertions land in disordered protein regions compared with only 23.4% in the case of non-trapping P-element insertions landing in coding sequence introns (p < 10(-4)). Interestingly, even in cases where protein domains are predicted, protein trap insertions frequently occur in regions encoding surface exposed areas that are likely to be functionally neutral. Considering the various biases observed, we predict that less than one third of intron-containing genes are likely to be amenable to trapping by the existing methods. Our analyses suggest that the utility of P-element vectors for protein trap screens

  7. A Protein Complex Network of Drosophila melanogaster

    PubMed Central

    Guruharsha, K. G.; Rual, J. -F.; Zhai, B.; Mintseris, J.; Vaidya, P.; Vaidya, N.; Beekman, C.; Wong, C.; Rhee, D. Y.; Cenaj, O.; McKillip, E.; Shah, S.; Stapleton, M.; Wan, K. H.; Yu, C.; Parsa, B.; Carlson, J. W.; Chen, X.; Kapadia, B.; VijayRaghavan, K.; Gygi, S. P.; Celniker, S. E.; Obar, R. A.; Artavanis-Tsakonas, S.

    2011-01-01

    SUMMARY Determining the composition of protein complexes is an essential step towards understanding the cell as an integrated system. Using co-affinity purification coupled to mass spectrometry analysis, we examined protein associations involving nearly five thousand individual, FLAG-HA epitope-tagged Drosophila proteins. Stringent analysis of these data, based on a novel statistical framework to define individual protein-protein interactions, led to the generation of a Drosophila Protein interaction Map (DPiM) encompassing 556 protein complexes. The high quality of DPiM and its usefulness as a paradigm for metazoan proteomes is apparent from the recovery of many known complexes, significant enrichment for shared functional attributes and validation in human cells. DPiM defines potential novel members for several important protein complexes and assigns functional links to 586 protein-coding genes lacking previous experimental annotation. DPiM represents, to our knowledge, the largest metazoan protein complex map and provides a valuable resource for analysis of protein complex evolution. PMID:22036573

  8. Exploring strategies for protein trapping in Drosophila

    SciTech Connect

    Quinones-Coello, Ana T.; Petrella, Lisa N.; Ayers, Kathleen; Melillo, Anthony; Mazzalupo, Stacy; Hudson, Andrew M.; Wang, Shu; Castiblanco, Claudia; Buszczak, Michael; Hoskins, Roger A.; Cooley, Lynn

    2006-12-18

    The use of fluorescent protein tags has had a huge impact oncell biological studies in virtually every experimental system.Incorporation of coding sequence for fluorescent proteins such as greenfluorescent protein (GFP) into genes at their endogenous chromosomalposition is especially useful for generating GFP-fusion proteins thatprovide accurate cellular and subcellular expression data. We testedmodifications of a transposon-based protein trap screening procedure inDrosophila to optimize the rate of recovering useful protein traps andtheir analysis. Transposons carrying the GFP-coding sequence flanked bysplice acceptor and donor sequences were mobilized, and new insertionsthat resulted in production of GFP were captured using an automatedembryo sorter. Individual stocks were established, GFP expression wasanalyzed during oogenesis, and insertion sites were determined bysequencing genomic DNA flanking the insertions. The resulting collectionincludes lines with protein traps in which GFP was spliced into mRNAs andembedded within endogenous proteins or enhancer traps in which GFPexpression depended on splicing into transposon-derived RNA. We report atotal of 335 genes associated with protein or enhancer traps and aweb-accessible database for viewing molecular information and expressiondata for these genes.

  9. Cubilin and amnionless mediate protein reabsorption in Drosophila nephrocytes.

    PubMed

    Zhang, Fujian; Zhao, Ying; Chao, Yufang; Muir, Katherine; Han, Zhe

    2013-02-01

    The insect nephrocyte and the mammalian glomerular podocyte are similar with regard to filtration, but it remains unclear whether there is an organ or cell type in flies that reabsorbs proteins. Here, we show that the Drosophila nephrocyte has molecular, structural, and functional similarities to the renal proximal tubule cell. We screened for genes required for nephrocyte function and identified two Drosophila genes encoding orthologs of mammalian cubilin and amnionless (AMN), two major receptors for protein reabsorption in the proximal tubule. In Drosophila, expression of dCubilin and dAMN is specific to nephrocytes, where they function as co-receptors for protein uptake. Targeted expression of human AMN in Drosophila nephrocytes was sufficient to rescue defective protein uptake induced by dAMN knockdown, suggesting evolutionary conservation of Cubilin/AMN co-receptors function from flies to humans. Furthermore, we found that Cubilin/AMN-mediated protein reabsorption is required for the maintenance of nephrocyte ultrastructure and fly survival under conditions of toxic stress. In conclusion, the insect nephrocyte combines filtration with protein reabsorption, using evolutionarily conserved genes and subcellular structures, suggesting that it can serve as a simplified model for both podocytes and the renal proximal tubule.

  10. Peptidoglycan recognition proteins in Drosophila immunity

    PubMed Central

    Kurata, Shoichiro

    2013-01-01

    Innate immunity is the front line of self-defense against infectious non-self in vertebrates and invertebrates. The innate immune system is mediated by germ-line encoding pattern recognition molecules (pathogen sensors) that recognize conserved molecular patterns present in the pathogens but absent in the host. Peptidoglycans (PGN) are essential cell wall components of almost all bacteria, except mycoplasma lacking a cell wall, which provides the host immune system an advantage for detecting invading bacteria. Several families of pattern recognition molecules that detect PGN and PGN-derived compounds have been indentified, and the role of PGRP family members in host defense is relatively well-chacterized in Drosophila. This review focuses on the role of PGRP family members in the recognition of invading bacteria and the activation and modulation of immune responses in Drosophila. PMID:23796791

  11. spenito is required for sex determination in Drosophila melanogaster

    PubMed Central

    Yan, Dong; Perrimon, Norbert

    2015-01-01

    Sex-lethal (Sxl) encodes the master regulator of the sex determination pathway in Drosophila and acts by controlling sex identity in both soma and germ line. In females Sxl maintains its own expression by controlling the alternative splicing of its own mRNA. Here, we identify a novel sex determination gene, spenito (nito) that encodes a SPEN family protein. Loss of nito activity results in stem cell tumors in the female germ line as well as female-to-male somatic transformations. We show that Nito is a ubiquitous nuclear protein that controls the alternative splicing of the Sxl mRNA by interacting with Sxl protein and pre-mRNA, suggesting that it is directly involved in Sxl auto-regulation. Given that SPEN family proteins are frequently mutated in cancers, our results suggest that these factors might be implicated in tumorigenesis through splicing regulation. PMID:26324914

  12. Transformation of Drosophila cell lines: an alternative approach to exogenous protein expression.

    PubMed

    Cherbas, Lucy; Cherbas, Peter

    2007-01-01

    Techniques and experimental applications are described for exogenous protein expression in Drosophila cell lines. Ways in which the Drosophila cell lines and the baculovirus expression vector system differ in their applications are emphasized.

  13. Evolution of Drosophila ribosomal protein gene core promoters

    PubMed Central

    Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

    2011-01-01

    The coordinated expression of ribosomal protein genes (RPGs) has been well documented in many species. Previous analyses of RPG promoters focus only on Fungi and mammals. Recognizing this gap and using a comparative genomics approach, we utilize a motif-finding algorithm that incorporates cross-species conservation to identify several significant motifs in Drosophila RPG promoters. As a result, significant differences of the enriched motifs in RPG promoter are found among Drosophila, Fungi, and mammals, demonstrating the evolutionary dynamics of the ribosomal gene regulatory network. We also report a motif present in similar numbers of RPGs among Drosophila species which does not appear to be conserved at the individual RPG gene level. A module-wise stabilizing selection theory is proposed to explain this observation. Overall, our results provide significant insight into the fast-evolving nature of transcriptional regulation in the RPG module. PMID:19059316

  14. Evolution of Drosophila ribosomal protein gene core promoters.

    PubMed

    Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

    2009-03-01

    The coordinated expression of ribosomal protein genes (RPGs) has been well documented in many species. Previous analyses of RPG promoters focus only on Fungi and mammals. Recognizing this gap and using a comparative genomics approach, we utilize a motif-finding algorithm that incorporates cross-species conservation to identify several significant motifs in Drosophila RPG promoters. As a result, significant differences of the enriched motifs in RPG promoter are found among Drosophila, Fungi, and mammals, demonstrating the evolutionary dynamics of the ribosomal gene regulatory network. We also report a motif present in similar numbers of RPGs among Drosophila species which does not appear to be conserved at the individual RPG gene level. A module-wise stabilizing selection theory is proposed to explain this observation. Overall, our results provide significant insight into the fast-evolving nature of transcriptional regulation in the RPG module.

  15. Transcriptional Repression by Drosophila Methyl-CpG-Binding Proteins

    PubMed Central

    Roder, Karim; Hung, Ming-Shiu; Lee, Tai-Lin; Lin, Tzu-Yang; Xiao, Hengyi; Isobe, Ken-Ichi; Juang, Jyh-Lyh; Shen, C.-K. James

    2000-01-01

    C methylation at genomic CpG dinucleotides has been implicated in the regulation of a number of genetic activities during vertebrate cell differentiation and embryo development. The methylated CpG could induce chromatin condensation through the recruitment of histone deacetylase (HDAC)-containing complexes by methyl-CpG-binding proteins. These proteins consist of the methylated-DNA binding domain (MBD). Unexpectedly, however, several studies have identified MBD-containing proteins encoded by genes of Drosophila melanogaster, an invertebrate species supposed to be void of detectable m5CpG. We now report the genomic structure of a Drosophila gene, dMBD2/3, that codes for two MBD-containing, alternatively spliced, and developmentally regulated isoforms of proteins, dMBD2/3 and dMBD2/3Δ. Interestingly, in vitro binding experiments showed that as was the case for vertebrate MBD proteins, dMBD2/3Δ could preferentially recognize m5CpG-containing DNA through its MBD. Furthermore, dMBD2/3Δ as well as one of its orthologs in mouse, MBD2b, could function in human cells as a transcriptional corepressor or repressor. The activities of HDACs appeared to be dispensable for transcriptional repression by dMBD2/3Δ. Finally, dMBD2/3Δ also could repress transcription effectively in transfected Drosophila cells. The surprisingly similar structures and characteristics of the MBD proteins as well as DNA cytosine (C-5) methyltransferase-related proteins in Drosophila and vertebrates suggest interesting scenarios for their roles in eukaryotic cellular functions. PMID:10982856

  16. A Drosophila gene encoding a protein resembling the human beta-amyloid protein precursor.

    PubMed Central

    Rosen, D R; Martin-Morris, L; Luo, L Q; White, K

    1989-01-01

    We have isolated genomic and cDNA clones for a Drosophila gene resembling the human beta-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human beta-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development. Images PMID:2494667

  17. A Drosophila gene encoding a protein resembling the human. beta. -amyloid protein precursor

    SciTech Connect

    Rosen, D.R.; Martin-Morris, L.; Luo, L.; White, K. )

    1989-04-01

    The authors have isolated genomic and cDNA clones for a Drosophila gene resembling the human {beta}-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human {beta}-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development.

  18. Developmental expression of Drosophila Wiskott-Aldrich Syndrome family proteins

    PubMed Central

    Rodriguez-Mesa, Evelyn; Abreu-Blanco, Maria Teresa; Rosales-Nieves, Alicia E.; Parkhurst, Susan M.

    2012-01-01

    Background Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. Results We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. Conclusion All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics. PMID:22275148

  19. Genetic analysis of a Drosophila microtubule-associated protein

    PubMed Central

    1992-01-01

    The 205-kD microtubule-associated protein (205K MAP) is one of the principal MAPs in Drosophila. 205K MAP is similar to the HeLa 210K/MAP4 family of MAPs since it shares the following biochemical properties: it is present in several isoforms, has a molecular mass of approximately 200 kD, and is thermostable. Furthermore, immuno-crossreactivity has been observed between mouse MAP4, HeLa 210K, and Drosophila 205K MAP. Currently, there is little information concerning the biological function of this group of nonmotor MAPs. We have used a classical genetic approach to try to identify the role of the 205K MAP in Drosophila by isolating mutations in the 205K MAP gene. An F2 lethal screen was used to acquire deficiencies of 100EF, the chromosomal location of the 205K MAP gene. Drosophila bearing a homozygous deficiency for the 205K MAP region are fully viable and show no obvious phenotype. A recently developed polymerase chain reaction screen was also used to recover five P-element insertions upstream from the 205K MAP gene. Western blot analysis has shown that these insertions result in hypomorphic mutations of the 205K MAP gene. As was seen with animals that have no 205K MAP, these mutations appear to have no phenotype. These data unambiguously demonstrate that the 205K MAP gene is inessential for development. These results also suggest that there may exist protein(s) with redundant function that can substitute for 205K MAP. PMID:1309812

  20. deadpan, an essential pan-neural gene encoding an HLH protein, acts as a denominator in Drosophila sex determination.

    PubMed

    Younger-Shepherd, S; Vaessin, H; Bier, E; Jan, L Y; Jan, Y N

    1992-09-18

    In Drosophila, sex is determined by the X:A ratio. One major numerator element on the X chromosome is sisterless-b (sis-b), also called scute, which encodes an HLH-type transcription factor. We report here that an essential pan-neural gene, the autosomal HLH gene deadpan (dpn), acts as a denominator element. As revealed by dosage-dependent dominant interactions, males die with too high a ratio of sc+ to dpn+, caused by misexpression of Sex lethal (Sxl) in embryos, and females die with too low a ratio of sc+ to dpn+, because of altered embryonic Sxl expression. In addition, we found that the HLH gene extramacrochaetae (emc), like daughterless (da), is needed maternally for proper communication of the X:A ratio, thus supporting the idea that a set of HLH genes comprises a functional cassette that makes a sensitive and stable genetic switch used in both neural determination and sex determination.

  1. Live imaging of GFP-labeled proteins in Drosophila oocytes.

    PubMed

    Pokrywka, Nancy Jo

    2013-03-29

    The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes. The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent and provides a useful system for investigating cytoskeleton function at these stages. Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

  2. Jupiter, a new Drosophila protein associated with microtubules.

    PubMed

    Karpova, Nina; Bobinnec, Yves; Fouix, Sylvaine; Huitorel, Philippe; Debec, Alain

    2006-05-01

    In this study we describe a novel Drosophila protein Jupiter, which shares properties with several structural microtubule-associated proteins (MAPs) including TAU, MAP2, MAP4. Jupiter is a soluble unfolded molecule with the high net positive charge, rich in Glycine. It possesses two degenerated repeats around the sequence PPGG, separated by a Serine-rich region. Jupiter associates with microtubules in vitro and, fused with the green fluorescent protein (GFP), is an excellent marker to follow microtubule dynamics in vivo. In a jupiter transgenic Drosophila strain generated by the "protein-trap" technique, Jupiter:GFP fusion protein localizes to the microtubule network through the cell cycle at the different stages of development. We found particularly high Jupiter:GFP concentrations in the young embryo, larval nervous system, precursors of eye photoreceptors and adult ovary. Moreover, from jupiter:gfp embryos we have established two permanent cell lines presenting strongly fluorescent microtubules during the whole cell cycle. In these cells, the distribution of the Jupiter:GFP fusion protein reproduces microtubule behavior upon treatment by the drugs colchicine and taxol. The jupiter cell lines and fly strain should be of wide interest for biologists interested in in vivo analysis of microtubule dynamics.

  3. Proteomics Reveals Novel Drosophila Seminal Fluid Proteins Transferred at Mating

    PubMed Central

    Findlay, Geoffrey D; Yi, Xianhua; MacCoss, Michael J; Swanson, Willie J

    2008-01-01

    Across diverse taxa, seminal fluid proteins (Sfps) transferred at mating affect the reproductive success of both sexes. Such reproductive proteins often evolve under positive selection between species; because of this rapid divergence, Sfps are hypothesized to play a role in speciation by contributing to reproductive isolation between populations. In Drosophila, individual Sfps have been characterized and are known to alter male sperm competitive ability and female post-mating behavior, but a proteomic-scale view of the transferred Sfps has been missing. Here we describe a novel proteomic method that uses whole-organism isotopic labeling to detect transferred Sfps in mated female D. melanogaster. We identified 63 proteins, which were previously unknown to function in reproduction, and confirmed the transfer of dozens of predicted Sfps. Relative quantification of protein abundance revealed that several of these novel Sfps are abundant in seminal fluid. Positive selection and tandem gene duplication are the prevailing forces of Sfp evolution, and comparative proteomics with additional species revealed lineage-specific changes in seminal fluid content. We also report a proteomic-based gene discovery method that uncovered 19 previously unannotated genes in D. melanogaster. Our results demonstrate an experimental method to identify transferred proteins in any system that is amenable to isotopic labeling, and they underscore the power of combining proteomic and evolutionary analyses to shed light on the complex process of Drosophila reproduction. PMID:18666829

  4. Vectors for the expression of tagged proteins in Drosophila.

    PubMed

    Parker, L; Gross, S; Alphey, L

    2001-12-01

    Regulated expression systems have been extremely useful in developmental studies, allowing the expression of specific proteins in defined spatial and temporal patterns. If these proteins are fused to an appropriate molecular tag, then they can be purified or visualized without the need to raise specific antibodies. If the tag is inherently fluorescent, then the proteins can even be visualized directly, in living tissue. We have constructed a series of P element-based transformation vectors for the most widely used expression system in Drosophila, GAL4/UAS. These vectors provide a series of useful tags for antibody detection, protein purification, and/or direct visualization, together with a convenient multiple cloning site into which the cDNA of interest can be inserted.

  5. The insulator protein CTCF regulates Drosophila steroidogenesis.

    PubMed

    Fresán, Ujué; Cuartero, Sergi; O'Connor, Michael B; Espinàs, M Lluisa

    2015-05-15

    The steroid hormone ecdysone is a central regulator of insect development. In this report we show that CTCF expression in the prothoracic gland is required for full transcriptional activation of the Halloween genes spookier, shadow and noppera-bo, which encode ecdysone biosynthetic enzymes, and for proper timing of ecdysone-responsive gene expression. Loss of CTCF results in delayed and less synchronized larval development that can only be rescued by feeding larvae with both, the steroid hormone 20-hydroxyecdysone and cholesterol. Moreover, CTCF-knockdown in prothoracic gland cells leads to increased lipid accumulation. In conclusion, the insulator protein CTCF is required for Halloween gene expression and cholesterol homeostasis in ecdysone-producing cells controlling steroidogenesis. © 2015. Published by The Company of Biologists Ltd.

  6. The insulator protein CTCF regulates Drosophila steroidogenesis

    PubMed Central

    Fresán, Ujué; Cuartero, Sergi; O'Connor, Michael B.; Espinàs, M. Lluisa

    2015-01-01

    ABSTRACT The steroid hormone ecdysone is a central regulator of insect development. In this report we show that CTCF expression in the prothoracic gland is required for full transcriptional activation of the Halloween genes spookier, shadow and noppera-bo, which encode ecdysone biosynthetic enzymes, and for proper timing of ecdysone-responsive gene expression. Loss of CTCF results in delayed and less synchronized larval development that can only be rescued by feeding larvae with both, the steroid hormone 20-hydroxyecdysone and cholesterol. Moreover, CTCF-knockdown in prothoracic gland cells leads to increased lipid accumulation. In conclusion, the insulator protein CTCF is required for Halloween gene expression and cholesterol homeostasis in ecdysone-producing cells controlling steroidogenesis. PMID:25979705

  7. [The role of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis].

    PubMed

    Nerusheva, O O; Dorogova, N V; Gubanova, N V; Omel'ianchuk, L V

    2008-09-01

    The cellular function of the gilgamesh mutation (89B9-12) of casein kinase gene in Drosophila spermatogenesis was studied. It was demonstrated that the sterility resulting from this mutation is connected with the abnormalities in spermatid individualization. A phylogenetic study of the protein sequences of casein kinases 1 from various organisms was conducted. The Gilgamesh protein was shown to be phylogenetically closer to the cytoplasmic casein kinase family, represented by the YCK3, YCK2, and YCK1 proteins of Saccharomyces cerevisiae and animal gamma-casein kinases. It is known that these yeast casein kinases are involved in vesicular trafficking, which, in turn, is related in its genetic control to the cell membrane remodeling during spermatid individualization. Thus, the data of phylogenetic analysis fit well the results obtained by studying the mutation phenotype.

  8. Drosophila Golgi membrane protein Ema promotes autophagosomal growth and function.

    PubMed

    Kim, Sungsu; Naylor, Sarah A; DiAntonio, Aaron

    2012-05-01

    Autophagy is a self-degradative process in which cellular material is enclosed within autophagosomes and trafficked to lysosomes for degradation. Autophagosomal biogenesis is well described; however mechanisms controlling the growth and ultimate size of autophagosomes are unclear. Here we demonstrate that the Drosophila membrane protein Ema is required for the growth of autophagosomes. In an ema mutant, autophagosomes form in response to starvation and developmental cues, and these autophagosomes can mature into autolysosomes; however the autophagosomes are very small, and autophagy is impaired. In fat body cells, Ema localizes to the Golgi complex and is recruited to the membrane of autophagosomes in response to starvation. The Drosophila Golgi protein Lva also is recruited to the periphery of autophagosomes in response to starvation, and this recruitment requires ema. Therefore, we propose that Golgi is a membrane source for autophagosomal growth and that Ema facilitates this process. Clec16A, the human ortholog of Ema, is a candidate autoimmune susceptibility locus. Expression of Clec16A can rescue the autophagosome size defect in the ema mutant, suggesting that regulation of autophagosome morphogenesis may be a fundamental function of this gene family.

  9. Role of Drosophila Amyloid Precursor Protein in Memory Formation

    PubMed Central

    Preat, Thomas; Goguel, Valérie

    2016-01-01

    The amyloid precursor protein (APP) is a membrane protein engaged in complex proteolytic pathways. APP and its derivatives have been shown to play a central role in Alzheimer’s disease (AD), a progressive neurodegenerative disease characterized by memory decline. Despite a huge effort from the research community, the primary cause of AD remains unclear, making it crucial to better understand the physiological role of the APP pathway in brain plasticity and memory. Drosophila melanogaster is a model system well-suited to address this issue. Although relatively simple, the fly brain is highly organized, sustains several forms of learning and memory, and drives numerous complex behaviors. Importantly, molecules and mechanisms underlying memory processes are conserved from flies to mammals. The fly encodes a single non-essential APP homolog named APP-Like (APPL). Using in vivo inducible RNA interference strategies, it was shown that APPL knockdown in the mushroom bodies (MB)—the central integrative brain structure for olfactory memory—results in loss of memory. Several APPL derivatives, such as secreted and full-length membrane APPL, may play different roles in distinct types of memory phases. Furthermore, overexpression of Drosophila amyloid peptide exacerbates the memory deficit caused by APPL knockdown, thus potentiating memory decline. Data obtained in the fly support the hypothesis that APP acts as a transmembrane receptor, and that disruption of its normal function may contribute to cognitive impairment during early AD. PMID:28008309

  10. Combgap contributes to recruitment of Polycomb group proteins in Drosophila

    PubMed Central

    Ray, Payal; De, Sandip; Mitra, Apratim; Bezstarosti, Karel; Demmers, Jeroen A. A.; Pfeifer, Karl; Kassis, Judith A.

    2016-01-01

    Polycomb group (PcG) proteins are responsible for maintaining the silenced transcriptional state of many developmentally regulated genes. PcG proteins are organized into multiprotein complexes that are recruited to DNA via cis-acting elements known as “Polycomb response elements” (PREs). In Drosophila, PREs consist of binding sites for many different DNA-binding proteins, some known and others unknown. Identification of these DNA-binding proteins is crucial to understanding the mechanism of PcG recruitment to PREs. We report here the identification of Combgap (Cg), a sequence-specific DNA-binding protein that is involved in recruitment of PcG proteins. Cg can bind directly to PREs via GTGT motifs and colocalizes with the PcG proteins Pleiohomeotic (Pho) and Polyhomeotic (Ph) at the majority of PREs in the genome. In addition, Cg colocalizes with Ph at a number of targets independent of Pho. Loss of Cg leads to decreased recruitment of Ph at only a subset of sites; some of these sites are binding sites for other Polycomb repressive complex 1 (PRC1) components, others are not. Our data suggest that Cg can recruit Ph in the absence of PRC1 and illustrate the diversity and redundancy of PcG protein recruitment mechanisms. PMID:27001825

  11. Functional dissection of Odorant binding protein genes in Drosophila melanogaster

    PubMed Central

    Swarup, S; Williams, T I; Anholt, R R H

    2011-01-01

    Most organisms rely on olfaction for survival and reproduction. The olfactory system of Drosophila melanogaster is one of the best characterized chemosensory systems and serves as a prototype for understanding insect olfaction. Olfaction in Drosophila is mediated by multigene families of odorant receptors and odorant binding proteins (OBPs). Although molecular response profiles of odorant receptors have been well documented, the contributions of OBPs to olfactory behavior remain largely unknown. Here, we used RNAi-mediated suppression of Obp gene expression and measurements of behavioral responses to 16 ecologically relevant odorants to systematically dissect the functions of 17 OBPs. We quantified the effectiveness of RNAi-mediated suppression by quantitative real-time polymerase chain reaction and used a proteomic liquid chromatography and tandem mass spectrometry procedure to show target-specific suppression of OBPs expressed in the antennae. Flies in which expression of a specific OBP is suppressed often show altered behavioral responses to more than one, but not all, odorants, in a sex-dependent manner. Similarly, responses to a specific odorant are frequently affected by suppression of expression of multiple, but not all, OBPs. These results show that OBPs are essential for mediating olfactory behavioral responses and suggest that OBP-dependent odorant recognition is combinatorial. PMID:21605338

  12. Deletion of endogenous Tau proteins is not detrimental in Drosophila

    PubMed Central

    Burnouf, Sylvie; Grönke, Sebastian; Augustin, Hrvoje; Dols, Jacqueline; Gorsky, Marianna Karina; Werner, Jennifer; Kerr, Fiona; Alic, Nazif; Martinez, Pedro; Partridge, Linda

    2016-01-01

    Human Tau (hTau) is a highly soluble and natively unfolded protein that binds to microtubules within neurons. Its dysfunction and aggregation into insoluble paired helical filaments is involved in the pathogenesis of Alzheimer’s disease (AD), constituting, together with accumulated β-amyloid (Aβ) peptides, a hallmark of the disease. Deciphering both the loss-of-function and toxic gain-of-function of hTau proteins is crucial to further understand the mechanisms leading to neurodegeneration in AD. As the fruit fly Drosophila melanogaster expresses Tau proteins (dTau) that are homologous to hTau, we aimed to better comprehend dTau functions by generating a specific tau knock-out (KO) fly line using homologous recombination. We observed that the specific removal of endogenous dTau proteins did not lead to overt, macroscopic phenotypes in flies. Indeed, survival, climbing ability and neuronal function were unchanged in tau KO flies. In addition, we did not find any overt positive or negative effect of dTau removal on human Aβ-induced toxicity. Altogether, our results indicate that the absence of dTau proteins has no major functional impact on flies, and suggests that our tau KO strain is a relevant model to further investigate the role of dTau proteins in vivo, thereby giving additional insights into hTau functions. PMID:26976084

  13. Analysis of Amyloid Precursor Protein Function in Drosophila melanogaster

    PubMed Central

    Cassar, Marlène; Kretzschmar, Doris

    2016-01-01

    The Amyloid precursor protein (APP) has mainly been investigated in connection with its role in Alzheimer’s Disease (AD) due to its cleavage resulting in the production of the Aβ peptides that accumulate in the plaques characteristic for this disease. However, APP is an evolutionary conserved protein that is not only found in humans but also in many other species, including Drosophila, suggesting an important physiological function. Besides Aβ, several other fragments are produced by the cleavage of APP; large secreted fragments derived from the N-terminus and a small intracellular C-terminal fragment. Although these fragments have received much less attention than Aβ, a picture about their function is finally emerging. In contrast to mammals, which express three APP family members, Drosophila expresses only one APP protein called APP-like or APPL. Therefore APPL functions can be studied in flies without the complication that other APP family members may have redundant functions. Flies lacking APPL are viable but show defects in neuronal outgrowth in the central and peripheral nervous system (PNS) in addition to synaptic changes. Furthermore, APPL has been connected with axonal transport functions. In the adult nervous system, APPL, and more specifically its secreted fragments, can protect neurons from degeneration. APPL cleavage also prevents glial death. Lastly, APPL was found to be involved in behavioral deficits and in regulating sleep/activity patterns. This review, will describe the role of APPL in neuronal development and maintenance and briefly touch on its emerging function in circadian rhythms while an accompanying review will focus on its role in learning and memory formation. PMID:27507933

  14. Purification and characterization of mRNA cap-binding protein from Drosophila melanogaster embryos.

    PubMed Central

    Maroto, F G; Sierra, J M

    1989-01-01

    A protein with specific affinity for the mRNA cap structure was purified both from the postribosomal supernatant and from the ribosomal high-salt wash of Drosophila melanogaster embryos by m7GTP-Sepharose chromatography. This protein had an apparent molecular mass of 35 kilodaltons (kDa) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size very different from those of the cap-binding proteins that have been characterized thus far. Drosophila 35-kDa cap-binding protein (CBP) could also be isolated from the ribosomal high-salt wash as part of a salt-stable protein complex consisting of polypeptides of 35, 72, and 140 to 180 kDa. Polyclonal antibodies against Drosophila 35-kDa CBP neither reacted with eucaryotic initiation factor 4E from rabbit reticulocytes nor affected mRNA translation in a rabbit reticulocyte cell-free system. However, in a cell-free system from Drosophila embryos, mRNA translation was specifically inhibited by these antibodies. The requirement of 35-kDa CBP for mRNA translation in Drosophila was diminished under ionic conditions in which the importance of mRNA cap structure recognition was reduced. Despite the structural differences between Drosophila 35-kDa CBP and mammalian initiation factor 4E, both proteins were functionally interchangeable in the in vitro translation system from Drosophila embryos. Images PMID:2501660

  15. Chromosomal Distribution of PcG Proteins during Drosophila Development

    PubMed Central

    Nègre, Nicolas; Hennetin, Jérôme; Sun, Ling V; Lavrov, Sergey; Bellis, Michel; White, Kevin P

    2006-01-01

    Polycomb group (PcG) proteins are able to maintain the memory of silent transcriptional states of homeotic genes throughout development. In Drosophila, they form multimeric complexes that bind to specific DNA regulatory elements named PcG response elements (PREs). To date, few PREs have been identified and the chromosomal distribution of PcG proteins during development is unknown. We used chromatin immunoprecipitation (ChIP) with genomic tiling path microarrays to analyze the binding profile of the PcG proteins Polycomb (PC) and Polyhomeotic (PH) across 10 Mb of euchromatin. We also analyzed the distribution of GAGA factor (GAF), a sequence-specific DNA binding protein that is found at most previously identified PREs. Our data show that PC and PH often bind to clustered regions within large loci that encode transcription factors which play multiple roles in developmental patterning and in the regulation of cell proliferation. GAF co-localizes with PC and PH to a limited extent, suggesting that GAF is not a necessary component of chromatin at PREs. Finally, the chromosome-association profile of PC and PH changes during development, suggesting that the function of these proteins in the regulation of some of their target genes might be more dynamic than previously anticipated. PMID:16613483

  16. Identification of Regions Interacting with Ovo(d) Mutations: Potential New Genes Involved in Germline Sex Determination or Differentiation in Drosophila Melanogaster

    PubMed Central

    Pauli, D.; Oliver, B.; Mahowald, A. P.

    1995-01-01

    Only a few Drosophila melanogaster germline sex determination genes are known, and there have been no systematic screens to identify new genes involved in this important biological process. The ovarian phenotypes produced by females mutant for dominant alleles of the ovo gene are modified in flies with altered doses of other loci involved in germline sex determination in Drosophila (Sex-lethal(+), sans fille(+) and ovarian tumor(+)). This observation constitutes the basis for a screen to identify additional genes required for proper establishment of germline sexual identity. We tested 300 deletions, which together cover ~58% of the euchromatic portion of the genome, for genetic interactions with ovo(D). Hemizygosity for more than a dozen small regions show interactions that either partially suppress or enhance the ovarian phenotypes of females mutant for one or more of the three dominant ovo mutations. These regions probably contain genes whose products act in developmental hierarchies that include ovo(+) protein. PMID:7713427

  17. Involvement of Drosophila uncoupling protein 5 in metabolism and aging.

    PubMed

    Sánchez-Blanco, Adolfo; Fridell, Yih-Woei C; Helfand, Stephen L

    2006-03-01

    A novel uncoupling protein, UCP5, has recently been characterized as a functional mitochondrial uncoupler in Drosophila. Here we demonstrate that UCP5 knockout (UCP5KO) flies are highly sensitive to starvation stress, a phenotype that can be reversed by ectopic neuronal expression of UCP5. UCP5KO flies live longer than controls on low-calorie diets, have a decreased level of fertility, and gain less weight than controls on high-calorie diets. However, isolated mitochondria from UCP5KO flies display the same respiration patterns as controls. Furthermore, total ATP levels in both UCP5KO and control flies are comparable. UCP5KO flies have a lower body composition of sugars, and during starvation stress their triglyceride reserves are depleted more rapidly than controls. Taken together, these data indicate that UCP5 is important to maintain metabolic homeostasis in the fly. We hypothesize that UCP5 influences hormonal control of metabolism.

  18. A Drosophila protein-tyrosine phosphatase associates with an adapter protein required for axonal guidance.

    PubMed

    Clemens, J C; Ursuliak, Z; Clemens, K K; Price, J V; Dixon, J E

    1996-07-19

    We have used the yeast two-hybrid system to isolate a novel Drosophila adapter protein, which interacts with the Drosophila protein-tyrosine phosphatase (PTP) dPTP61F. Absence of this protein in Drosophila causes the mutant photoreceptor axon phenotype dreadlocks (dock) (Garrity, P. A., Rao, Y., Salecker, I., and Zipursky, S. L.(1996) Cell 85, 639-650). Dock is similar to the mammalian oncoprotein Nck and contains three Src homology 3 (SH3) domains and one Src homology 2 (SH2) domain. The interaction of dPTP61F with Dock was confirmed in vivo by immune precipitation experiments. A sequence containing five PXXP motifs from the non-catalytic domain of the PTP is sufficient for interaction with Dock. This suggests that binding to the PTP is mediated by one or more of the SH3 domains of Dock. Immune precipitations of Dock also co-precipitate two tyrosine-phosphorylated proteins having molecular masses of 190 and 145 kDa. Interactions between Dock and these tyrosine-phosphorylated proteins are likely mediated by the Dock SH2 domain. These findings identify potential signal-transducing partners of Dock and propose a role for dPTP61F and the unidentified phosphoproteins in axonal guidance.

  19. A Systematic Cell-Based Analysis of Localization of Predicted Drosophila Peroxisomal Proteins.

    PubMed

    Baron, Matthew N; Klinger, Christen M; Rachubinski, Richard A; Simmonds, Andrew J

    2016-05-01

    Peroxisomes are membrane-bound organelles found in almost all eukaryotic cells. They perform specialized biochemical functions that vary with organism, tissue or cell type. Mutations in human genes required for the assembly of peroxisomes result in a spectrum of diseases called the peroxisome biogenesis disorders. A previous sequence-based comparison of the predicted proteome of Drosophila melanogaster (the fruit fly) to human proteins identified 82 potential homologues of proteins involved in peroxisomal biogenesis, homeostasis or metabolism. However, the subcellular localization of these proteins relative to the peroxisome was not determined. Accordingly, we tested systematically the localization and selected functions of epitope-tagged proteins in Drosophila Schneider 2 cells to determine the subcellular localization of 82 potential Drosophila peroxisomal protein homologues. Excluding the Pex proteins, 34 proteins localized primarily to the peroxisome, 8 showed dual localization to the peroxisome and other structures, and 26 localized exclusively to organelles other than the peroxisome. Drosophila is a well-developed laboratory animal often used for discovery of gene pathways, including those linked to human disease. Our work establishes a basic understanding of peroxisome protein localization in Drosophila. This will facilitate use of Drosophila as a genetically tractable, multicellular model system for studying key aspects of human peroxisome disease. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Drosophila Vps13 Is Required for Protein Homeostasis in the Brain

    PubMed Central

    Vonk, Jan J.; Lahaye, Liza L.; Kanon, Bart; van der Zwaag, Marianne; Velayos-Baeza, Antonio; Freire, Raimundo; van IJzendoorn, Sven C.; Grzeschik, Nicola A.; Sibon, Ody C. M.

    2017-01-01

    Chorea-Acanthocytosis is a rare, neurodegenerative disorder characterized by progressive loss of locomotor and cognitive function. It is caused by loss of function mutations in the Vacuolar Protein Sorting 13A (VPS13A) gene, which is conserved from yeast to human. The consequences of VPS13A dysfunction in the nervous system are still largely unspecified. In order to study the consequences of VPS13A protein dysfunction in the ageing central nervous system we characterized a Drosophila melanogaster Vps13 mutant line. The Drosophila Vps13 gene encoded a protein of similar size as human VPS13A. Our data suggest that Vps13 is a peripheral membrane protein located to endosomal membranes and enriched in the fly head. Vps13 mutant flies showed a shortened life span and age associated neurodegeneration. Vps13 mutant flies were sensitive to proteotoxic stress and accumulated ubiquitylated proteins. Levels of Ref(2)P, the Drosophila orthologue of p62, were increased and protein aggregates accumulated in the central nervous system. Overexpression of the human Vps13A protein in the mutant flies partly rescued apparent phenotypes. This suggests a functional conservation of human VPS13A and Drosophila Vps13. Our results demonstrate that Vps13 is essential to maintain protein homeostasis in the larval and adult Drosophila brain. Drosophila Vps13 mutants are suitable to investigate the function of Vps13 in the brain, to identify genetic enhancers and suppressors and to screen for potential therapeutic targets for Chorea-Acanthocytosis. PMID:28107480

  1. Regulation of Drosophila yolk protein genes by an ovary-specific GATA factor

    SciTech Connect

    Lossky, M.; Wensink, P.C.

    1995-12-01

    This report investigates the expression of the genes for yolk protein of Drosophila melanogaster and the tissue specific function of the regulatory element which activates transcription in vivo. 70 refs., 8 figs.

  2. High carbohydrate-low protein consumption maximizes Drosophila lifespan

    PubMed Central

    Bruce, Kimberley D.; Hoxha, Sany; Carvalho, Gil B.; Yamada, Ryuichi; Wang, Horng-Dar; Karayan, Paul; He, Shan; Brummel, Ted; Kapahi, Pankaj; Ja, William W.

    2013-01-01

    Dietary restriction extends lifespan in a variety of organisms, but the key nutritional components driving this process and how they interact remain uncertain. In Drosophila, while a substantial body of research suggests that protein is the major dietary component affecting longevity, recent studies claim that carbohydrates also play a central role. To clarify how nutritional factors influence longevity, nutrient consumption and lifespan were measured on a series of diets with varying yeast and sugar content. We show that optimal lifespan requires both high carbohydrate and low protein consumption, but neither nutrient by itself entirely predicts lifespan. Increased dietary carbohydrate or protein concentration does not always result in reduced feeding—the regulation of food consumption is best described by a constant daily caloric intake target. Moreover, due to differences in food intake, increased concentration of a nutrient within the diet does not necessarily result in increased consumption of that particular nutrient. Our results shed light on the issue of dietary effects on lifespan and highlight the need for accurate measures of nutrient intake in dietary manipulation studies. PMID:23403040

  3. Dual fluorescence detection of protein and RNA in Drosophila tissues

    PubMed Central

    Toledano, Hila; D’Alterio, Cecilia; Loza-Coll, Mariano; Jones, D Leanne

    2015-01-01

    Detection of RNAs by in situ hybridization (ISH) is a well-established technique that permits the study of specific RNA expression patterns in tissues; however, not all tissues are equally amenable to staining using the same procedure. Here we describe a protocol that combines whole-mount immunofluorescence (IF) and fluorescence in situ hybridization (FISH) for the simultaneous detection of specific RNA transcripts and proteins, greatly enhancing the spatial resolution of RNA expression in complex, intact fly tissues. To date, we have successfully used this protocol in adult testis, larval male gonads, adult intestine and Malpighian tubules. IF is conducted in RNase-free solutions, prior to the harsh conditions of FISH, in order to preserve protein antigenicity within dissected tissues. Separate protocols are described for mRNA and miRNA detection, which are based on robust digoxigenin (DIG) RNA and locked nucleic acid (LNA) probes, respectively. The combined IF-FISH procedure can be completed in 2 d for miRNA detection and 4 d for mRNA detection. Although optimized for Drosophila, this IF-FISH protocol should be adaptable to a wide variety of organisms, tissues, antibodies and probes, thus providing a reliable and simple means to compare RNA and protein abundance and localization. PMID:22976352

  4. The GAGA protein of Drosophila is phosphorylated by CK2.

    PubMed

    Bonet, Carles; Fernández, Irene; Aran, Xavier; Bernués, Jordi; Giralt, Ernest; Azorín, Fernando

    2005-08-19

    The GAGA factor of Drosophila is a sequence-specific DNA-binding protein that contributes to multiple processes from the regulation of gene expression to the structural organisation of heterochromatin and chromatin remodelling. GAGA is known to interact with various other proteins (tramtrack, pipsqueak, batman and dSAP18) and protein complexes (PRC1, NURF and FACT). GAGA functions are likely regulated at the level of post-translational modifications. Little is known, however, about its actual pattern of modification. It was proposed that GAGA can be O-glycosylated. Here, we report that GAGA519 isoform is a phosphoprotein that is phosphorylated by CK2 at the region of the DNA-binding domain. Our results indicate that phosphorylation occurs at S388 and, to a lesser extent, at S378. These two residues are located in a region of the DNA-binding domain that makes no direct contact with DNA, being dispensable for sequence-specific recognition. Phosphorylation at these sites does not abolish DNA binding but reduces the affinity of the interaction. These results are discussed in the context of the various functions and interactions that GAGA supports.

  5. A Low Protein Diet Increases the Hypoxic Tolerance in Drosophila

    PubMed Central

    Vigne, Paul; Frelin, Christian

    2006-01-01

    Dietary restriction is well known to increase the life span of a variety of organisms from yeast to mammals, but the relationships between nutrition and the hypoxic tolerance have not yet been considered. Hypoxia is a major cause of cell death in myocardial infarction and stroke. Here we forced hypoxia-related death by exposing one-day-old male Drosophila to chronic hypoxia (5% O2) and analysed their survival. Chronic hypoxia reduced the average life span from 33.6 days to 6.3 days when flies were fed on a rich diet. A demographic analysis indicated that chronic hypoxia increased the slope of the mortality trajectory and not the short-term risk of death. Dietary restriction produced by food dilution, by yeast restriction, or by amino acid restriction partially reversed the deleterious action of hypoxia. It increased the life span of hypoxic flies up to seven days, which represented about 25% of the life time of an hypoxic fly. Maximum survival of hypoxic flies required only dietary sucrose, and it was insensitive to drugs such as rapamycin and resveratrol, which increase longevity of normoxic animals. The results thus uncover a new link between protein nutrition, nutrient signalling, and resistance to hypoxic stresses. PMID:17183686

  6. A low protein diet increases the hypoxic tolerance in Drosophila.

    PubMed

    Vigne, Paul; Frelin, Christian

    2006-12-20

    Dietary restriction is well known to increase the life span of a variety of organisms from yeast to mammals, but the relationships between nutrition and the hypoxic tolerance have not yet been considered. Hypoxia is a major cause of cell death in myocardial infarction and stroke. Here we forced hypoxia-related death by exposing one-day-old male Drosophila to chronic hypoxia (5% O(2)) and analysed their survival. Chronic hypoxia reduced the average life span from 33.6 days to 6.3 days when flies were fed on a rich diet. A demographic analysis indicated that chronic hypoxia increased the slope of the mortality trajectory and not the short-term risk of death. Dietary restriction produced by food dilution, by yeast restriction, or by amino acid restriction partially reversed the deleterious action of hypoxia. It increased the life span of hypoxic flies up to seven days, which represented about 25% of the life time of an hypoxic fly. Maximum survival of hypoxic flies required only dietary sucrose, and it was insensitive to drugs such as rapamycin and resveratrol, which increase longevity of normoxic animals. The results thus uncover a new link between protein nutrition, nutrient signalling, and resistance to hypoxic stresses.

  7. Network of protein interactions within the Drosophila inner kinetochore

    PubMed Central

    Richter, Magdalena M.; Poznanski, Jaroslaw; Zdziarska, Anna; Czarnocki-Cieciura, Mariusz; Dadlez, Michal; Glover, David M.

    2016-01-01

    The kinetochore provides a physical connection between microtubules and the centromeric regions of chromosomes that is critical for their equitable segregation. The trimeric Mis12 sub-complex of the Drosophila kinetochore binds to the mitotic centromere using CENP-C as a platform. However, knowledge of the precise connections between Mis12 complex components and CENP-C has remained elusive despite the fundamental importance of this part of the cell division machinery. Here, we employ hydrogen–deuterium exchange coupled with mass spectrometry to reveal that Mis12 and Nnf1 form a dimer maintained by interacting coiled-coil (CC) domains within the carboxy-terminal parts of both proteins. Adjacent to these interacting CCs is a carboxy-terminal domain that also interacts with Nsl1. The amino-terminal parts of Mis12 and Nnf1 form a CENP-C-binding surface, which docks the complex and thus the entire kinetochore to mitotic centromeres. Mutational analysis confirms these precise interactions are critical for both structure and function of the complex. Thus, we conclude the organization of the Mis12–Nnf1 dimer confers upon the Mis12 complex a bipolar, elongated structure that is critical for kinetochore function. PMID:26911623

  8. Ciliary Phosphoinositide Regulates Ciliary Protein Trafficking in Drosophila.

    PubMed

    Park, Jina; Lee, Nayoung; Kavoussi, Adriana; Seo, Jeong Taeg; Kim, Chul Hoon; Moon, Seok Jun

    2015-12-29

    Cilia are highly specialized antennae-like cellular organelles. Inositol polyphosphate 5-phosphatase E (INPP5E) converts PI(4,5)P2 into PI4P and is required for proper ciliary function. Although Inpp5e mutations are associated with ciliopathies in humans and mice, the precise molecular role INPP5E plays in cilia remains unclear. Here, we report that Drosophila INPP5E (dINPP5E) regulates ciliary protein trafficking by controlling the phosphoinositide composition of ciliary membranes. Mutations in dInpp5e lead to hearing deficits due to the mislocalization of dTULP and mechanotransduction channels, Inactive and NOMPC, in chordotonal cilia. Both loss of dINPP5E and ectopic expression of the phosphatidylinositol-4-phosphate 5-kinase Skittles increase PI(4,5)P2 levels in the ciliary base. The fact that Skittles expression phenocopies the dInpp5e mutants confirms a central role for PI(4,5)P2 in the regulation of dTULP, Inactive, and NOMPC localization. These data suggest that the spatial localization and levels of PI(4,5)P2 in ciliary membranes are important regulators of ciliary trafficking and function. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Organization and function of Drosophila odorant binding proteins

    PubMed Central

    Larter, Nikki K; Sun, Jennifer S; Carlson, John R

    2016-01-01

    Odorant binding proteins (Obps) are remarkable in their number, diversity, and abundance, yet their role in olfactory coding remains unclear. They are widely believed to be required for transporting hydrophobic odorants through an aqueous lymph to odorant receptors. We construct a map of the Drosophila antenna, in which the abundant Obps are mapped to olfactory sensilla with defined functions. The results lay a foundation for an incisive analysis of Obp function. The map identifies a sensillum type that contains a single abundant Obp, Obp28a. Surprisingly, deletion of the sole abundant Obp in these sensilla does not reduce the magnitude of their olfactory responses. The results suggest that this Obp is not required for odorant transport and that this sensillum does not require an abundant Obp. The results further suggest a novel role for this Obp in buffering changes in the odor environment, perhaps providing a molecular form of gain control. DOI: http://dx.doi.org/10.7554/eLife.20242.001 PMID:27845621

  10. Nuclear and Cytoplasmic Soluble Proteins Extraction from a Small Quantity of Drosophila's Whole Larvae and Tissues.

    PubMed

    Lo Piccolo, Luca; Bonaccorso, Rosa; Onorati, Maria Cristina

    2015-06-01

    The identification and study of protein's function in several model organisms is carried out using both nuclear and cytoplasmic extracts. For a long time, Drosophila's embryos have represented the main source for protein extractions, although in the last year, the importance of collecting proteins extracts also from larval tissues has also been understood. Here we report a very simple protocol, improved by a previously developed method, to produce in a single extraction both highly stable nuclear and cytoplasmic protein extracts from a small quantity of whole Drosophila's larvae or tissues, suitable for biochemical analyses like co-immunoprecipitation.

  11. Intron retention in the Drosophila melanogaster Rieske iron sulphur protein gene generated a new protein

    PubMed Central

    Gontijo, Alisson M.; Miguela, Veronica; Whiting, Michael F.; Woodruff, R.C.; Dominguez, Maria

    2011-01-01

    Genomes can encode a variety of proteins with unrelated architectures and activities. It is known that protein-coding genes of de novo origin have significantly contributed to this diversity. However, the molecular mechanisms and evolutionary processes behind these originations are still poorly understood. Here we show that the last 102 codons of a novel gene, Noble, assembled directly from non-coding DNA following an intronic deletion that induced alternative intron retention at the Drosophila melanogaster Rieske Iron Sulphur Protein (RFeSP) locus. A systematic analysis of the evolutionary processes behind the origin of Noble showed that its emergence was strongly biased by natural selection on and around the RFeSP locus. Noble mRNA is shown to encode a bona fide protein that lacks an iron sulphur domain and localizes to mitochondria. Together, these results demonstrate the generation of a novel protein at a naturally selected site. PMID:21610726

  12. The ribosomal protein genes and Minute loci of Drosophila melanogaster

    PubMed Central

    Marygold, Steven J; Roote, John; Reuter, Gunter; Lambertsson, Andrew; Ashburner, Michael; Millburn, Gillian H; Harrison, Paul M; Yu, Zhan; Kenmochi, Naoya; Kaufman, Thomas C; Leevers, Sally J; Cook, Kevin R

    2007-01-01

    Background Mutations in genes encoding ribosomal proteins (RPs) have been shown to cause an array of cellular and developmental defects in a variety of organisms. In Drosophila melanogaster, disruption of RP genes can result in the 'Minute' syndrome of dominant, haploinsufficient phenotypes, which include prolonged development, short and thin bristles, and poor fertility and viability. While more than 50 Minute loci have been defined genetically, only 15 have so far been characterized molecularly and shown to correspond to RP genes. Results We combined bioinformatic and genetic approaches to conduct a systematic analysis of the relationship between RP genes and Minute loci. First, we identified 88 genes encoding 79 different cytoplasmic RPs (CRPs) and 75 genes encoding distinct mitochondrial RPs (MRPs). Interestingly, nine CRP genes are present as duplicates and, while all appear to be functional, one member of each gene pair has relatively limited expression. Next, we defined 65 discrete Minute loci by genetic criteria. Of these, 64 correspond to, or very likely correspond to, CRP genes; the single non-CRP-encoding Minute gene encodes a translation initiation factor subunit. Significantly, MRP genes and more than 20 CRP genes do not correspond to Minute loci. Conclusion This work answers a longstanding question about the molecular nature of Minute loci and suggests that Minute phenotypes arise from suboptimal protein synthesis resulting from reduced levels of cytoribosomes. Furthermore, by identifying the majority of haplolethal and haplosterile loci at the molecular level, our data will directly benefit efforts to attain complete deletion coverage of the D. melanogaster genome. PMID:17927810

  13. Inducible DamID systems for genomic mapping of chromatin proteins in Drosophila

    PubMed Central

    Pindyurin, Alexey V.; Pagie, Ludo; Kozhevnikova, Elena N.; van Arensbergen, Joris; van Steensel, Bas

    2016-01-01

    Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity, it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila. Here, we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress possible toxic effects of Dam. In addition, we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue. These new DamID tools will be valuable for the mapping of binding patterns of chromatin proteins in Drosophila tissues and especially in cell lineages. PMID:27001518

  14. Two Drosophila melanogaster proteins related to intermediate filament proteins of vertebrate cells

    PubMed Central

    1981-01-01

    Monoclonal antibodies were prepared against a 46,000 mol wt major cytoplasmic protein from Drosophila melanogaster Kc cells. These antibodies reacted with the 46,000 and a 40,000 mol wt protein from Kc cells. Some antibodies showed cross-reaction with 55,000 (vimentin) and 52,000 mol wt (desmin) proteins from baby hamster kidney (BHK) cells that form intermediate sized filaments in vertebrate cells. In indirect immunofluorescence, the group of cross reacting antibodies stained a filamentous meshwork in the cytoplasm of vertebrate cells. In Kc cells the fluorescence seemed to be localized in a filamentous meshwork that became more obvious after the cells had flattened out on a surface. These cytoskeletal structures are heat-labile; the proteins in Kc or BHK cells rearrange after a brief heat shock, forming juxtanuclear cap structures. PMID:6795212

  15. Fluorescent protein tagging confirms the presence of ribosomal proteins at Drosophila polytene chromosomes

    PubMed Central

    Ramanathan, Preethi; Matina, Tina; Wen, Jikai

    2013-01-01

    Most ribosomal proteins (RPs) are stoichiometrically incorporated into ribosomal subunits and play essential roles in ribosome biogenesis and function. However, a number of RPs appear to have non-ribosomal functions, which involve direct association with pre-mRNA and transcription factors at transcription sites. The consensus is that the RPs found at these sites are off ribosomal subunits, but observation that different RPs are usually found together suggests that ribosomal or ribosomal-like subunits might be present. Notably, it has previously been reported that antibodies against 20 different RPs stain the same Pol II transcription sites in Drosophila polytene chromosomes. Some concerns, however, were raised about the specificity of the antibodies. To investigate further whether RPs are present at transcription sites in Drosophila, we have generated several transgenic flies expressing RPs (RpS2, RpS5a, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, and RpL36) tagged with either green or red fluorescent protein. Imaging of salivary gland cells showed that these proteins are, as expected, abundant in the cytoplasm as well as in the nucleolus. However, these RPs are also apparent in the nucleus in the region occupied by the chromosomes. Indeed, polytene chromosome immunostaining of a representative subset of tagged RPs confirms the association with transcribed loci. Furthermore, characterization of a strain expressing RpL41 functionally tagged at its native genomic locus with YFP, also showed apparent nuclear accumulation and chromosomal association, suggesting that such a nuclear localization pattern might be a shared feature of RPs and is biologically important. We anticipate that the transgenes described here should provide a useful research tool to visualize ribosomal subunits in Drosophila tissues and to study the non-ribosomal functions of RPs. PMID:23638349

  16. Drosophila FIT is a protein-specific satiety hormone essential for feeding control

    PubMed Central

    Sun, Jinghan; Liu, Chang; Bai, Xiaobing; Li, Xiaoting; Li, Jingyun; Zhang, Zhiping; Zhang, Yunpeng; Guo, Jing; Li, Yan

    2017-01-01

    Protein homeostasis is critical for health and lifespan of animals. However, the mechanisms for controlling protein feeding remain poorly understood. Here we report that in Drosophila, protein intake-induced feeding inhibition (PIFI) is specific to protein-containing food, and this effect is mediated by a fat body (FB) peptide named female-specific independent of transformer (FIT). Upon consumption of protein food, FIT expression is greatly elevated. Secreted FIT peptide in the fly haemolymph conveys this metabolic message to the brain, thereby promoting the release of Drosophila insulin-like peptide 2 (DILP2) and suppressing further protein intake. Interestingly, Fit is a sexually dimorphic gene, and consequently protein consumption-induced insulin release, as well as protein feeding behaviour, are also dimorphic between sexes. Thus, our findings reveal a protein-specific satiety hormone, providing important insights into the complex regulation of feeding decision, as well as the sexual dimorphism in feeding behaviour. PMID:28102207

  17. Evolutionary conservation and predicted structure of the Drosophila extra sex combs repressor protein.

    PubMed Central

    Ng, J; Li, R; Morgan, K; Simon, J

    1997-01-01

    The Drosophila extra sex combs (esc) protein, a member of the Polycomb group (PcG), is a transcriptional repressor of homeotic genes. Genetic studies have shown that esc protein is required in early embryos at about the time that other PcG proteins become engaged in homeotic gene repression. The esc protein consists primarily of multiple copies of the WD repeat, a motif that has been implicated in protein-protein interaction. To further investigate the domain organization of esc protein, we have isolated and characterized esc homologs from divergent insect species. We report that esc protein is highly conserved in housefly (72% identical to Drosophila esc), butterfly (55% identical), and grasshopper (56% identical). We show that the butterfly homolog provides esc function in Drosophila, indicating that the sequence similarities reflect functional conservation. Homology modeling using the crystal structure of another WD repeat protein, the G-protein beta-subunit, predicts that esc protein adopts a beta-propeller structure. The sequence comparisons and modeling suggest that there are seven WD repeats in esc protein which together form a seven-bladed beta-propeller. We locate the conserved regions in esc protein with respect to this predicted structure. Site-directed mutagenesis of specific loops, predicted to extend from the propeller surface, identifies conserved parts of esc protein required for function in vivo. We suggest that these regions might mediate physical interaction with esc partner proteins. PMID:9343430

  18. A ribosome-inactivating protein in a Drosophila defensive symbiont

    PubMed Central

    Hamilton, Phineas T.; Peng, Fangni; Boulanger, Martin J.; Perlman, Steve J.

    2016-01-01

    Vertically transmitted symbionts that protect their hosts against parasites and pathogens are well known from insects, yet the underlying mechanisms of symbiont-mediated defense are largely unclear. A striking example of an ecologically important defensive symbiosis involves the woodland fly Drosophila neotestacea, which is protected by the bacterial endosymbiont Spiroplasma when parasitized by the nematode Howardula aoronymphium. The benefit of this defense strategy has led to the rapid spread of Spiroplasma throughout the range of D. neotestacea, although the molecular basis for this protection has been unresolved. Here, we show that Spiroplasma encodes a ribosome-inactivating protein (RIP) related to Shiga-like toxins from enterohemorrhagic Escherichia coli and that Howardula ribosomal RNA (rRNA) is depurinated during Spiroplasma-mediated protection of D. neotestacea. First, we show that recombinant Spiroplasma RIP catalyzes depurination of 28S rRNAs in a cell-free assay, as well as Howardula rRNA in vitro at the canonical RIP target site within the α-sarcin/ricin loop (SRL) of 28S rRNA. We then show that Howardula parasites in Spiroplasma-infected flies show a strong signal of rRNA depurination consistent with RIP-dependent modification and large decreases in the proportion of 28S rRNA intact at the α-sarcin/ricin loop. Notably, host 28S rRNA is largely unaffected, suggesting targeted specificity. Collectively, our study identifies a novel RIP in an insect defensive symbiont and suggests an underlying RIP-dependent mechanism in Spiroplasma-mediated defense. PMID:26712000

  19. Cross-Species Interaction between Rapidly Evolving Telomere-Specific Drosophila Proteins

    PubMed Central

    Vedelek, Balázs; Blastyák, András; Boros, Imre M.

    2015-01-01

    Telomere integrity in Drosophila melanogaster is maintained by a putative multisubunit complex called terminin that is believed to act in analogy to the mammalian shelterin complex in protecting chromosome ends from being recognized as sites of DNA damage. The five proteins supposed to form the terminin complex are HP1-ORC associated protein, HP1-HOAP interacting protein, Verrocchio, Drosophila Telomere Loss/Modigliani and Heterochromatic Protein 1. Four of these proteins evolve rapidly within the Drosophila genus. The accelerated evolution of terminin components may indicate the involvement of these proteins in the process by which new species arise, as the resulting divergence of terminin proteins might prevent hybrid formation, thus driving speciation. However, terminin is not an experimentally proven entity, and no biochemical studies have been performed to investigate its assembly and action in detail. Motivated by these facts in order to initiate biochemical studies on terminin function, we attempted to reconstitute terminin by co-expressing its subunits in bacteria and investigated the possible role of the fast-evolving parts of terminin components in complex assembly. Our results suggest formation of stable subcomplexes of terminin, but not of the whole complex in vitro. We found that the accelerated evolution is restricted to definable regions of terminin components, and that the divergence of D. melanogaster Drosophila Telomere Loss and D. yakuba Verrocchio proteins does not preclude their stable interaction. PMID:26566042

  20. Cross-Species Interaction between Rapidly Evolving Telomere-Specific Drosophila Proteins.

    PubMed

    Vedelek, Balázs; Blastyák, András; Boros, Imre M

    2015-01-01

    Telomere integrity in Drosophila melanogaster is maintained by a putative multisubunit complex called terminin that is believed to act in analogy to the mammalian shelterin complex in protecting chromosome ends from being recognized as sites of DNA damage. The five proteins supposed to form the terminin complex are HP1-ORC associated protein, HP1-HOAP interacting protein, Verrocchio, Drosophila Telomere Loss/Modigliani and Heterochromatic Protein 1. Four of these proteins evolve rapidly within the Drosophila genus. The accelerated evolution of terminin components may indicate the involvement of these proteins in the process by which new species arise, as the resulting divergence of terminin proteins might prevent hybrid formation, thus driving speciation. However, terminin is not an experimentally proven entity, and no biochemical studies have been performed to investigate its assembly and action in detail. Motivated by these facts in order to initiate biochemical studies on terminin function, we attempted to reconstitute terminin by co-expressing its subunits in bacteria and investigated the possible role of the fast-evolving parts of terminin components in complex assembly. Our results suggest formation of stable subcomplexes of terminin, but not of the whole complex in vitro. We found that the accelerated evolution is restricted to definable regions of terminin components, and that the divergence of D. melanogaster Drosophila Telomere Loss and D. yakuba Verrocchio proteins does not preclude their stable interaction.

  1. DMAP-85: a tau-like protein from Drosophila melanogaster larvae.

    PubMed

    Cambiazo, V; González, M; Maccioni, R B

    1995-03-01

    Microtubule-associated proteins (MAPs) play major regulatory roles in the organization and integrity of the cytoskeletal network. Our main interest in this study was the identification and the analysis of structural and functional aspects of Drosophila melanogaster MAPs. A novel MAP with a relative molecular mass of 85 kDa from Drosophila larvae was found associated with taxol-polymerized microtubules. In addition, this protein bound to mammalian tubulin in an overlay assay and coassembled with purified bovine brain tubulin in microtubule sedimentation experiments. The estimated stoichiometry of 85-kDa protein versus tubulin in the polymers was 1:5.3 +/- 0.2 mol/mol. It was shown that the 85-kDa protein bound specifically to an affinity column of Sepharose-beta II-(422-434) tubulin peptide, which contains the sequence of the MAP binding domain on beta II-tubulin. Affinity-purified 85-kDa protein enhanced microtubule assembly in a concentration-dependent manner. This effect was significantly decreased by the presence of the beta II-(422-434) peptide in the assembly assays, thus confirming the specificity of the 85-kDa protein interaction with the C-terminal domain on tubulin. Furthermore, this protein also exhibited a strong affinity for calmodulin, based on affinity chromatographic assays. Monoclonal and polyclonal anti-tau antibodies, including sequence-specific probes that recognize repeated microtubule-binding motifs on tau, MAP-2, and MAP-4 and specific N-terminal sequences of tau, cross-reacted with the 85-kDa protein from Drosophila larvae. These results suggest that tau and Drosophila 85-kDa protein share common functional and structural epitopes. We have named this protein as DMAP-85 for Drosophila MAP.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Drosophila Protein Kinase CK2: Genetics, Regulatory Complexity and Emerging Roles during Development

    PubMed Central

    Bandyopadhyay, Mohna; Arbet, Scott; Bishop, Clifton P.; Bidwai, Ashok P.

    2016-01-01

    CK2 is a Ser/Thr protein kinase that is highly conserved amongst all eukaryotes. It is a well-known oncogenic kinase that regulates vital cell autonomous functions and animal development. Genetic studies in the fruit fly Drosophila are providing unique insights into the roles of CK2 in cell signaling, embryogenesis, organogenesis, neurogenesis, and the circadian clock, and are revealing hitherto unknown complexities in CK2 functions and regulation. Here, we review Drosophila CK2 with respect to its structure, subunit diversity, potential mechanisms of regulation, developmental abnormalities linked to mutations in the gene encoding CK2 subunits, and emerging roles in multiple aspects of eye development. We examine the Drosophila CK2 “interaction map” and the eye-specific “transcriptome” databases, which raise the prospect that this protein kinase has many additional targets in the developing eye. We discuss the possibility that CK2 functions during early retinal neurogenesis in Drosophila and mammals bear greater similarity than has been recognized, and that this conservation may extend to other developmental programs. Together, these studies underscore the immense power of the Drosophila model organism to provide new insights and avenues to further investigate developmentally relevant targets of this protein kinase. PMID:28036067

  3. Organizational analysis of elav gene and functional analysis of ELAV protein of Drosophila melanogaster and Drosophila virilis

    SciTech Connect

    Yao, Kwokming; White, K. )

    1991-06-01

    Drosophila virilis genomic DNA corresponding analysis of a 3.8-kb genomic piece allowed identification of (1) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (2) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identify at the amino acid level, indicating a strong structural constraint for this functional domain. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.

  4. The comparative study of five sex-determining proteins across insects unveils high rates of evolution at basal components of the sex determination cascade.

    PubMed

    Eirín-López, José M; Sánchez, Lucas

    2015-01-01

    In insects, the sex determination cascade is composed of genes that interact with each other in a strict hierarchical manner, constituting a coadapted gene complex built in reverse order from bottom to top. Accordingly, ancient elements at the bottom are expected to remain conserved ensuring the correct functionality of the cascade. In the present work, we have studied the levels of variation displayed by five key components of the sex determination cascade across 59 insect species, including Sex-lethal, transformer, transformer-2, fruitless, doublesex, and sister-of-Sex-lethal (a paralog of Sxl encompassing sex-independent functions). Surprisingly, our results reveal that basal components of the cascade (doublesex, fruitless) seem to evolve more rapidly than previously suspected. Indeed, in the case of Drosophila, these proteins evolve more rapidly than the master regulator Sex-lethal. These results agree with the notion suggesting that genes involved in early aspects of development will be more constrained due to the large deleterious pleiotropic effects of mutations, resulting in increased levels of purifying selection at top positions of the cascade. The analyses of the selective episodes involved in the recruitment of Sxl into sex-determining functions further support this idea, suggesting the presence of bursts of adaptive selection in the common ancestor of drosophilids, followed by the onset of purifying selection preserving the master regulatory role of this protein on top of the Drosophila sex determination cascade. Altogether, these results underscore the importance of the position of sex determining genes in the cascade, constituting a major constraint shaping the molecular evolution of the insect sex determination pathway.

  5. Small nuclear ribonucleoproteins of Drosophila: Identification of U1 RNA-associated proteins and their behavior during heat shock

    SciTech Connect

    Wieben, E.D.; Pederson, T.

    1982-08-01

    In Drosophila, two nuclear proteins of approximately 26,000 and 14,000 molecular weight are recognized by a human autoimmune antibody for mammalian ribonucleoprotein (RNP) particles that contain U1 small nuclear RNA. The antibody-selected Drosophila RNP contains, in addition to these two proteins, a single RNA species that has been identified as U1 by hybridization with a cloned Drosophila U1 DNA probe. Small nuclear RNP isolated from human cells under the same conditions as used for Drosophila and selected by the anti-U1 RNP-specific antibody contains eight proteins, two of which are similar in molecular weight to the two Drosophila U1 RNP proteins. Thus, even though the nucleotide sequences of Drosophila and human U1 RNA are about 72% homologous, and the corresponding RNPs are both recognized by the same human autoantibody, Drosophila U1 RNP appears to have a simpler protein complement that its mammalian counterpart. The two Drosophila U1 RNA-associated proteins are synthesized at normal or slightly increased rates during the heat shock response and are incorporated into antibody-recognizable RNP complexes. This raises the possibility that U1 RNP is an indispensable nuclear element for cell survival during heat shock.

  6. Corolla Is a Novel Protein That Contributes to the Architecture of the Synaptonemal Complex of Drosophila

    PubMed Central

    Collins, Kimberly A.; Unruh, Jay R.; Slaughter, Brian D.; Yu, Zulin; Lake, Cathleen M.; Nielsen, Rachel J.; Box, Kimberly S.; Miller, Danny E.; Blumenstiel, Justin P.; Perera, Anoja G.; Malanowski, Kathryn E.; Hawley, R. Scott

    2014-01-01

    In most organisms the synaptonemal complex (SC) connects paired homologs along their entire length during much of meiotic prophase. To better understand the structure of the SC, we aim to identify its components and to determine how each of these components contributes to SC function. Here, we report the identification of a novel SC component in Drosophila melanogaster female oocytes, which we have named Corolla. Using structured illumination microscopy, we demonstrate that Corolla is a component of the central region of the SC. Consistent with its localization, we show by yeast two-hybrid analysis that Corolla strongly interacts with Cona, a central element protein, demonstrating the first direct interaction between two inner-synaptonemal complex proteins in Drosophila. These observations help provide a more complete model of SC structure and function in Drosophila females. PMID:24913682

  7. Immunohistochemical analysis of a novel dehydroepiandrosterone sulfotransferase-like protein in Drosophila neural circuits.

    PubMed

    Liu, Tzu-An; Liu, Ming-Cheh; Yang, Yuh-Shyong

    2008-02-29

    Sulfotransferase (ST)-catalyzed sulfation plays an important role in various neuronal functions such as homeostasis of catecholamine neurotransmitters and hormones. Drosophila is a popular model for the study of memory and behavioral manifestations because it is able to mimic the intricate neuroregulation and recognition in humans. However, there has been no evidence indicating that cytosolic ST(s) is(are) present in Drosophila. The aim of this study is to investigate whether or not cytosolic ST(s) is(are) expressed in the Drosophila nervous system. Immunoblot analysis demonstrated the presence of dehydroepiandrosterone (DHEA) ST-like protein in Drosophila brain and a sensitive fluorometric assay revealed its sulfating activity toward DHEA. Immunohistochemical staining demonstrated this DHEA ST-like protein to be abundant in specific neurons as well as in several bundles of nerve fibers in Drosophila. Clarification of a possible link between ST and a neurotransmitter-mediated effect may eventually aid in designing approaches for alleviating neuronal disorders in humans.

  8. Stable Drosophila Cell Lines: An Alternative Approach to Exogenous Protein Expression.

    PubMed

    Backovic, Marija; Krey, Thomas

    2016-01-01

    Recombinant protein production has become an indispensable tool for various research directions and biotechnological applications in the past decades. Among the numerous reported expression systems, insect cells provide the possibility to produce complex target proteins that require posttranslational modifications. Stable expression in Drosophila S2 cells represents an attractive alternative to the widely used baculovirus expression system, offering important advantages in particular for difficult-to-express proteins, e.g., membrane proteins or heavily glycosylated multi-domain proteins that are stabilized by a complex disulfide pattern. Here we present the methodology that is required for the generation of stable Drosophila S2 cell transfectants and for production of recombinant proteins using those transfectants.

  9. Cbl-associated protein regulates assembly and function of two tension-sensing structures in Drosophila

    PubMed Central

    Bharadwaj, Rajnish; Roy, Madhuparna; Ohyama, Tomoko; Sivan-Loukianova, Elena; Delannoy, Michael; Lloyd, Thomas E.; Zlatic, Marta; Eberl, Daniel F.; Kolodkin, Alex L.

    2013-01-01

    Cbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, its physiological roles remain unknown. Here, we demonstrate that Drosophila CAP regulates the organization of two actin-rich structures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, which form an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrant junctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse of scolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. We investigate the roles of different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of the CAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopy Drosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at the membrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin protein complex in stretch-sensitive organ assembly and function. PMID:23293294

  10. [Ecological imprinting and protein biosynthesis. Experiments with Drosophila melanogaster Meigen].

    PubMed

    Laudien, H; Iken, H H

    1977-06-01

    According to the "host selection principle", butterflies and other herbivorous insects preferentially lay their eggs on those plant races that they fed on when young. This is also true for karpophagic and parasitic insects. The selection of specific chemical conditions could be either inherited or acquired. If learned information determines host selection, we have a case of imprinting, as a) reception and use of the information are not simultaneous, b) there is no reward. In experiments with Drosophila melanogaster we marked the egg deposition medium with ethanol, acetic acid, peppermint oil, or benzaldehyd. The flies spontaneously prefer mediums with ethanol and acetic acid, and reject peppermint oil and benzaldehyd. If they are reared in one of these media, the preference for it is increased, or the rejection rate lowered. Rearing with actinomycin C neutralizes the effect of the other markers. It is concluded that actinomycin C blocks imprinting on the egg deposition substrate in Drosophila melanogaster.

  11. Male accessory gland secretory protein polymorphism in natural populations of Drosophila nasuta nasuta and Drosophila sulfurigaster neonasuta.

    PubMed

    Ravi Ram, K; Ramesh, S R

    2007-12-01

    Male accessory gland secretory protein polymorphism was analysed in natural populations of Drosophila nasuta nasuta and D. sulfurigaster neonasuta for the first time, using SDS-PAGE to score polymorphism of these proteins in 2788 individuals of D. n. nasuta and 2232 individuals of D. s. neonasuta from 12 different populations from southern India. A total of 25 and 18 variant protein phenotypes were identified in D. n. nasuta and D. s. neonasuta, respectively. Protein fractions of group III were more polymorphic than those from groups I and II. The results show that accessory gland secretory proteins show high levels of polymorphism, irrespective of species or habitat. Moreover, we have used the variation in the accessory gland proteins to assess the extent of divergence between the species and to infer their population structure. The study suggests that though both D. n. nasuta and D. s. neonasuta belong to the same subgroup, they differ in population structure, as far as accessory gland protein polymorphism is concerned.

  12. Drosophila SMN complex proteins Gemin2, Gemin3, and Gemin5 are components of U bodies

    SciTech Connect

    Cauchi, Ruben J.; Sanchez-Pulido, Luis; Liu, Ji-Long

    2010-08-15

    Uridine-rich small nuclear ribonucleoproteins (U snRNPs) play key roles in pre-mRNA processing in the nucleus. The assembly of most U snRNPs takes place in the cytoplasm and is facilitated by the survival motor neuron (SMN) complex. Discrete cytoplasmic RNA granules called U bodies have been proposed to be specific sites for snRNP assembly because they contain U snRNPs and SMN. U bodies invariably associate with P bodies, which are involved in mRNA decay and translational control. However, it remains unknown whether other SMN complex proteins also localise to U bodies. In Drosophila there are four SMN complex proteins, namely SMN, Gemin2/CG10419, Gemin3 and Gemin5/Rigor mortis. Drosophila Gemin3 was originally identified as the Drosophila orthologue of human and yeast Dhh1, a component of P bodies. Through an in silico analysis of the DEAD-box RNA helicases we confirmed that Gemin3 is the bona fide Drosophila orthologue of vertebrate Gemin3 whereas the Drosophila orthologue of Dhh1 is Me31B. We then made use of the Drosophila egg chamber as a model system to study the subcellular distribution of the Gemin proteins as well as Me31B. Our cytological investigations show that Gemin2, Gemin3 and Gemin5 colocalise with SMN in U bodies. Although they are excluded from P bodies, as components of U bodies, Gemin2, Gemin3 and Gemin5 are consistently found associated with P bodies, wherein Me31B resides. In addition to a role in snRNP biogenesis, SMN complexes residing in U bodies may also be involved in mRNP assembly and/or transport.

  13. Supplementation with major royal jelly proteins increases lifespan, feeding and fecundity in Drosophila

    USDA-ARS?s Scientific Manuscript database

    The major royal-jelly proteins (MRJPs) are the main constituents responsible for the specific physiological role of royal jelly (RJ) in honeybees. Male and female Drosophila flies were fed diets containing either no MRJPs (A) or casein (B) at 1.25% (w/w) of diet or MRJPs at 1.25% (C), 2.50% (D), or ...

  14. SOLO: a meiotic protein required for centromere cohesion, coorientation, and SMC1 localization in Drosophila melanogaster

    PubMed Central

    Yan, Rihui; Thomas, Sharon E.; Tsai, Jui-He; Yamada, Yukihiro

    2010-01-01

    Sister chromatid cohesion is essential to maintain stable connections between homologues and sister chromatids during meiosis and to establish correct centromere orientation patterns on the meiosis I and II spindles. However, the meiotic cohesion apparatus in Drosophila melanogaster remains largely uncharacterized. We describe a novel protein, sisters on the loose (SOLO), which is essential for meiotic cohesion in Drosophila. In solo mutants, sister centromeres separate before prometaphase I, disrupting meiosis I centromere orientation and causing nondisjunction of both homologous and sister chromatids. Centromeric foci of the cohesin protein SMC1 are absent in solo mutants at all meiotic stages. SOLO and SMC1 colocalize to meiotic centromeres from early prophase I until anaphase II in wild-type males, but both proteins disappear prematurely at anaphase I in mutants for mei-S332, which encodes the Drosophila homologue of the cohesin protector protein shugoshin. The solo mutant phenotypes and the localization patterns of SOLO and SMC1 indicate that they function together to maintain sister chromatid cohesion in Drosophila meiosis. PMID:20142422

  15. SOLO: a meiotic protein required for centromere cohesion, coorientation, and SMC1 localization in Drosophila melanogaster.

    PubMed

    Yan, Rihui; Thomas, Sharon E; Tsai, Jui-He; Yamada, Yukihiro; McKee, Bruce D

    2010-02-08

    Sister chromatid cohesion is essential to maintain stable connections between homologues and sister chromatids during meiosis and to establish correct centromere orientation patterns on the meiosis I and II spindles. However, the meiotic cohesion apparatus in Drosophila melanogaster remains largely uncharacterized. We describe a novel protein, sisters on the loose (SOLO), which is essential for meiotic cohesion in Drosophila. In solo mutants, sister centromeres separate before prometaphase I, disrupting meiosis I centromere orientation and causing nondisjunction of both homologous and sister chromatids. Centromeric foci of the cohesin protein SMC1 are absent in solo mutants at all meiotic stages. SOLO and SMC1 colocalize to meiotic centromeres from early prophase I until anaphase II in wild-type males, but both proteins disappear prematurely at anaphase I in mutants for mei-S332, which encodes the Drosophila homologue of the cohesin protector protein shugoshin. The solo mutant phenotypes and the localization patterns of SOLO and SMC1 indicate that they function together to maintain sister chromatid cohesion in Drosophila meiosis.

  16. Mutations in the Drosophila gene encoding ribosomal protein S6 cause tissue overgrowth.

    PubMed Central

    Stewart, M J; Denell, R

    1993-01-01

    We have characterized two P-element-induced, lethal mutations in Drosophila melanogaster which affect the larval hemocytes, mediators of the insect immune response. Each mutant displays larval melanotic tumors characteristic of mutations affecting the insect cellular immune system, and the moribund animals develop grossly hypertrophied hematopoietic organs because of increased cell proliferation and extra rounds of endoreduplication in some hematopoietic cells. Surprisingly, these mutations are due to P element insertions in the 5' regulatory region of the Drosophila gene encoding ribosomal protein S6 and cause a reduction of S6 transcript abundance in mutant larvae. Images PMID:8384310

  17. Protein kinase D regulates several aspects of development in Drosophila melanogaster.

    PubMed

    Maier, Dieter; Nagel, Anja C; Gloc, Helena; Hausser, Angelika; Kugler, Sabrina J; Wech, Irmgard; Preiss, Anette

    2007-06-25

    Protein Kinase D (PKD) is an effector of diacylglycerol-regulated signaling pathways. Three isoforms are known in mammals that have been linked to diverse cellular functions including regulation of cell proliferation, differentiation, motility and secretory transport from the trans-Golgi network to the plasma membrane. In Drosophila, there is a single PKD orthologue, whose broad expression implicates a more general role in development. We have employed tissue specific overexpression of various PKD variants as well as tissue specific RNAi, in order to investigate the function of the PKD gene in Drosophila. Apart from a wild type (WT), a kinase dead (kd) and constitutively active (SE) Drosophila PKD variant, we also analyzed two human isoforms hPKD2 and hPKD3 for their capacity to substitute PKD activity in the fly. Overexpression of either WT or kd-PKD variants affected primarily wing vein development. However, overexpression of SE-PKD and PKD RNAi was deleterious. We observed tissue loss, wing defects and degeneration of the retina. The latter phenotype conforms to a role of PKD in the regulation of cytoskeletal dynamics. Strongest phenotypes were larval to pupal lethality. RNAi induced phenotypes could be rescued by a concurrent overexpression of Drosophila wild type PKD or either human isoform hPKD2 and hPKD3. Our data confirm the hypothesis that Drosophila PKD is a multifunctional kinase involved in diverse processes such as regulation of the cytoskeleton, cell proliferation and death as well as differentiation of various fly tissues.

  18. Enhancement of presynaptic performance in transgenic Drosophila overexpressing heat shock protein Hsp70.

    PubMed

    Karunanithi, Shanker; Barclay, Jeffrey W; Brown, Ian R; Robertson, R Meldrum; Atwood, Harold L

    2002-04-01

    Prior heat shock confers protection to Drosophila synapses during subsequent heat stress by stabilizing quantal size and reducing the decline of quantal emission at individual synaptic boutons. The major heat shock protein Hsp70, which is strongly induced by high temperatures in Drosophila, may be responsible for this synaptic protection. To test this hypothesis, we investigated synaptic protection and stabilization at larval neuromuscular junctions of transgenic Drosophila which produce more than the normal amount of Hsp70 in response to heat shock. Overexpression of Hsp70 coincides with enhanced protection of presynaptic performance, assayed by measuring mean quantal content and percentage success of transmission. Quantal size was not selectively altered, indicating no effects of overexpression on postsynaptic performance. Thus, presynaptic mechanisms can be protected by manipulating levels of Hsp70, which would provide stability to neural circuits otherwise susceptible to heat stress. Copyright 2002 Wiley-Liss, Inc.

  19. The Drosophila Tis11 Protein and Its Effects on mRNA Expression in Flies*

    PubMed Central

    Choi, Youn-Jeong; Lai, Wi S.; Fedic, Robert; Stumpo, Deborah J.; Huang, Weichun; Li, Leping; Perera, Lalith; Brewer, Brandy Y.; Wilson, Gerald M.; Mason, James M.; Blackshear, Perry J.

    2014-01-01

    Members of the mammalian tristetraprolin family of CCCH tandem zinc finger proteins can bind to certain AU-rich elements (AREs) in mRNAs, leading to their deadenylation and destabilization. Mammals express three or four members of this family, but Drosophila melanogaster and other insects appear to contain a single gene, Tis11. We found that recombinant Drosophila Tis11 protein could bind to ARE-containing RNA oligonucleotides with low nanomolar affinity. Remarkably, co-expression in mammalian cells with “target” RNAs demonstrated that Tis11 could promote destabilization of ARE-containing mRNAs and that this was partially dependent on a conserved C-terminal sequence resembling the mammalian NOT1 binding domain. Drosophila Tis11 promoted both deadenylation and decay of a target transcript in this heterologous cell system. We used chromosome deletion/duplication and P element insertion to produce two types of Tis11 deficiency in adult flies, both of which were viable and fertile. To address the hypothesis that Tis11 deficiency would lead to the abnormal accumulation of potential target transcripts, we analyzed gene expression in adult flies by deep mRNA sequencing. We identified 69 transcripts from 56 genes that were significantly up-regulated more than 1.5-fold in both types of Tis11-deficient flies. Ten of the up-regulated transcripts encoded probable proteases, but many other functional classes of proteins were represented. Many of the up-regulated transcripts contained potential binding sites for tristetraprolin family member proteins that were conserved in other Drosophila species. Tis11 is thus an ARE-binding, mRNA-destabilizing protein that may play a role in post-transcriptional gene expression in Drosophila and other insects. PMID:25342740

  20. Structure and expression of the Drosophila ubiquitin-52-amino-acid fusion-protein gene.

    PubMed Central

    Cabrera, H L; Barrio, R; Arribas, C

    1992-01-01

    Ubiquitin belongs to a multigene family. In Drosophila two members of this family have been previously described. We report here the organization and expression of a third member, the DUb52 gene, isolated by screening a Drosophila melanogaster genomic library. This gene encodes an ubiquitin monomer fused to a 52-amino acid extension protein. There are no introns interrupting the coding sequence. Recently, it has been described that this extension encodes a ribosomal protein in Saccharomyces, Dictyostelium, and Arabidopsis. The present results show that the 5' regulatory region of DUb52 shares common features with the ribosomal protein genes of Drosophila, Xenopus and mouse, including GC- and pyrimidine-rich regions. Moreover, sequences similar to the consensus Ribo-box in Neurospora crassa have been identified. Furthermore, a sequence has been found that is similar to the binding site for the TFIIIA distal element factor from Xenopus laevis. The DUb52 gene is transcribed to a 0.9 kb mRNA that is expressed constitutively throughout development and is particularly abundant in ovaries. In addition, the DUb52 gene has been found to be preferentially transcribed in exponentially growing Drosophila cells. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1381584

  1. The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

    PubMed

    Buszczak, Michael; Paterno, Shelley; Lighthouse, Daniel; Bachman, Julia; Planck, Jamie; Owen, Stephenie; Skora, Andrew D; Nystul, Todd G; Ohlstein, Benjamin; Allen, Anna; Wilhelm, James E; Murphy, Terence D; Levis, Robert W; Matunis, Erika; Srivali, Nahathai; Hoskins, Roger A; Spradling, Allan C

    2007-03-01

    Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.

  2. Drosophila Orb2 targets genes involved in neuronal growth, synapse formation, and protein turnover

    PubMed Central

    Mastushita-Sakai, Tomoko; White-Grindley, Erica; Samuelson, Jessica; Seidel, Chris; Si, Kausik

    2010-01-01

    In the study of long-term memory, how memory persists is a fundamental and unresolved question. What are the molecular components of the long-lasting memory trace? Previous studies in Aplysia and Drosophila have found that a neuronal variant of a RNA-binding protein with a self-perpetuating prion-like property, cytoplasmic polyadenylation element binding protein, is required for the persistence of long-term synaptic facilitation in the snail and long-term memory in the fly. In this study, we have identified the mRNA targets of the Drosophila neuronal cytoplasmic polyadenylation element binding protein, Orb2. These Orb2 targets include genes involved in neuronal growth, synapse formation, and intriguingly, protein turnover. These targets suggest that the persistent form of the memory trace might be comprised of molecules that maintain a sustained, permissive environment for synaptic growth in an activated synapse. PMID:20547833

  3. Expression and cellular localization of the Toucan protein during Drosophila oogenesis.

    PubMed

    Grammont, M; Berson, G; Dastugue, B; Couderc, J L

    2000-02-01

    The toucan (toc) gene is required in the germline for somatic cell patterning during Drosophila oogenesis. To better understand the function of toc, we performed a detailed analysis of the distribution of the Toucan protein during oogenesis. Toc expression is restricted to the germline cells and shows a dynamic distribution pattern throughout follicle development. Mislocalization of the Toc protein in mutant follicles in which the microtubule network is altered indicates that microtubules play a role in Toc localization during oogenesis.

  4. RNA-guided genome editing in Drosophila with the purified Cas9 protein.

    PubMed

    Lee, Jeong-Soo; Kwak, Su-Jin; Kim, Jungeun; Kim, Ae-Kyeong; Noh, Hae Min; Kim, Jin-Soo; Yu, Kweon

    2014-05-28

    We report a method for generating Drosophila germline mutants effectively via injection of the complex of the purified Cas9 protein, tracrRNA, and gene-specific crRNAs, which may reduce delayed mutations because of the transient activity of the Cas9 protein, combined with the simple mutation detection in GO founders by the T7E1 assay. Copyright © 2014 Lee et al.

  5. The Drosophila retinoblastoma protein, Rbf1, induces a Debcl- and Drp1-dependent mitochondrial apoptosis.

    PubMed

    Clavier, Amandine; Ruby, Vincent; Rincheval-Arnold, Aurore; Mignotte, Bernard; Guénal, Isabelle

    2015-09-01

    In accordance with its tumor suppressor role, the retinoblastoma protein pRb can ensure pro-apoptotic functions. Rbf1, the Drosophila homolog of Rb, also displays a pro-apoptotic activity in proliferative cells. We have previously shown that the Rbf1 pro-apoptotic activity depends on its ability to decrease the level of anti-apoptotic proteins such as the Bcl-2 family protein Buffy. Buffy often acts in an opposite manner to Debcl, the other Drosophila Bcl-2-family protein. Both proteins can localize at the mitochondrion, but the way they control apoptosis still remains unclear. Here, we demonstrate that Debcl and the pro-fission gene Drp1 are necessary downstream of Buffy to trigger a mitochondrial fragmentation during Rbf1-induced apoptosis. Interestingly, Rbf1-induced apoptosis leads to a Debcl- and Drp1-dependent reactive oxygen species production, which in turn activates the Jun Kinase pathway to trigger cell death. Moreover, we show that Debcl and Drp1 can interact and that Buffy inhibits this interaction. Notably, Debcl modulates Drp1 mitochondrial localization during apoptosis. These results provide a mechanism by which Drosophila Bcl-2 family proteins can control apoptosis, and shed light on a link between Rbf1 and mitochondrial dynamics in vivo.

  6. Apocrine Secretion in Drosophila Salivary Glands: Subcellular Origin, Dynamics, and Identification of Secretory Proteins

    PubMed Central

    Farkaš, Robert; Ďatková, Zuzana; Mentelová, Lucia; Löw, Péter; Beňová-Liszeková, Denisa; Beňo, Milan; Sass, Miklós; Řehulka, Pavel; Řehulková, Helena; Raška, Otakar; Kováčik, Lubomír; Šmigová, Jana; Raška, Ivan; Mechler, Bernard M.

    2014-01-01

    In contrast to the well defined mechanism of merocrine exocytosis, the mechanism of apocrine secretion, which was first described over 180 years ago, remains relatively uncharacterized. We identified apocrine secretory activity in the late prepupal salivary glands of Drosophila melanogaster just prior to the execution of programmed cell death (PCD). The excellent genetic tools available in Drosophila provide an opportunity to dissect for the first time the molecular and mechanistic aspects of this process. A prerequisite for such an analysis is to have pivotal immunohistochemical, ultrastructural, biochemical and proteomic data that fully characterize the process. Here we present data showing that the Drosophila salivary glands release all kinds of cellular proteins by an apocrine mechanism including cytoskeletal, cytosolic, mitochondrial, nuclear and nucleolar components. Surprisingly, the apocrine release of these proteins displays a temporal pattern with the sequential release of some proteins (e.g. transcription factor BR-C, tumor suppressor p127, cytoskeletal β-tubulin, non-muscle myosin) earlier than others (e.g. filamentous actin, nuclear lamin, mitochondrial pyruvate dehydrogenase). Although the apocrine release of proteins takes place just prior to the execution of an apoptotic program, the nuclear DNA is never released. Western blotting indicates that the secreted proteins remain undegraded in the lumen. Following apocrine secretion, the salivary gland cells remain quite vital, as they retain highly active transcriptional and protein synthetic activity. PMID:24732043

  7. Assembly of Drosophila Centromeric Chromatin Proteins during Mitosis

    PubMed Central

    Mellone, Barbara G.; Bowers, Sarion R.; Oderberg, Isaac; Karpen, Gary H.

    2011-01-01

    Semi-conservative segregation of nucleosomes to sister chromatids during DNA replication creates gaps that must be filled by new nucleosome assembly. We analyzed the cell-cycle timing of centromeric chromatin assembly in Drosophila, which contains the H3 variant CID (CENP-A in humans), as well as CENP-C and CAL1, which are required for CID localization. Pulse-chase experiments show that CID and CENP-C levels decrease by 50% at each cell division, as predicted for semi-conservative segregation and inheritance, whereas CAL1 displays higher turnover. Quench-chase-pulse experiments demonstrate that there is a significant lag between replication and replenishment of centromeric chromatin. Surprisingly, new CID is recruited to centromeres in metaphase, by a mechanism that does not require an intact mitotic spindle, but does require proteasome activity. Interestingly, new CAL1 is recruited to centromeres before CID in prophase. Furthermore, CAL1, but not CENP-C, is found in complex with pre-nucleosomal CID. Finally, CENP-C displays yet a different pattern of incorporation, during both interphase and mitosis. The unusual timing of CID recruitment and unique dynamics of CAL1 identify a distinct centromere assembly pathway in Drosophila and suggest that CAL1 is a key regulator of centromere propagation. PMID:21589899

  8. Analysis of Thioester-Containing Proteins during the Innate Immune Response of Drosophila melanogaster

    PubMed Central

    Bou Aoun, Richard; Hetru, Charles; Troxler, Laurent; Doucet, Daniel; Ferrandon, Dominique; Matt, Nicolas

    2010-01-01

    Thioester-containing proteins (TEPs) are conserved proteins among insects that are thought to be involved in innate immunity. In Drosophila, the Tep family is composed of 6 genes named Tep1–Tep6. In this study, we investigated the phylogeny, expression pattern and roles of these genes in the host defense of Drosophila. Protostomian Tep genes are clustered in 3 distinct branches, 1 of which is specific to mosquitoes. Most D. melanogaster Tep genes are expressed in hemocytes, can be induced in the fat body, and are expressed in specific regions of the hypodermis. This expression pattern is consistent with a role in innate immunity. However, we find that TEP1, TEP2, and TEP4 are not strictly required in the body cavity to fight several bacterial and fungal infections. One possibility is that Drosophila TEPs act redundantly or that their absence can be compensated by other components of the immune response. TEPs may thus provide a subtle selective advantage during evolution. Alternatively, they may be required in host defense against specific as yet unidentified natural pathogens of Drosophila. PMID:21063077

  9. Exploring the Conserved Role of MANF in the Unfolded Protein Response in Drosophila melanogaster

    PubMed Central

    Lindström, Riitta; Lindholm, Päivi; Kallijärvi, Jukka; Palgi, Mari; Saarma, Mart; Heino, Tapio I.

    2016-01-01

    Disturbances in the homeostasis of endoplasmic reticulum (ER) referred to as ER stress is involved in a variety of human diseases. ER stress activates unfolded protein response (UPR), a cellular mechanism the purpose of which is to restore ER homeostasis. Previous studies show that Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is an important novel component in the regulation of UPR. In vertebrates, MANF is upregulated by ER stress and protects cells against ER stress-induced cell death. Biochemical studies have revealed an interaction between mammalian MANF and GRP78, the major ER chaperone promoting protein folding. In this study we discovered that the upregulation of MANF expression in response to drug-induced ER stress is conserved between Drosophila and mammals. Additionally, by using a genetic in vivo approach we found genetic interactions between Drosophila Manf and genes encoding for Drosophila homologues of GRP78, PERK and XBP1, the key components of UPR. Our data suggest a role for Manf in the regulation of Drosophila UPR. PMID:26975047

  10. The zinc finger proteins Pannier and GATA4 function as cardiogenic factors in Drosophila.

    PubMed

    Gajewski, K; Fossett, N; Molkentin, J D; Schulz, R A

    1999-12-01

    The regulation of cardiac gene expression by GATA zinc finger transcription factors is well documented in vertebrates. However, genetic studies in mice have failed to demonstrate a function for these proteins in cardiomyocyte specification. In Drosophila, the existence of a cardiogenic GATA factor has been implicated through the analysis of a cardial cell enhancer of the muscle differentiation gene D-mef2. We show that the GATA gene pannier is expressed in the dorsal mesoderm and required for cardial cell formation while repressing a pericardial cell fate. Ectopic expression of Pannier results in cardial cell overproduction, while co-expression of Pannier and the homeodomain protein Tinman synergistically activate cardiac gene expression and induce cardial cells. The related GATA4 protein of mice likewise functions as a cardiogenic factor in Drosophila, demonstrating an evolutionarily conserved function between Pannier and GATA4 in heart development.

  11. Sex, stem cells and tumors in the Drosophila ovary.

    PubMed

    Salz, Helen K

    2013-01-01

    The Drosophila Sex-lethal (Sxl) gene encodes a female-specific RNA binding protein that in somatic cells globally regulates all aspects of female-specific development and behavior. Sxl also has a critical, but less well understood, role in female germ cells. Germ cells without Sxl protein can adopt a stem cell fate when housed in a normal ovary, but fail to successfully execute the self-renewal differentiation fate switch. The failure to differentiate is accompanied by the inappropriate expression of a set of male specific markers, continued proliferation, and formation of a tumor. The findings in Chau et al., (2012) identify the germline stem cell maintenance factor nanos as one of its target genes, and suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional downregulation of nanos expression. These studies provide the basis for a new model in which Sxl directly couples sexual identity with the self-renewal differentiation decision and raises several interesting questions about the genesis of the tumor phenotype.

  12. sry h-1, a new Drosophila melanogaster multifingered protein gene showing maternal and zygotic expression.

    PubMed Central

    Vincent, A; Kejzlarovà-Lepesant, J; Segalat, L; Yanicostas, C; Lepesant, J A

    1988-01-01

    Low-stringency hybridization of the Drosophila serendipity (sry) finger-coding sequences revealed copies of homologous DNA sequences in the genomes of members of the family Drosophilidae and higher vertebrates. sry h-1, a new Drosophila finger protein-coding gene isolated on the basis of this homology, encodes a 3.2-kilobase (kb) mRNA accumulating in eggs and abundant in early embryos. The predicted sry h-1 protein product, starting at an internal initiation site of translation, is a 868-amino-acid basic polypeptide containing eight TFIIIA-like fingers encoded by three separate exons. Links separating individual fingers in the sry h-1 protein are variable in length and sequence, in contrast with the invariant H/C link found in most multi-fingered proteins. The similarity of the developmental pattern of transcription of sry h-1 with that of several other Drosophila finger protein genes suggests the existence of a complex set of such genes encoding an information which is, at least partly, maternally provided to the embryo and required for activation of gene transcription in early embryos or maintenance of gene activity during subsequent development. Images PMID:3141791

  13. The HIV-1 Vpu protein induces apoptosis in Drosophila via activation of JNK signaling.

    PubMed

    Marchal, Christelle; Vinatier, Gérald; Sanial, Matthieu; Plessis, Anne; Pret, Anne-Marie; Limbourg-Bouchon, Bernadette; Théodore, Laurent; Netter, Sophie

    2012-01-01

    The genome of the human immunodeficiency virus type 1 (HIV-1) encodes the canonical retroviral proteins, as well as additional accessory proteins that enhance the expression of viral genes, the infectivity of the virus and the production of virions. The accessory Viral Protein U (Vpu), in particular, enhances viral particle production, while also promoting apoptosis of HIV-infected human T lymphocytes. Some Vpu effects rely on its interaction with the ubiquitin-proteasome protein degradation system, but the mechanisms responsible for its pro-apoptotic effects in vivo are complex and remain largely to be elucidated.We took advantage of the Drosophila model to study the effects of Vpu activity in vivo. Expression of Vpu in the developing Drosophila wing provoked tissue loss due to caspase-dependent apoptosis. Moreover, Vpu induced expression of the pro-apoptotic gene reaper, known to down-regulate Inhibitor of Apoptosis Proteins (IAPs) which are caspase-antagonizing E3 ubiquitin ligases. Indeed, Vpu also reduced accumulation of Drosophila IAP1 (DIAP1). Though our results demonstrate a physical interaction between Vpu and the proteasome-addressing SLIMB/β-TrCP protein, as in mammals, both SLIMB/βTrCP-dependent and -independent Vpu effects were observed in the Drosophila wing. Lastly, the pro-apoptotic effect of Vpu in this tissue was abrogated upon inactivation of the c-Jun N-terminal Kinase (JNK) pathway. Our results in the fly thus provide the first functional evidence linking Vpu pro-apoptotic effects to activation of the conserved JNK pathway.

  14. The HIV-1 Vpu Protein Induces Apoptosis in Drosophila via Activation of JNK Signaling

    PubMed Central

    Marchal, Christelle; Vinatier, Gérald; Sanial, Matthieu; Plessis, Anne; Pret, Anne-Marie; Limbourg-Bouchon, Bernadette; Théodore, Laurent; Netter, Sophie

    2012-01-01

    The genome of the human immunodeficiency virus type 1 (HIV-1) encodes the canonical retroviral proteins, as well as additional accessory proteins that enhance the expression of viral genes, the infectivity of the virus and the production of virions. The accessory Viral Protein U (Vpu), in particular, enhances viral particle production, while also promoting apoptosis of HIV-infected human T lymphocytes. Some Vpu effects rely on its interaction with the ubiquitin–proteasome protein degradation system, but the mechanisms responsible for its pro-apoptotic effects in vivo are complex and remain largely to be elucidated. We took advantage of the Drosophila model to study the effects of Vpu activity in vivo. Expression of Vpu in the developing Drosophila wing provoked tissue loss due to caspase-dependent apoptosis. Moreover, Vpu induced expression of the pro-apoptotic gene reaper, known to down-regulate Inhibitor of Apoptosis Proteins (IAPs) which are caspase-antagonizing E3 ubiquitin ligases. Indeed, Vpu also reduced accumulation of Drosophila IAP1 (DIAP1). Though our results demonstrate a physical interaction between Vpu and the proteasome-addressing SLIMB/β-TrCP protein, as in mammals, both SLIMB/βTrCP-dependent and -independent Vpu effects were observed in the Drosophila wing. Lastly, the pro-apoptotic effect of Vpu in this tissue was abrogated upon inactivation of the c-Jun N-terminal Kinase (JNK) pathway. Our results in the fly thus provide the first functional evidence linking Vpu pro-apoptotic effects to activation of the conserved JNK pathway. PMID:22479597

  15. The twisted Gene Encodes Drosophila Protein O-Mannosyltransferase 2 and Genetically Interacts With the rotated abdomen Gene Encoding Drosophila Protein O-Mannosyltransferase 1

    PubMed Central

    Lyalin, Dmitry; Koles, Kate; Roosendaal, Sigrid D.; Repnikova, Elena; Van Wechel, Laura; Panin, Vladislav M.

    2006-01-01

    The family of mammalian O-mannosyltransferases includes two enzymes, POMT1 and POMT2, which are thought to be essential for muscle and neural development. Similar to mammalian organisms, Drosophila has two O-mannosyltransferase genes, rotated abdomen (rt) and DmPOMT2, encoding proteins with high homology to their mammalian counterparts. The previously reported mutant phenotype of the rt gene includes a clockwise rotation of the abdomen and defects in embryonic muscle development. No mutants have been described so far for the DmPOMT2 locus. In this study, we determined that the mutation in the twisted (tw) locus, tw1, corresponds to a DmPOMT2 mutant. The twisted alleles represent a complementation group of recessive mutations that, similar to the rt mutants, exhibit a clockwise abdomen rotation phenotype. Several tw alleles were isolated in the past; however, none of them was molecularly characterized. We used an expression rescue approach to confirm that tw locus represents DmPOMT2 gene. We found that the tw1 allele represents an amino acid substitution within the conserved PMT domain of DmPOMT2 (TW) protein. Immunostaining experiments revealed that the protein products of both rt and tw genes colocalize within Drosophila cells where they reside in the ER subcellular compartment. In situ hybridization analysis showed that both genes have essentially overlapping patterns of expression throughout most of embryogenesis (stages 8–17), while only the rt transcript is present at early embryonic stages (5 and 6), suggesting its maternal origin. Finally, we analyzed the genetic interactions between rt and tw using several mutant alleles, RNAi, and ectopic expression approaches. Our data suggest that the two Drosophila O-mannosyltransferase genes, rt and tw, have nonredundant functions within the same developmental cascade and that their activities are required simultaneously for possibly the same biochemical process. Our results establish the possibility of using

  16. A novel muscle LIM-only protein is generated from the paxillin gene locus in Drosophila

    PubMed Central

    Yagi, Ryohei; Ishimaru, Satoshi; Yano, Hajime; Gaul, Ulrike; Hanafusa, Hidesaburo; Sabe, Hisataka

    2001-01-01

    Paxillin is a protein containing four LIM domains, and functions in integrin signaling. We report here that two transcripts are generated from the paxillin gene locus in Drosophila; one encodes a protein homolog of the vertebrate Paxillin (DPxn37), and the other a protein with only three LIM domains, partly encoded by its own specific exon (PDLP). At the myotendinous junctions of Drosophila embryos where integrins play important roles, both DPxn37 and PDLP are highly expressed with different patterns; DPxn37 is predominantly concentrated at the center of the junctions, whereas PDLP is highly enriched at neighboring sides of the junction centers, primarily expressed in the mesodermal myotubes. Northern blot analysis revealed that DPxn37 is ubiquitously expressed throughout the life cycle, whereas PDLP expression exhibits a biphasic pattern during development, largely concomitant with muscle generation and remodeling. Our results collectively reveal that a unique system exists in Drosophila for the generation of a novel type of LIM-only protein, highly expressed in the embryonic musclature, largely utilizing the Paxillin LIM domains. PMID:11520860

  17. Heterochromatin protein 1, a known suppressor of position-effect variegation, is highly conserved in Drosophila.

    PubMed Central

    Clark, R F; Elgin, S C

    1992-01-01

    The Su(var)205 gene of Drosophila melanogaster encodes heterochromatin protein 1 (HP1), a protein located preferentially within beta-heterochromatin. Mutation of this gene has been associated with dominant suppression of position-effect variegation. We have cloned and sequenced the gene encoding HP1 from Drosophila virilis, a distantly related species. Comparison of the predicted amino acid sequence with Drosophila melanogaster HP1 shows two regions of strong homology, one near the N-terminus (57/61 amino acids identical) and the other near the C-terminus (62/68 amino acids identical) of the protein. Little homology is seen in the 5' and 3' untranslated portions of the gene, as well as in the intronic sequences, although intron/exon boundaries are generally conserved. A comparison of the deduced amino acid sequences of HP1-like proteins from other species shows that the cores of the N-terminal and C-terminal domains have been conserved from insects to mammals. The high degree of conservation suggests that these N- and C-terminal domains could interact with other macromolecules in the formation of the condensed structure of heterochromatin. Images PMID:1461737

  18. NAP-1, Nucleosome assembly protein 1, a histone chaperone involved in Drosophila telomeres.

    PubMed

    López-Panadès, Elisenda; Casacuberta, Elena

    2016-03-01

    Telomere elongation is a function that all eukaryote cells must accomplish in order to guarantee, first, the stability of the end of the chromosomes and second, to protect the genetic information from the inevitable terminal erosion. The targeted transposition of the telomere transposons HeT-A, TART and TAHRE perform this function in Drosophila, while the telomerase mechanism elongates the telomeres in most eukaryotes. In order to integrate telomere maintenance together with cell cycle and metabolism, different components of the cell interact, regulate, and control the proteins involved in telomere elongation. Different partners of the telomerase mechanism have already been described, but in contrast, very few proteins have been related with assisting the telomere transposons of Drosophila. Here, we describe for the first time, the implication of NAP-1 (Nucleosome assembly protein 1), a histone chaperone that has been involved in nuclear transport, transcription regulation, and chromatin remodeling, in telomere biology. We find that Nap-1 and HeT-A Gag, one of the major components of the Drosophila telomeres, are part of the same protein complex. We also demonstrate that their close interaction is necessary to guarantee telomere stability in dividing cells. We further show that NAP-1 regulates the transcription of the HeT-A retrotransposon, pointing to a positive regulatory role of NAP-1 in telomere expression. All these results facilitate the understanding of the transposon telomere maintenance mechanism, as well as the integration of telomere biology with the rest of the cell metabolism.

  19. Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins

    PubMed Central

    Huang, Da Wei; Fanti, Laura; Pak, Daniel T.S.; Botchan, Michael R.; Pimpinelli, Sergio; Kellum, Rebecca

    1998-01-01

    The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae. PMID:9679132

  20. Kinetics of doubletime kinase-dependent degradation of the Drosophila period protein.

    PubMed

    Syed, Sheyum; Saez, Lino; Young, Michael W

    2011-08-05

    Robust circadian oscillations of the proteins PERIOD (PER) and TIMELESS (TIM) are hallmarks of a functional clock in the fruit fly Drosophila melanogaster. Early morning phosphorylation of PER by the kinase Doubletime (DBT) and subsequent PER turnover is an essential step in the functioning of the Drosophila circadian clock. Here using time-lapse fluorescence microscopy we study PER stability in the presence of DBT and its short, long, arrhythmic, and inactive mutants in S2 cells. We observe robust PER degradation in a DBT allele-specific manner. With the exception of doubletime-short (DBT(S)), all mutants produce differential PER degradation profiles that show direct correspondence with their respective Drosophila behavioral phenotypes. The kinetics of PER degradation with DBT(S) in cell culture resembles that with wild-type DBT and posits that, in flies DBT(S) likely does not modulate the clock by simply affecting PER degradation kinetics. For all the other tested DBT alleles, the study provides a simple model in which the changes in Drosophila behavioral rhythms can be explained solely by changes in the rate of PER degradation.

  1. Protein kinase A inhibits a consolidated form of memory in Drosophila.

    PubMed

    Horiuchi, Junjiro; Yamazaki, Daisuke; Naganos, Shintaro; Aigaki, Toshiro; Saitoe, Minoru

    2008-12-30

    Increasing activity of the cAMP/protein kinase A (PKA) pathway has often been proposed as an approach to improve memory in various organisms. However, here we demonstrate that single-point mutations, which decrease PKA activity, dramatically improve aversive olfactory memory in Drosophila. These mutations do not affect formation of early memory phases or of protein synthesis-dependent long-term memory but do cause a significant increase in a specific consolidated form of memory, anesthesia-resistant memory. Significantly, heterozygotes of null mutations in PKA are sufficient to cause this memory increase. Expressing a PKA transgene in the mushroom bodies, brain structures critical for memory formation in Drosophila, reduces memory back to wild-type levels. These results indicate that although PKA is critical for formation of several memory phases, it also functions to inhibit at least one memory phase.

  2. Neuron-specific protein interactions of Drosophila CASK-β are revealed by mass spectrometry

    PubMed Central

    Mukherjee, Konark; Slawson, Justin B.; Christmann, Bethany L.; Griffith, Leslie C.

    2014-01-01

    Modular scaffolding proteins are designed to have multiple interactors. CASK, a member of the membrane-associated guanylate kinase (MAGUK) superfamily, has been shown to have roles in many tissues, including neurons and epithelia. It is likely that the set of proteins it interacts with is different in each of these diverse tissues. In this study we asked if within the Drosophila central nervous system, there were neuron-specific sets of CASK-interacting proteins. A YFP-tagged CASK-β transgene was expressed in genetically defined subsets of neurons in the Drosophila brain known to be important for CASK function, and proteins present in an anti-GFP immunoprecipitation were identified by mass spectrometry. Each subset of neurons had a distinct set of interacting proteins, suggesting that CASK participates in multiple protein networks and that these networks may be different in different neuronal circuits. One common set of proteins was associated with mitochondria, and we show here that endogenous CASK-β co-purifies with mitochondria. We also determined CASK-β posttranslational modifications for one cell type, supporting the idea that this technique can be used to assess cell- and circuit-specific protein modifications as well as protein interaction networks. PMID:25071438

  3. The hypoparathyroidism-associated mutation in Drosophila Gcm compromises protein stability and glial cell formation

    PubMed Central

    Xi, Xiao; Lu, Lu; Zhuge, Chun-Chun; Chen, Xuebing; Zhai, Yuanfen; Cheng, Jingjing; Mao, Haian; Yang, Chang-Ching; Tan, Bertrand Chin-Ming; Lee, Yi-Nan; Chien, Cheng-Ting; Ho, Margaret S.

    2017-01-01

    Differentiated neurons and glia are acquired from immature precursors via transcriptional controls exerted by factors such as proteins in the family of Glial Cells Missing (Gcm). Mammalian Gcm proteins mediate neural stem cell induction, placenta and parathyroid development, whereas Drosophila Gcm proteins act as a key switch to determine neuronal and glial cell fates and regulate hemocyte development. The present study reports a hypoparathyroidism-associated mutation R59L that alters Drosophila Gcm (Gcm) protein stability, rendering it unstable, and hyperubiquitinated via the ubiquitin-proteasome system (UPS). GcmR59L interacts with the Slimb-based SCF complex and Protein Kinase C (PKC), which possibly plays a role in its phosphorylation, hence altering ubiquitination. Additionally, R59L causes reduced Gcm protein levels in a manner independent of the PEST domain signaling protein turnover. GcmR59L proteins bind DNA, functionally activate transcription, and induce glial cells, yet at a less efficient level. Finally, overexpression of either wild-type human Gcmb (hGcmb) or hGcmb carrying the conserved hypoparathyroidism mutation only slightly affects gliogenesis, indicating differential regulatory mechanisms in human and flies. Taken together, these findings demonstrate the significance of this disease-associated mutation in controlling Gcm protein stability via UPS, hence advance our understanding on how glial formation is regulated. PMID:28051179

  4. G-rich, a Drosophila selenoprotein, is a Golgi-resident type III membrane protein

    SciTech Connect

    Chen, Chang Lan; Shim, Myoung Sup; Chung, Jiyeol; Yoo, Hyun-Seung; Ha, Ji Min; Kim, Jin Young; Choi, Jinmi; Zang, Shu Liang; Hou, Xiao; Carlson, Bradley A.; Hatfield, Dolph L.; Lee, Byeong Jae . E-mail: imbglmg@plaza.snu.ac.kr

    2006-10-06

    G-rich is a Drosophila melanogaster selenoprotein, which is a homologue of human and mouse SelK. Subcellular localization analysis using GFP-tagged G-rich showed that G-rich was localized in the Golgi apparatus. The fusion protein was co-localized with the Golgi marker proteins but not with an endoplasmic reticulum (ER) marker protein in Drosophila SL2 cells. Bioinformatic analysis of G-rich suggests that this protein is either type II or type III transmembrane protein. To determine the type of transmembrane protein experimentally, GFP-G-rich in which GFP was tagged at the N-terminus of G-rich, or G-rich-GFP in which GFP was tagged at the C-terminus of G-rich, were expressed in SL2 cells. The tagged proteins were then digested with trypsin, and analyzed by Western blot analysis. The results showed that the C-terminus of the G-rich protein was exposed to the cytoplasm indicating it is a type III microsomal membrane protein. G-rich is First selenoprotein identified in the Golgi apparatus.

  5. A genome-wide resource for the analysis of protein localisation in Drosophila.

    PubMed

    Sarov, Mihail; Barz, Christiane; Jambor, Helena; Hein, Marco Y; Schmied, Christopher; Suchold, Dana; Stender, Bettina; Janosch, Stephan; K J, Vinay Vikas; Krishnan, R T; Krishnamoorthy, Aishwarya; Ferreira, Irene R S; Ejsmont, Radoslaw K; Finkl, Katja; Hasse, Susanne; Kämpfer, Philipp; Plewka, Nicole; Vinis, Elisabeth; Schloissnig, Siegfried; Knust, Elisabeth; Hartenstein, Volker; Mann, Matthias; Ramaswami, Mani; VijayRaghavan, K; Tomancak, Pavel; Schnorrer, Frank

    2016-02-20

    The Drosophila genome contains >13000 protein-coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here, we present a genome-wide fosmid library of 10000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins, we created transgenic lines, and for a total of 207 lines, we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests, we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts.

  6. Isolation of Drosophila genes encoding G protein-coupled receptor kinases.

    PubMed Central

    Cassill, J A; Whitney, M; Joazeiro, C A; Becker, A; Zuker, C S

    1991-01-01

    G protein-coupled receptors are regulated via phosphorylation by a variety of protein kinases. Recently, termination of the active state of two such receptors, the beta-adrenergic receptor and rhodopsin, has been shown to be mediated by agonist- or light-dependent phosphorylation of the receptor by members of a family of protein-serine/threonine kinases (here referred to as G protein-coupled receptor kinases). We now report the isolation of a family of genes encoding a set of Drosophila protein kinases that appear to code for G protein-coupled receptor kinases. These proteins share a high degree of sequence homology with the bovine beta-adrenergic receptor kinase. The presence of a conserved family of G protein-coupled receptor kinases in vertebrates and invertebrates points to the central role of these kinases in signal transduction cascades. Images PMID:1662381

  7. Cell surface proteins Nasrat and Polehole stabilize the Torso-like extracellular determinant in Drosophila oogenesis

    PubMed Central

    Jiménez, Gerardo; González-Reyes, Acaimo; Casanova, Jordi

    2002-01-01

    Structural cell-surface and extracellular-matrix proteins modulate intercellular signaling events during development, but how this is achieved remains largely unknown. Here we identify a novel family of Drosophila proteins, Nasrat and Polehole, that coat the oocyte surface and play two roles: They mediate assembly of the eggshell, and act in the Torso RTK signaling pathway that specifies the terminal regions of the embryo. Nasrat and Polehole are essential for extracellular accumulation of Torso-like, a factor secreted during oogenesis that initiates Torso receptor activation. Stabilization of secreted factors by specialized pericellular proteins may be a general mechanism during signaling and developmental patterning. PMID:11959840

  8. A Drosophila orthologue of larp protein family is required for multiple processes in male meiosis.

    PubMed

    Ichihara, Keiko; Shimizu, Hanako; Taguchi, Osamu; Yamaguchi, Masamitsu; Inoue, Yoshihiro H

    2007-01-01

    It is important for the proper execution of cell division in both mitosis and meiosis that the chromosome segregation, cytokinesis, and partition of cell organelles progress in smooth coordination. We show here that the mitochondria inheritance is closely linked with microtubules during meiotic divisions in Drosophila males. They are first clustered in a cell equator at metaphase associated with astral microtubules and then distributed along central spindle microtubules after anaphase. The molecular mechanism for the microtubule-dependent inheritance of mitochondria in male meiosis has not been demonstrated yet. We first isolated mutations for a larp gene that is highly conserved among eukaryotes and showed that these mutant males exhibited multiple meiotic phenotypes such as a failure of chromosome segregation, cytokinesis, and mitochondrial partition. Our cytological examination revealed that the mutants showed defects in spindle pole organization and spindle formation. The larp encodes a Drosophila orthologue of a La-related protein containing a domain exhibiting an outstanding homology with a La type RNA-binding protein. Surprisingly, the dLarp protein is localized in the cytoplasm of the male germ line cells, as observed by its distinct co-localization with mitochondria in early spermatocytes and during meiotic divisions. We discuss here the essential role that dLarp plays in multiple processes in Drosophila male meiosis.

  9. The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation.

    PubMed

    Scherfer, Christoph; Qazi, Mousumi R; Takahashi, Kuniaki; Ueda, Ryu; Dushay, Mitchell S; Theopold, Ulrich; Lemaitre, Bruno

    2006-07-01

    Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.

  10. Regulation of synaptic development and function by the Drosophila PDZ protein Dyschronic.

    PubMed

    Jepson, James E C; Shahidullah, Mohammed; Liu, Die; le Marchand, Sylvain J; Liu, Sha; Wu, Mark N; Levitan, Irwin B; Dalva, Matthew B; Koh, Kyunghee

    2014-12-01

    Synaptic scaffold proteins control the localization of ion channels and receptors, and facilitate molecular associations between signaling components that modulate synaptic transmission and plasticity. Here, we define novel roles for a recently described scaffold protein, Dsychronic (DYSC), at the Drosophila larval neuromuscular junction. DYSC is the Drosophila homolog of whirlin/DFNB31, a PDZ domain protein linked to Usher syndrome, the most common form of human deaf-blindness. We show that DYSC is expressed presynaptically and is often localized adjacent to the active zone, the site of neurotransmitter release. Loss of DYSC results in marked alterations in synaptic morphology and cytoskeletal organization. Moreover, active zones are frequently enlarged and misshapen in dysc mutants. Electrophysiological analyses further demonstrate that dysc mutants exhibit substantial increases in both evoked and spontaneous synaptic transmission. We have previously shown that DYSC binds to and regulates the expression of the Slowpoke (SLO) BK potassium channel. Consistent with this, slo mutant larvae exhibit similar alterations in synapse morphology, active zone size and neurotransmission, and simultaneous loss of dysc and slo does not enhance these phenotypes, suggesting that dysc and slo act in a common genetic pathway to modulate synaptic development and output. Our data expand our understanding of the neuronal functions of DYSC and uncover non-canonical roles for the SLO potassium channel at Drosophila synapses. © 2014. Published by The Company of Biologists Ltd.

  11. Regulation of mammalian Gli proteins by Costal 2 and PKA in Drosophila reveals Hedgehog pathway conservation.

    PubMed

    Marks, Steven A; Kalderon, Daniel

    2011-06-01

    Hedgehog (Hh) signaling activates full-length Ci/Gli family transcription factors and prevents Ci/Gli proteolytic processing to repressor forms. In the absence of Hh, Ci/Gli processing is initiated by direct Pka phosphorylation. Despite those fundamental similarities between Drosophila and mammalian Hh pathways, the differential reliance on cilia and some key signal transduction components had suggested a major divergence in the mechanisms that regulate Ci/Gli protein activities, including the role of the kinesin-family protein Costal 2 (Cos2), which directs Ci processing in Drosophila. Here, we show that Cos2 binds to three regions of Gli1, just as for Ci, and that Cos2 functions to silence mammalian Gli1 in Drosophila in a Hh-regulated manner. Cos2 and the mammalian kinesin Kif7 can also direct Gli3 and Ci processing in fly, underscoring a fundamental conserved role for Cos2 family proteins in Hh signaling. We also show that direct PKA phosphorylation regulates the activity, rather than the proteolysis of Gli in Drosophilia, and we provide evidence for an analogous action of PKA on Ci.

  12. Regulation of Heart Rate in Drosophila via Fragile X Mental Retardation Protein

    PubMed Central

    Novak, Stefanie Mares; Joardar, Archi; Gregorio, Carol C.; Zarnescu, Daniela C.

    2015-01-01

    RNA binding proteins play a pivotal role in post-transcriptional gene expression regulation, however little is understood about their role in cardiac function. The Fragile X (FraX) family of RNA binding proteins is most commonly studied in the context of neurological disorders, as mutations in Fragile X Mental Retardation 1 (FMR1) are the leading cause of inherited mental retardation. More recently, alterations in the levels of Fragile X Related 1 protein, FXR1, the predominant FraX member expressed in vertebrate striated muscle, have been linked to structural and functional defects in mice and zebrafish models. FraX proteins are established regulators of translation and are known to regulate specific targets in different tissues. To decipher the direct role of FraX proteins in the heart in vivo, we turned to Drosophila, which harbors a sole, functionally conserved and ubiquitously expressed FraX protein, dFmr1. Using classical loss of function alleles as well as muscle specific RNAi knockdown, we show that Drosophila FMRP, dFmr1, is required for proper heart rate during development. Functional analyses in the context of cardiac-specific dFmr1 knockdown by RNAi demonstrate that dFmr1 is required cell autonomously in cardiac cells for regulating heart rate. Interestingly, these functional defects are not accompanied by any obvious structural abnormalities, suggesting that dFmr1 may regulate a different repertoire of targets in Drosophila than in vertebrates. Taken together, our findings support the hypothesis that dFmr1 protein is essential for proper cardiac function and establish the fly as a new model for studying the role(s) of FraX proteins in the heart. PMID:26571124

  13. Regulation of Heart Rate in Drosophila via Fragile X Mental Retardation Protein.

    PubMed

    Novak, Stefanie Mares; Joardar, Archi; Gregorio, Carol C; Zarnescu, Daniela C

    2015-01-01

    RNA binding proteins play a pivotal role in post-transcriptional gene expression regulation, however little is understood about their role in cardiac function. The Fragile X (FraX) family of RNA binding proteins is most commonly studied in the context of neurological disorders, as mutations in Fragile X Mental Retardation 1 (FMR1) are the leading cause of inherited mental retardation. More recently, alterations in the levels of Fragile X Related 1 protein, FXR1, the predominant FraX member expressed in vertebrate striated muscle, have been linked to structural and functional defects in mice and zebrafish models. FraX proteins are established regulators of translation and are known to regulate specific targets in different tissues. To decipher the direct role of FraX proteins in the heart in vivo, we turned to Drosophila, which harbors a sole, functionally conserved and ubiquitously expressed FraX protein, dFmr1. Using classical loss of function alleles as well as muscle specific RNAi knockdown, we show that Drosophila FMRP, dFmr1, is required for proper heart rate during development. Functional analyses in the context of cardiac-specific dFmr1 knockdown by RNAi demonstrate that dFmr1 is required cell autonomously in cardiac cells for regulating heart rate. Interestingly, these functional defects are not accompanied by any obvious structural abnormalities, suggesting that dFmr1 may regulate a different repertoire of targets in Drosophila than in vertebrates. Taken together, our findings support the hypothesis that dFmr1 protein is essential for proper cardiac function and establish the fly as a new model for studying the role(s) of FraX proteins in the heart.

  14. Localization of Drosophila retinal degeneration B, a membrane- associated phosphatidylinositol transfer protein

    PubMed Central

    1993-01-01

    The Drosophila retinal degeneration B (rdgB) mutation causes abnormal photoreceptor response and light-enhanced retinal degeneration. Immunoblots using polyclonal anti-rdgB serum showed that rdgB is a 160- kD membrane protein. The antiserum localized the rdgB protein in photoreceptors, antennae, and regions of the Drosophila brain, indicating that the rdgB protein functions in many sensory and neuronal cells. In photoreceptors, the protein localized adjacent to the rhabdomeres, in the vicinity of the subrhabdomeric cisternae. The rdgB protein's amino-terminal 281 residues are > 40% identical to the rat brain phosphatidylinositol transfer protein (PI-TP). A truncated rdgB protein, which contains only this amino-terminal domain, possesses a phosphatidylinositol transfer activity in vitro. The remaining 773 carboxyl terminal amino acids have additional functional domains. Nitrocellulose overlay experiments reveal that an acidic amino acid domain, adjacent to the PI transfer domain, binds 45Ca+2. Six hydrophobic segments are found in the middle of the putative translation product and likely function as membrane spanning domains. These results suggest that the rdgB protein, unlike the small soluble PI-TPs, is a membrane-associated PI-TP, which may be directly regulated by light-induced changes in intracellular calcium. PMID:8354691

  15. Protein and Genetic Composition of Four Chromatin Types in Drosophila melanogaster Cell Lines.

    PubMed

    Boldyreva, Lidiya V; Goncharov, Fyodor P; Demakova, Olga V; Zykova, Tatyana Yu; Levitsky, Victor G; Kolesnikov, Nikolay N; Pindyurin, Alexey V; Semeshin, Valeriy F; Zhimulev, Igor F

    2017-04-01

    Recently, we analyzed genome-wide protein binding data for the Drosophila cell lines S2, Kc, BG3 and Cl.8 (modENCODE Consortium) and identified a set of 12 proteins enriched in the regions corresponding to interbands of salivary gland polytene chromosomes. Using these data, we developed a bioinformatic pipeline that partitioned the Drosophila genome into four chromatin types that we hereby refer to as aquamarine, lazurite, malachite and ruby. Here, we describe the properties of these chromatin types across different cell lines. We show that aquamarine chromatin tends to harbor transcription start sites (TSSs) and 5' untranslated regions (5'UTRs) of the genes, is enriched in diverse "open" chromatin proteins, histone modifications, nucleosome remodeling complexes and transcription factors. It encompasses most of the tRNA genes and shows enrichment for non-coding RNAs and miRNA genes. Lazurite chromatin typically encompasses gene bodies. It is rich in proteins involved in transcription elongation. Frequency of both point mutations and natural deletion breakpoints is elevated within lazurite chromatin. Malachite chromatin shows higher frequency of insertions of natural transposons. Finally, ruby chromatin is enriched for proteins and histone modifications typical for the "closed" chromatin. Ruby chromatin has a relatively low frequency of point mutations and is essentially devoid of miRNA and tRNA genes. Aquamarine and ruby chromatin types are highly stable across cell lines and have contrasting properties. Lazurite and malachite chromatin types also display characteristic protein composition, as well as enrichment for specific genomic features. We found that two types of chromatin, aquamarine and ruby, retain their complementary protein patterns in four Drosophila cell lines.

  16. Functional dissection of the Hox protein Abdominal-B in Drosophila cell culture

    SciTech Connect

    Zhai, Zongzhao; Yang, Xingke; Lohmann, Ingrid

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer ct340 CRM was identified to be the posterior spiracle enhancer of gene cut. Black-Right-Pointing-Pointer ct340 is under the direct transcriptional control of Hox protein Abd-B. Black-Right-Pointing-Pointer An efficient cloning system was developed to assay protein-DNA interaction. Black-Right-Pointing-Pointer New features of Abd-B dependent target gene regulation were detected. -- Abstract: Hox transcription factors regulate the morphogenesis along the anterior-posterior (A/P) body axis through the interaction with small cis-regulatory modules (CRMs) of their target gene, however so far very few Hox CRMs are known and have been analyzed in detail. In this study we have identified a new Hox CRM, ct340, which guides the expression of the cell type specification gene cut (ct) in the posterior spiracle under the direct control of the Hox protein Abdominal-B (Abd-B). Using the ct340 enhancer activity as readout, an efficient cloning system to generate VP16 activation domain fusion protein was developed to unambiguously test protein-DNA interaction in Drosophila cell culture. By functionally dissecting the Abd-B protein, new features of Abd-B dependent target gene regulation were detected. Due to its easy adaptability, this system can be generally used to map functional domains within sequence-specific transcriptional factors in Drosophila cell culture, and thus provide preliminary knowledge of the protein functional domain structure for further in vivo analysis.

  17. Protein phosphatase 4 mediates localization of the Miranda complex during Drosophila neuroblast asymmetric divisions.

    PubMed

    Sousa-Nunes, Rita; Chia, William; Somers, W Greg

    2009-02-01

    Asymmetric localization of cell fate determinants is a crucial step in neuroblast asymmetric divisions. Whereas several protein kinases have been shown to mediate this process, no protein phosphatase has so far been implicated. In a clonal screen of larval neuroblasts we identified the evolutionarily conserved Protein Phosphatase 4 (PP4) regulatory subunit PP4R3/Falafel (Flfl) as a key mediator specific for the localization of Miranda (Mira) and associated cell fate determinants during both interphase and mitosis. Flfl is predominantly nuclear during interphase/prophase and cytoplasmic after nuclear envelope breakdown. Analyses of nuclear excluded as well as membrane targeted versions of the protein suggest that the asymmetric cortical localization of Mira and its associated proteins during mitosis depends on cytoplasmic/membrane-associated Flfl, whereas nuclear Flfl is required to exclude the cell fate determinant Prospero (Pros), and consequently Mira, from the nucleus during interphase/prophase. Attenuating the function of either the catalytic subunit of PP4 (PP4C; Pp4-19C in Drosophila) or of another regulatory subunit, PP4R2 (PPP4R2r in Drosophila), leads to similar defects in the localization of Mira and associated proteins. Flfl is capable of directly interacting with Mira, and genetic analyses indicate that flfl acts in parallel to or downstream from the tumor suppressor lethal (2) giant larvae (lgl). Our findings suggest that Flfl may target PP4 to the MIra protein complex to facilitate dephosphorylation step(s) crucial for its cortical association/asymmetric localization.

  18. The DOCK protein sponge binds to ELMO and functions in Drosophila embryonic CNS development.

    PubMed

    Biersmith, Bridget; Liu, Ze Cindy; Bauman, Kenneth; Geisbrecht, Erika R

    2011-01-25

    Cell morphogenesis, which requires rearrangement of the actin cytoskeleton, is essential to coordinate the development of tissues such as the musculature and nervous system during normal embryonic development. One class of signaling proteins that regulate actin cytoskeletal rearrangement is the evolutionarily conserved CDM (C. elegansCed-5, human DOCK180, DrosophilaMyoblast city, or Mbc) family of proteins, which function as unconventional guanine nucleotide exchange factors for the small GTPase Rac. This CDM-Rac protein complex is sufficient for Rac activation, but is enhanced upon the association of CDM proteins with the ELMO/Ced-12 family of proteins. We identified and characterized the role of Drosophila Sponge (Spg), the vertebrate DOCK3/DOCK4 counterpart as an ELMO-interacting protein. Our analysis shows Spg mRNA and protein is expressed in the visceral musculature and developing nervous system, suggesting a role for Spg in later embryogenesis. As maternal null mutants of spg die early in development, we utilized genetic interaction analysis to uncover the role of Spg in central nervous system (CNS) development. Consistent with its role in ELMO-dependent pathways, we found genetic interactions with spg and elmo mutants exhibited aberrant axonal defects. In addition, our data suggests Ncad may be responsible for recruiting Spg to the membrane, possibly in CNS development. Our findings not only characterize the role of a new DOCK family member, but help to further understand the role of signaling downstream of N-cadherin in neuronal development.

  19. Lipid droplet subset targeting of the Drosophila protein CG2254/dmLdsdh1.

    PubMed

    Thul, Peter J; Tschapalda, Kirsten; Kolkhof, Petra; Thiam, Abdou Rachid; Oberer, Monika; Beller, Mathias

    2017-08-03

    Lipid droplets (LDs) are the principal organelles of lipid storage. They consist of a hydrophobic core of storage lipids, surrounded by a phospholipid monolayer with proteins attached. While some of these proteins are essential to regulate cellular and organismic lipid metabolism, key questions concerning LD protein function, such as their targeting to LDs, are still unanswered. Intriguingly, some proteins are restricted to LD subsets by an as yet unknown mechanism. This finding makes LD targeting even more complex.Here, we characterize the Drosophila protein CG2254 which targets LD subsets in cultured cells and different larval Drosophila tissues, where the prevalence of LD subsets appears highly dynamic. We find that an amphipathic amino acid stretch mediates CG2254 LD localization. Additionally, we identified a juxtaposed sequence stretch limiting CG2254 localization to LD subsets. This sequence is sufficient to restrict a chimeric protein - consisting of the subset targeting sequence introduced to an otherwise pan LD localized protein sequence - to LD subsets. Based on its subcellular localization and annotated function, we suggest to rename CG2254 to Lipid droplet subset dehydrogenase 1 (Ldsdh1). © 2017. Published by The Company of Biologists Ltd.

  20. Nanodiamonds Mediate Oral Delivery of Proteins for Stem Cell Activation and Intestinal Remodeling in Drosophila.

    PubMed

    Hu, Xingjie; Li, Xiaojiao; Yin, Min; Li, Ping; Huang, Ping; Wang, Lihua; Jiang, Yiguo; Wang, Hui; Chen, Nan; Fan, Chunhai; Song, Haiyun

    2017-06-07

    Introduction of exogenous biomacromolecules into living systems is of great interest in genome editing, cancer immunotherapy, and stem cell reprogramming. Whereas current strategies generally depend on nucleic acids transfection, direct delivery of functional proteins that provides enhanced specificity, increased safety, and fast and temporal regulation is highly desirable. Nevertheless, intracellular delivery of intact and bioactive proteins, especially in vivo, remains poorly explored. In this study, we developed a nanodiamonds (NDs)-based protein delivery system in cultured cells and in Drosophila that showed high adsorption, high efficiency, and effective cytosolic release of fully functional proteins. Through live-cell imaging, we observed a novel phenomenon wherein a substantial amount of internalized NDs-protein complex rejected fusion with the early endosome, thereby evading protein degradation in the lysosome. More significantly, we demonstrated that dietary NDs-RNase induced apoptosis in enterocytes, stimulating regenerative divisions in intestinal stem cells and increasing the number of stem cells and precursor cells in Drosophila intestine. As stem cells are poorly accessible by exogenous agents in vivo, NDs-mediated oral delivery of proteins provides a new approach to modulate the stem cell microenvironment for intestinal remodeling, which has important implications for colorectal cancer therapy and regenerative medicine.

  1. Drosophila Torsin Protein Regulates Motor Control and Stress Sensitivity and Forms a Complex with Fragile-X Mental Retardation Protein

    PubMed Central

    Ahn, Hyo-Min; Koh, Young Ho

    2016-01-01

    We investigated unknown in vivo functions of Torsin by using Drosophila as a model. Downregulation of Drosophila Torsin (DTor) by DTor-specific inhibitory double-stranded RNA (RNAi) induced abnormal locomotor behavior and increased susceptibility to H2O2. In addition, altered expression of DTor significantly increased the numbers of synaptic boutons. One important biochemical consequence of DTor-RNAi expression in fly brains was upregulation of alcohol dehydrogenase (ADH). Altered expression of ADH has also been reported in Drosophila Fragile-X mental retardation protein (DFMRP) mutant flies. Interestingly, expression of DFMRP was altered in DTor mutant flies, and DTor and DFMRP were present in the same protein complexes. In addition, DTor and DFMRP immunoreactivities were partially colocalized in several cellular organelles in larval muscles. Furthermore, there were no significant differences between synaptic morphologies of dfmrp null mutants and dfmrp mutants expressing DTor-RNAi. Taken together, our evidences suggested that DTor and DFMRP might be present in the same signaling pathway regulating synaptic plasticity. In addition, we also found that human Torsin1A and human FMRP were present in the same protein complexes, suggesting that this phenomenon is evolutionarily conserved. PMID:27313903

  2. Drosophila CTCF tandemly aligns with other insulator proteins at the borders of H3K27me3 domains

    PubMed Central

    Van Bortle, Kevin; Ramos, Edward; Takenaka, Naomi; Yang, Jingping; Wahi, Jessica E.; Corces, Victor G.

    2012-01-01

    Several multiprotein DNA complexes capable of insulator activity have been identified in Drosophila melanogaster, yet only CTCF, a highly conserved zinc finger protein, and the transcription factor TFIIIC have been shown to function in mammals. CTCF is involved in diverse nuclear activities, and recent studies suggest that the proteins with which it associates and the DNA sequences that it targets may underlie these various roles. Here we show that the Drosophila homolog of CTCF (dCTCF) aligns in the genome with other Drosophila insulator proteins such as Suppressor of Hairy wing [SU(HW)] and Boundary Element Associated Factor of 32 kDa (BEAF-32) at the borders of H3K27me3 domains, which are also enriched for associated insulator proteins and additional cofactors. RNAi depletion of dCTCF and combinatorial knockdown of gene expression for other Drosophila insulator proteins leads to a reduction in H3K27me3 levels within repressed domains, suggesting that insulators are important for the maintenance of appropriate repressive chromatin structure in Polycomb (Pc) domains. These results shed new insights into the roles of insulators in chromatin domain organization and support recent models suggesting that insulators underlie interactions important for Pc-mediated repression. We reveal an important relationship between dCTCF and other Drosophila insulator proteins and speculate that vertebrate CTCF may also align with other nuclear proteins to accomplish similar functions. PMID:22722341

  3. Drosophila CTCF tandemly aligns with other insulator proteins at the borders of H3K27me3 domains.

    PubMed

    Van Bortle, Kevin; Ramos, Edward; Takenaka, Naomi; Yang, Jingping; Wahi, Jessica E; Corces, Victor G

    2012-11-01

    Several multiprotein DNA complexes capable of insulator activity have been identified in Drosophila melanogaster, yet only CTCF, a highly conserved zinc finger protein, and the transcription factor TFIIIC have been shown to function in mammals. CTCF is involved in diverse nuclear activities, and recent studies suggest that the proteins with which it associates and the DNA sequences that it targets may underlie these various roles. Here we show that the Drosophila homolog of CTCF (dCTCF) aligns in the genome with other Drosophila insulator proteins such as Suppressor of Hairy wing [SU(HW)] and Boundary Element Associated Factor of 32 kDa (BEAF-32) at the borders of H3K27me3 domains, which are also enriched for associated insulator proteins and additional cofactors. RNAi depletion of dCTCF and combinatorial knockdown of gene expression for other Drosophila insulator proteins leads to a reduction in H3K27me3 levels within repressed domains, suggesting that insulators are important for the maintenance of appropriate repressive chromatin structure in Polycomb (Pc) domains. These results shed new insights into the roles of insulators in chromatin domain organization and support recent models suggesting that insulators underlie interactions important for Pc-mediated repression. We reveal an important relationship between dCTCF and other Drosophila insulator proteins and speculate that vertebrate CTCF may also align with other nuclear proteins to accomplish similar functions.

  4. Altered heterochromatin binding by a hybrid sterility protein in Drosophila sibling species.

    PubMed

    Bayes, Joshua J; Malik, Harmit S

    2009-12-11

    Hybrid sterility of the heterogametic sex is one of the first postzygotic reproductive barriers to evolve during speciation, yet the molecular basis of hybrid sterility is poorly understood. We show that the hybrid male sterility gene Odysseus-site homeobox (OdsH) encodes a protein that localizes to evolutionarily dynamic loci within heterochromatin and leads to their decondensation. In Drosophila mauritiana x Drosophila simulans male hybrids, OdsH from D. mauritiana (OdsHmau) acts as a sterilizing factor by associating with the heterochromatic Y chromosome of D. simulans, whereas D. simulans OdsH (OdsHsim) does not. Characterization of sterile hybrid testes revealed that OdsH abundance and localization in the premeiotic phases of spermatogenesis differ between species. These results reveal that rapid heterochromatin evolution affects the onset of hybrid sterility.

  5. Debra, a protein mediating lysosomal degradation, is required for long-term memory in Drosophila.

    PubMed

    Kottler, Benjamin; Lampin-Saint-Amaux, Aurélie; Comas, Daniel; Preat, Thomas; Goguel, Valérie

    2011-01-01

    A central goal of neuroscience is to understand how neural circuits encode memory and guide behavior changes. Many of the molecular mechanisms underlying memory are conserved from flies to mammals, and Drosophila has been used extensively to study memory processes. To identify new genes involved in long-term memory, we screened Drosophila enhancer-trap P(Gal4) lines showing Gal4 expression in the mushroom bodies, a specialized brain structure involved in olfactory memory. This screening led to the isolation of a memory mutant that carries a P-element insertion in the debra locus. debra encodes a protein involved in the Hedgehog signaling pathway as a mediator of protein degradation by the lysosome. To study debra's role in memory, we achieved debra overexpression, as well as debra silencing mediated by RNA interference. Experiments conducted with a conditional driver that allowed us to specifically restrict transgene expression in the adult mushroom bodies led to a long-term memory defect. Several conclusions can be drawn from these results: i) debra levels must be precisely regulated to support normal long-term memory, ii) the role of debra in this process is physiological rather than developmental, and iii) debra is specifically required for long-term memory, as it is dispensable for earlier memory phases. Drosophila long-term memory is the only long-lasting memory phase whose formation requires de novo protein synthesis, a process underlying synaptic plasticity. It has been shown in several organisms that regulation of proteins at synapses occurs not only at translation level of but also via protein degradation, acting in remodeling synapses. Our work gives further support to a role of protein degradation in long-term memory, and suggests that the lysosome plays a role in this process.

  6. The Drosophila melanogaster sir2+ gene is nonessential and has only minor effects on position-effect variegation.

    PubMed Central

    Aström, Stefan U; Cline, Thomas W; Rine, Jasper

    2003-01-01

    Five Drosophila melanogaster genes belong to the highly conserved sir2 family, which encodes NAD(+)-dependent protein deacetylases. Of these five, dsir2(+) (CG5216) is most similar to the Saccharomyces cerevisiae SIR2 gene, which has profound effects on chromatin structure and life span. Four independent Drosophila strains were found with P-element insertions near the dsir2 transcriptional start site as well as extraneous linked recessive lethal mutations. Imprecise excision of one of these P elements (PlacW07223) from a chromosome freed of extraneous lethal mutations produced dsir2(17), a null intragenic deletion allele that generates no DSIR2 protein. Contrary to expectations from the report by Rosenberg and Parkhurst on their P-mobilization allele dSir2(ex10), homozygosity for dsir2(17) had no apparent deleterious effects on viability, developmental rate, or sex ratio, and it fully complemented sir2(ex10). Moreover, through a genetic test, we ruled out the reported effect of dSir2(ex10) on Sex-lethal expression. We did observe a modest, strictly recessive suppression of white(m4) position-effect variegation and a shortening of life span in dsir2 homozygous mutants, suggesting that dsir2 has some functions in common with yeast SIR2. PMID:12663533

  7. A survey of conservation of sea spider and Drosophila Hox protein activities.

    PubMed

    Saadaoui, Mehdi; Litim-Mecheri, Isma; Macchi, Meiggie; Graba, Yacine; Maurel-Zaffran, Corinne

    2015-11-01

    Hox proteins have well-established functions in development and evolution, controlling the final morphology of bilaterian animals. The common phylogenetic origin of Hox proteins and the associated evolutionary diversification of protein sequences provide a unique framework to explore the relationship between changes in protein sequence and function. In this study, we aimed at questioning how sequence variation within arthropod Hox proteins influences function. This was achieved by exploring the functional impact of sequence conservation/divergence of the Hox genes, labial, Sex comb reduced, Deformed, Ultrabithorax and abdominalA from two distant arthropods, the sea spider and the well-studied Drosophila. Results highlight a correlation between sequence conservation within the homeodomain and the degree of functional conservation, and identify a novel functional domain in the Labial protein.

  8. Recombineering-mediated tagging of Drosophila genomic constructs for in vivo localization and acute protein inactivation

    PubMed Central

    Venken, Koen J. T.; Kasprowicz, Jaroslaw; Kuenen, Sabine; Yan, Jiekun; Hassan, Bassem A.; Verstreken, Patrik

    2008-01-01

    Studying gene function in the post-genome era requires methods to localize and inactivate proteins in a standardized fashion in model organisms. While genome-wide gene disruption and over-expression efforts are well on their way to vastly expand the repertoire of Drosophila tools, a complementary method to efficiently and quickly tag proteins expressed under endogenous control does not exist for fruit flies. Here, we describe the development of an efficient procedure to generate protein fusions at either terminus in an endogenous genomic context using recombineering. We demonstrate that the fluorescent protein tagged constructs, expressed under the proper control of regulatory elements, can rescue the respective mutations and enable the detection of proteins in vivo. Furthermore, we also adapted our method for use of the tetracysteine tag that tightly binds the fluorescent membrane-permeable FlAsH ligand. This technology allows us to acutely inactivate any tagged protein expressed under native control using fluorescein-assisted light inactivation and we provide proof of concept by demonstrating that acute loss of clathrin heavy chain function in the fly eye leads to synaptic transmission defects in photoreceptors. Our tagging technology is efficient and versatile, adaptable to any tag desired and paves the way to genome-wide gene tagging in Drosophila. PMID:18676454

  9. Tissue distribution of PEBBLE RNA and pebble protein during Drosophila embryonic development.

    PubMed

    Prokopenko, S N; Saint, R; Bellen, H J

    2000-02-01

    pebble (pbl) is required for cytokinesis during postblastoderm mitoses (Hime, G., Saint, R., 1992. Zygotic expression of the pebble locus is required for cytokinesis during the postblastoderm mitoses of Drosophila. Development 114, 165-171; Lehner, C.F., 1992. The pebble gene is required for cytokinesis in Drosophila. J. Cell Sci. 103, 1021-1030) and encodes a putative guanine nucleotide exchange factor (RhoGEF) for Rho1 GTPase (Prokopenko, S.N., Brumby, A., O'Keefe, L., Prior, L., He, Y., Saint, R., Bellen, H.J., 1999. A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila. Genes Dev. 13, 2301-2314). Mutations in pbl result in the absence of a contractile ring leading to a failure of cytokinesis and formation of polyploid multinucleate cells. Analysis of the subcellular distribution of PBL demonstrated that during mitosis, PBL accumulates at the cleavage furrow at the anaphase to telophase transition when assembly of a contractile ring is initiated (Prokopenko, S.N., Brumby, A., O'Keefe, L., Prior, L., He, Y., Saint, R., Bellen, H.J., 1999. A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila. Genes Dev. 13, 2301-2314). In addition, levels of PBL protein cycle during each round of cell division with the highest levels of PBL found in telophase and interphase nuclei. Here, we report the expression pattern of pbl during embryonic development. We show that PEBBLE RNA and PBL protein have a similar tissue distribution and are expressed in a highly dynamic pattern throughout embryogenesis. We show that PBL is strongly enriched in dividing nuclei in syncytial embryos and in pole cells as well as in nuclei of dividing cells in postblastoderm embryos. Our expression data correlate well with the phenotypes observed in pole cells and, particularly, with the absence of cytokinesis after cellular blastoderm formation in pbl mutants.

  10. The Iroquois homeodomain proteins are required to specify body wall identity in Drosophila

    PubMed Central

    del Corral, Ruth Diez; Aroca, Pilar; Gómez-Skarmeta, José Luis; Cavodeassi, Florencia; Modolell, Juan

    1999-01-01

    The Iroquois complex (Iro-C) homeodomain proteins allow cells at the proximal part of the Drosophila imaginal wing disc to form mesothoracic body wall (notum). Cells lacking these proteins form wing hinge structures instead (tegula and axillary sclerites). Moreover, the mutant cells impose on neighboring wild-type cells more distal developmental fates, like lateral notum or wing hinge. These findings support a tergal phylogenetic origin for the most proximal part of the wing and provide evidence for a novel pattern organizing center at the border between the apposed notum (Iro-C-expressing) and hinge (Iro-C-nonexpressing) cells. This border is not a cell lineage restriction boundary. PMID:10398687

  11. The Drosophila EKC/KEOPS complex: roles in protein synthesis homeostasis and animal growth.

    PubMed

    Rojas-Benítez, Diego; Ibar, Consuelo; Glavic, Álvaro

    2013-01-01

    The TOR signaling pathway is crucial in the translation of nutritional inputs into the protein synthesis machinery regulation, allowing animal growth. We recently identified the Bud32 (yeast)/PRPK (human) ortholog in Drosophila, Prpk (p53-related protein kinase), and found that it is required for TOR kinase activity. Bud32/PRPK is an ancient and atypical kinase conserved in evolution from Archeae to humans, being essential for Archeae. It has been linked with p53 stabilization in human cell culture and its absence in yeast causes a slow-growth phenotype. This protein has been associated to KEOPS (kinase, putative endopeptidase and other proteins of small size) complex together with Kae1p (ATPase), Cgi-121 and Pcc1p. This complex has been implicated in telomere maintenance, transcriptional regulation, bud site selection and chemical modification of tRNAs (tRNAs). Bud32p and Kae1p have been related with N6-threonylcarbamoyladenosine (t (6)A) synthesis, a particular chemical modification that occurs at position 37 of tRNAs that pair A-starting codons, required for proper translation in most species. Lack of this modification causes mistranslations and open reading frame shifts in yeast. The core constituents of the KEOPS complex are present in Drosophila, but their physical interaction has not been reported yet. Here, we present a review of the findings regarding the function of this complex in different organisms and new evidence that extends our recent observations of Prpk function in animal growth showing that depletion of Kae1 or Prpk, in accordance with their role in translation in yeast, is able to induce the unfolded protein response (UPR) in Drosophila. We suggest that EKC/KEOPS complex could be integrating t (6)A-modified tRNA availability with translational rates, which are ultimately reflected in animal growth.

  12. Expression of the Drosophila Secreted Cuticle Protein 73 (dsc73) Requires Shavenbaby

    PubMed Central

    Andrew, Deborah J.; Baker, Bruce S.

    2010-01-01

    Low stringency genomic library screens with genomic fragments from the sex determination gene doublesex identified the Drosophila secreted cuticle protein 73 (dsc73) gene, which encodes an 852-residue protein with an N-terminal signal sequence. In embryos, dsc73 RNA and protein are expressed to high levels in the epidermal cells that secrete the larval cuticle as well as in other cuticle-secreting tissues such as the trachea and salivary duct. Embryonic expression of dsc73 requires Shavenbaby, a transcription factor regulating cuticle formation. Double-labeling experiments with αCrb and αSAS reveal that, as with chitin and other known cuticle proteins, Dsc73 is secreted apically. Zygotic loss of dsc73 results in larval lethality but loss does not result in overt patterning defects or overt morphological defects in the embryonic tissues in which it is expressed. Thus, dsc73 encodes a novel secreted protein, and it is conserved within the Drosophila group. dsc73 may serve as a useful embryonic marker for cuticular patterning. PMID:18351665

  13. The Drosophila Hrb87F gene encodes a new member of the A and B hnRNP protein group.

    PubMed Central

    Haynes, S R; Johnson, D; Raychaudhuri, G; Beyer, A L

    1991-01-01

    Nascent premessenger RNA transcripts are packaged into heterogeneous nuclear ribonucleoprotein (hnRNP) complexes containing specific nuclear proteins, the hnRNP proteins. The A and B group proteins constitute a major class of small basic proteins found in mammalian hnRNP complexes. We have previously characterized the Drosophila melanogaster Hrb98DE gene, which is alternatively spliced to encode four protein isoforms closely related to the A and B proteins. We report here that the Drosophila genome contains a family of genes related to the Hrb98DE gene. One member of the family, Hrb87F, is very homologous to Hrb98DE in both sequence and structure. The Hrb87F transcripts (1.7 and 2.2 kb) utilize two alternative polyadenylation sites, are abundant in ovaries and early embryos, and are present in lesser amounts throughout development. In one wildtype strain of Drosophila there is a naturally-occurring polymorphism in this gene due to the insertion of a 412 transposable element in the 3' untranslated region. The larger transcript is not produced in these files and thus is not required for viability. Sequence identities among the Drosophila Hrb proteins and the vertebrate A and B hnRNP proteins suggest that these proteins may form a distinct subfamily within the larger family of related RNA binding proteins. Images PMID:1849257

  14. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    SciTech Connect

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; Graveley, Brenton R.; Brenner, Steven E.

    2015-08-20

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. In conclusion, this large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.

  15. Stage-specific phosphorylation of the fushi tarazu protein during Drosophila development.

    PubMed Central

    Krause, H M; Gehring, W J

    1989-01-01

    The regulatory protein encoded by the fushi tarazu (ftz) gene is expressed during three different stages of Drosophila embryogenesis in three different developing tissues. Previously, we demonstrated that ftz protein ectopically expressed throughout developing embryos under the control of an hsp70 heat shock promoter is heavily modified. Here we show that these negatively charged isoforms of the protein are the result of phosphorylation at as many as 16 sites. Phosphate groups could be removed in vitro by treatment with various phosphatases and could be added in vivo by incubating embryo-derived cells or nuclei in the presence of [32P]-orthophosphate. Phosphoamino acid analysis of immunoprecipitated ftz protein yielded both phosphoserines and phosphothreonines at a ratio of approximately 1:1. Interestingly we find that the endogenous ftz protein is also phosphorylated at multiple sites and that different subsets of the phosphoisoforms occur during different stages of development. Images PMID:2743978

  16. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    PubMed Central

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; Graveley, Brenton R.; Brenner, Steven E.

    2015-01-01

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. This large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs. PMID:26294686

  17. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    DOE PAGES

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; ...

    2015-08-20

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected themore » splicing of pre-mRNAs encoding other splicing regulators. In conclusion, this large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.« less

  18. Supplementation with Major Royal-Jelly Proteins Increases Lifespan, Feeding, and Fecundity in Drosophila.

    PubMed

    Xin, Xiao-Xuan; Chen, Yong; Chen, Di; Xiao, Fa; Parnell, Laurence D; Zhao, Jing; Liu, Liang; Ordovas, Jose M; Lai, Chao-Qiang; Shen, Li-Rong

    2016-07-27

    The major royal-jelly proteins (MRJPs) are the main constituents responsible for the specific physiological role of royal jelly (RJ) in honeybees. Male and female Drosophila flies were fed diets containing either no MRJPs (A) or casein (B) at 1.25% (w/w) of diet or MRJPs at 1.25% (C), 2.50% (D), or 5.00% (E). Diets B, C, D, and E increased mean lifespan by 4.3%, 9.0%, 12.4%, and 13.9% in males and by 5.8%, 9.7%, 20.0%, and 11.8% in females in comparison to results from diet A, respectively. The diet supplemented with 2.50% MRJPs seems to have the optimal dose to improve both physiological and biochemical measures related to aging in both sexes. Interestingly, lifespan extension by MRJPs in Drosophila was positively associated with feeding and fecundity and up-regulation of copper and zinc-superoxide dismutase (CuZn-SOD) and the Egfr-mediated signaling pathway. This study provides strong evidence that MRJPs are important components of RJ for prolonging lifespan in Drosophila.

  19. Drosophila Low Temperature Viability Protein 1 (LTV1) Is Required for Ribosome Biogenesis and Cell Growth Downstream of Drosophila Myc (dMyc)*

    PubMed Central

    Kim, Wonho; Kim, Hag Dong; Jung, Youjin; Kim, Joon; Chung, Jongkyeong

    2015-01-01

    During animal development, various signaling pathways converge to regulate cell growth. In this study, we identified LTV1 as a novel cell growth regulator in Drosophila. LTV1 mutant larvae exhibited developmental delays and lethality at the second larval stage. Using biochemical studies, we discovered that LTV1 interacted with ribosomal protein S3 and co-purified with free 40S ribosome subunits. We further demonstrated that LTV1 is crucial for ribosome biogenesis through 40S ribosome subunit synthesis and preribosomal RNA processing, suggesting that LTV1 is required for cell growth by regulating protein synthesis. We also demonstrated that Drosophila Myc (dMyc) directly regulates LTV1 transcription and requires LTV1 to stimulate ribosome biogenesis. Importantly, the loss of LTV1 blocked the cell growth and endoreplication induced by dMyc. Combined, these results suggest that LTV1 is a key downstream factor of dMyc-induced cell growth by properly maintaining ribosome biogenesis. PMID:25858587

  20. An RNAi Screen To Identify Protein Phosphatases That Function Within the Drosophila Circadian Clock.

    PubMed

    Agrawal, Parul; Hardin, Paul E

    2016-12-07

    Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. A well-characterized example of such a transcriptional feedback loop is in Drosophila, where CLOCK-CYCLE (CLK-CYC) complexes activate transcription of period (per) and timeless (tim) genes, rising levels of PER-TIM complexes feed-back to repress CLK-CYC activity, and degradation of PER and TIM permits the next cycle of CLK-CYC transcription. The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Previous behavioral screens identified several kinases that control CLK, PER, and TIM levels, subcellular localization, and/or activity, but two phosphatases that function within the clock were identified through the analysis of candidate genes from other pathways or model systems. To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. This screen identified 19 protein phosphatases that lengthened or shortened the circadian period by ≥1 hr (p ≤ 0.05 compared to controls) or were arrhythmic. Additional RNAi lines, transposon inserts, overexpression, and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. Based on genetic validation and molecular analysis, 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans.

  1. An RNAi Screen To Identify Protein Phosphatases That Function Within the Drosophila Circadian Clock

    PubMed Central

    Agrawal, Parul; Hardin, Paul E.

    2016-01-01

    Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. A well-characterized example of such a transcriptional feedback loop is in Drosophila, where CLOCK-CYCLE (CLK-CYC) complexes activate transcription of period (per) and timeless (tim) genes, rising levels of PER-TIM complexes feed-back to repress CLK-CYC activity, and degradation of PER and TIM permits the next cycle of CLK-CYC transcription. The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Previous behavioral screens identified several kinases that control CLK, PER, and TIM levels, subcellular localization, and/or activity, but two phosphatases that function within the clock were identified through the analysis of candidate genes from other pathways or model systems. To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. This screen identified 19 protein phosphatases that lengthened or shortened the circadian period by ≥1 hr (p ≤ 0.05 compared to controls) or were arrhythmic. Additional RNAi lines, transposon inserts, overexpression, and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. Based on genetic validation and molecular analysis, 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans. PMID:27784754

  2. Neuronal overexpression of APPL, the Drosophila homologue of the amyloid precursor protein (APP), disrupts axonal transport.

    PubMed

    Torroja, L; Chu, H; Kotovsky, I; White, K

    1999-05-06

    The two pathological hallmarks of Alzheimer's disease, amyloid plaques and neurofibrillary tangles, involve two apparently unrelated proteins, the amyloid precursor protein (APP) and Tau. Although it is known that aberrant processing of APP is associated with Alzheimer's disease, the definitive role of APP in neurons is not yet clear. Tau regulates microtubule stabilization and assembly in axons and is, thus, an essential component of the microtubule-associated organelle transport machinery. Although several groups have reported physical interaction between APP and Tau, and induction of Tau phosphorylation by APP and beta-amyloid peptide, the functional connection between APP and Tau is unclear. To explore the possibility that the functions of these two proteins may somehow converge on the same cellular process, we overexpressed APPL, the Drosophila homologue of APP, along with Tau in Drosophila neurons. Panneural coexpression of APPL and Tau resulted in adults that, upon eclosion, failed to expand wings and harden the cuticle, which is suggestive of neuroendocrine dysfunction. We analyzed axonal transport when Tau and APPL were coexpressed and found that transport of axonal cargo was disrupted, as evidenced by increased retention of synaptic proteins in axons and scarcity of neuropeptide-containing vesicles in the distal processes of peptidergic neurons. In an independent approach, we demonstrated genetic interaction and phenotypic similarity between APPL overexpression and mutations in the Kinesin heavy chain (Khc) gene, the product of which is a motor for anterograde vesicle trafficking.

  3. Multi-step control of muscle diversity by Hox proteins in the Drosophila embryo

    PubMed Central

    Enriquez, Jonathan; Boukhatmi, Hadi; Dubois, Laurence; Philippakis, Anthony A.; Bulyk, Martha L.; Michelson, Alan M.; Crozatier, Michèle; Vincent, Alain

    2010-01-01

    Summary Hox transcription factors control many aspects of animal morphogenetic diversity. The segmental pattern of Drosophila larval muscles shows stereotyped variations along the anteroposterior body axis. Each muscle is seeded by a founder cell and the properties specific to each muscle reflect the expression by each founder cell of a specific combination of ‘identity’ transcription factors. Founder cells originate from asymmetric division of progenitor cells specified at fixed positions. Using the dorsal DA3 muscle lineage as a paradigm, we show here that Hox proteins play a decisive role in establishing the pattern of Drosophila muscles by controlling the expression of identity transcription factors, such as Nautilus and Collier (Col), at the progenitor stage. High-resolution analysis, using newly designed intron-containing reporter genes to detect primary transcripts, shows that the progenitor stage is the key step at which segment-specific information carried by Hox proteins is superimposed on intrasegmental positional information. Differential control of col transcription by the Antennapedia and Ultrabithorax/Abdominal-A paralogs is mediated by separate cis-regulatory modules (CRMs). Hox proteins also control the segment-specific number of myoblasts allocated to the DA3 muscle. We conclude that Hox proteins both regulate and contribute to the combinatorial code of transcription factors that specify muscle identity and act at several steps during the muscle-specification process to generate muscle diversity. PMID:20056681

  4. Distinct modes of centromere protein dynamics during cell cycle progression in Drosophila S2R+ cells.

    PubMed

    Lidsky, Peter V; Sprenger, Frank; Lehner, Christian F

    2013-10-15

    Centromeres are specified epigenetically in animal cells. Therefore, faithful chromosome inheritance requires accurate maintenance of epigenetic centromere marks during progression through the cell cycle. Clarification of the mechanisms that control centromere protein behavior during the cell cycle should profit from the relatively simple protein composition of Drosophila centromeres. Thus we have analyzed the dynamics of the three key players Cid/Cenp-A, Cenp-C and Cal1 in S2R+ cells using quantitative microscopy and fluorescence recovery after photobleaching, in combination with novel fluorescent cell cycle markers. As revealed by the observed protein abundances and mobilities, centromeres proceed through at least five distinct states during the cell cycle, distinguished in part by unexpected Cid behavior. In addition to the predominant Cid loading onto centromeres during G1, a considerable but transient increase was detected during early mitosis. A low level of Cid loading was detected in late S and G2, starting at the reported time of centromere DNA replication. Our results reveal the complexities of Drosophila centromere protein dynamics and its intricate coordination with cell cycle progression.

  5. Genetic control and expression of the major ejaculatory bulb protein (PEB-me) in Drosophila melanogaster.

    PubMed

    Ludwig, M Z; Uspensky, I I; Ivanov, A I; Kopantseva, M R; Dianov, C M; Tamarina, N A; Korochkin, L I

    1991-06-01

    PEB-me is a predominant protein of mature Drosophila melanogaster ejaculatory bulbs. It is resolved into four or five closely spaced subfractions (apparent molecular weight 35-39 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four electrophoretic variants of PEB-me differing in apparent molecular weight by 200-800 daltons were found. These appear to be controlled by four alleles of a gene (peb) located by recombination and deletion mapping to the 60F1-2 region of chromosome 2. A minor ejaculatory bulb protein of ca. 80 kD (hPEB) was found to be immunochemically related to PEB and possibly encoded by peb. PEB is not detected by immunoblotting techniques in virgin females, in male tissues other than the ejaculatory bulb, or during developmental stages preceding the formation of this organ. The results of transplantations of genital imaginal discs and of immature ejaculatory bulbs between two strains having different PEB alleles suggest that the ejaculatory bulb is the site of PEB synthesis. In flies mutant for tra, tra-2, dsx, or ix, tissue specificity of PEB localization is retained and the protein is found whenever the ejaculatory bulb is formed, regardless of the chromosomal sex of the fly. The protein is transferred into the female genital duct during mating, where it can be detected for up to 12 hr. Possible functions of PEB in Drosophila reproduction are discussed.

  6. Structure and expression of the Drosophila ubiquitin-80-amino-acid fusion-protein gene.

    PubMed Central

    Barrio, R; del Arco, A; Cabrera, H L; Arribas, C

    1994-01-01

    In the fruitfly Drosophila, as in all eukaryotes examined so far, some ubiquitin-coding sequences appear fused to unrelated open reading frames. Two of these fusion genes have been previously described (the homologues of UBI1-UBI2 and UBI4 in yeast), and we report here the organization and expression of a third one, the DUb80 gene (the homologue of UBI3 in yeast). This gene encodes a ubiquitin monomer fused to an 80-amino-acid extension which is homologous with the ribosomal protein encoded by the UB13 gene. The 5' regulatory region of DUb80 shares common features with another ubiquitin fusion gene, DUb52, and with the ribosomal protein genes of Drosophila, Xenopus and mouse. We also find helix-loop-helix protein-binding sequences (E-boxes). The DUb80 gene is transcribed to a 0.9 kb mRNA which is particularly abundant under conditions of high protein synthesis, such as in ovaries and exponentially growing cells. Images Figure 3 Figure 4 PMID:8068011

  7. Developmental and cell biological functions of the Drosophila DEAD-box protein abstrakt.

    PubMed

    Irion, U; Leptin, M

    1999-12-02

    DEAD-box proteins are a large family of proteins found in bacteria, plants and animals, but only few have been analysed functionally. They are involved in the regulation of various aspects of RNA processing and metabolism, including splicing, transport and translation. The study of their function in multicellular organisms has been restricted to a few special cases, such as the Vasa protein in the fruit fly Drosophila. We show that abstrakt, a gene originally identified genetically by its effect on axon outgrowth and fasciculation of the Bolwig nerve, encodes a new Drosophila DEAD-box protein of which the closest homologue is a human gene of unknown function. Using temperature-sensitive alleles to assay its function, we found that abstrakt is essential for survival at all stages throughout the life cycle of the fly. Mutants show specific defects in many developmental processes, including cell-shape changes, localisation of RNA and apoptosis. Abstrakt is not globally required for RNA splicing, transport, subcellular localisation or translation. Nevertheless, there is a widespread requirement for Abstrakt during post-transcriptional gene expression. Abstrakt must affect processing of specific subsets of RNAs, suggesting that differential post-translational control during development is more common than previously suspected.

  8. Multifunctional RNA Processing Protein SRm160 Induces Apoptosis and Regulates Eye and Genital Development in Drosophila

    PubMed Central

    Fan, Yu-Jie; Gittis, Aryn H.; Juge, François; Qiu, Chen; Xu, Yong-Zhen; Rabinow, Leonard

    2014-01-01

    SRm160 is an SR-like protein implicated in multiple steps of RNA processing and nucleocytoplasmic export. Although its biochemical functions have been extensively described, its genetic interactions and potential participation in signaling pathways remain largely unknown, despite the fact that it is highly phosphorylated in both mammalian cells and Drosophila. To begin elucidating the functions of the protein in signaling and its potential role in developmental processes, we characterized mutant and overexpression SRm160 phenotypes in Drosophila and their interactions with the locus encoding the LAMMER protein kinase, Doa. SRm160 mutations are recessive lethal, while its overexpression generates phenotypes including roughened eyes and highly disorganized internal eye structure, which are due at least in part to aberrantly high levels of apoptosis. SRm160 is required for normal somatic sex determination, since its alleles strongly enhance a subtle sex transformation phenotype induced by Doa kinase alleles. Moreover, modification of SRm160 by DOA kinase appears to be necessary for its activity, since Doa alleles suppress phenotypes induced by SRm160 overexpression in the eye and enhance those in genital discs. Modification of SRm160 may occur through direct interaction because DOA kinase phosphorylates it in vitro. Remarkably, SRm160 protein was concentrated in the nuclei of precellular embryos but was very rapidly excluded from nuclei or degraded coincident with cellularization. Also of interest, transcripts are restricted almost exclusively to the developing nervous system in mature embryos. PMID:24907259

  9. Expression of dengue virus NS3 protein in Drosophila alters its susceptibility to infection

    PubMed Central

    Querenet, Matthieu; Danjoy, Marie-Laure; Mollereau, Bertrand; Davoust, Nathalie

    2015-01-01

    We developed a Drosophila model in which the dengue virus NS3 protein is expressed in a tissue specific and inducible manner. Dengue virus NS3 is a multifunctional protein playing a major role during viral replication. Both protease and helicase domains of NS3 are interacting with human and insect host proteins including innate immune components of the host machinery. We characterized the NS3 transgenic flies showing that NS3 expression did not affect fly development. To further study the links between NS3 and the innate immune response, we challenge the flies with gram-positive and gram-negative bacteria. Interestingly, the Drosophila transgenic flies expressing NS3 were more susceptible to bacterial infections than control flies. However ubiquitous or immune-specific NS3 expression affected neither the life span nor the response to a non-infectious stress of the flies. In conclusion, we generated a new in vivo system to study the functional impact of DENV NS3 protein on the innate immune response. PMID:26267447

  10. The Drosophila suppressor of underreplication protein binds to late-replicating regions of polytene chromosomes.

    PubMed Central

    Makunin, I V; Volkova, E I; Belyaeva, E S; Nabirochkina, E N; Pirrotta, V; Zhimulev, I F

    2002-01-01

    In many late-replicating euchromatic regions of salivary gland polytene chromosomes, DNA is underrepresented. A mutation in the SuUR gene suppresses underreplication and leads to normal levels of DNA polytenization in these regions. We identified the SuUR gene and determined its structure. In the SuUR mutant stock a 6-kb insertion was found in the fourth exon of the gene. A single SuUR transcript is present at all stages of Drosophila development and is most abundant in adult females and embryos. The SuUR gene encodes a protein of 962 amino acids whose putative sequence is similar to the N-terminal part of SNF2/SWI2 proteins. Staining of salivary gland polytene chromosomes with antibodies directed against the SuUR protein shows that the protein is localized mainly in late-replicating regions and in regions of intercalary and pericentric heterochromatin. PMID:11901119

  11. Drosophila photoreceptor axon guidance and targeting requires the dreadlocks SH2/SH3 adapter protein.

    PubMed

    Garrity, P A; Rao, Y; Salecker, I; McGlade, J; Pawson, T; Zipursky, S L

    1996-05-31

    Mutations in the Drosophila gene dreadlocks (dock) disrupt photoreceptor cell (R cell) axon guidance and targeting. Genetic mosaic analysis and cell-type-specific expression of dock transgenes demonstrate dock is required in R cells for proper innervation. Dock protein contains one SH2 and three SH3 domains, implicating it in tyrosine kinase signaling, and is highly related to the human proto-oncogene Nck. Dock expression is detected in R cell growth cones in the target region. We propose Dock transmits signals in the growth cone in response to guidance and targeting cues. These findings provide an important step for dissection of signaling pathways regulating growth cone motility.

  12. Characterization of the major hnRNP proteins from Drosophila melanogaster

    PubMed Central

    1992-01-01

    To better understand the role(s) of hnRNP proteins in the process of mRNA formation, we have identified and characterized the major nuclear proteins that interact with hnRNAs in Drosophila melanogaster. cDNA clones of several D. melanogaster hnRNP proteins have been isolated and sequenced, and the genes encoding these proteins have been mapped cytologically on polytene chromosomes. These include the hnRNP proteins hrp36, hrp40, and hrp48, which together account for the major proteins of hnRNP complexes in D. melanogaster (Matunis et al., 1992, accompanying paper). All of the proteins described here contain two amino-terminal RNP consensus sequence RNA-binding domains and a carboxyl-terminal glycine-rich domain. We refer to this configuration, which is also found in the hnRNP A/B proteins of vertebrates, as 2 x RBD-Gly. The sequences of the D. melanogaster hnRNP proteins help define both highly conserved and variable amino acids within each RBD and support the suggestion that each RBD in multiple RBD-containing proteins has been conserved independently and has a different function. Although 2 x RBD-Gly proteins from evolutionarily distant organisms are conserved in their general structure, we find a surprising diversity among the members of this family of proteins. A mAb to the hrp40 proteins crossreacts with the human A/B and G hnRNP proteins and detects immunologically related proteins in divergent organisms from yeast to man. These data establish 2 x RBD-Gly as a prevalent hnRNP protein structure across eukaryotes. This information about the composition of hnRNP complexes and about the structure of hnRNA-binding proteins will facilitate studies of the functions of these proteins. PMID:1730754

  13. The Drosophila Homologue of the Amyloid Precursor Protein Is a Conserved Modulator of Wnt PCP Signaling

    PubMed Central

    Soldano, Alessia; Okray, Zeynep; Janovska, Pavlina; Tmejová, Kateřina; Reynaud, Elodie; Claeys, Annelies; Yan, Jiekun; Atak, Zeynep Kalender; De Strooper, Bart; Dura, Jean-Maurice; Bryja, Vítězslav; Hassan, Bassem A.

    2013-01-01

    Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. We therefore investigated the role of Drosophila APPL during brain development. We find that APPL is involved in the development of the Mushroom Body αβ neurons and, in particular, is required cell-autonomously for the β-axons and non-cell autonomously for the α-axons growth. Moreover, we find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover, we show that APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) by Abelson kinase (Abl). Taken together our data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth. PMID:23690751

  14. The Arf family G protein Arl1 is required for secretory granule biogenesis in Drosophila

    PubMed Central

    Torres, Isabel L.; Rosa-Ferreira, Cláudia; Munro, Sean

    2014-01-01

    ABSTRACT The small G protein Arf like 1 (Arl1) is found at the Golgi complex, and its GTP-bound form recruits several effectors to the Golgi including GRIP-domain-containing coiled-coil proteins, and the Arf1 exchange factors Big1 and Big2. To investigate the role of Arl1, we have characterised a loss-of-function mutant of the Drosophila Arl1 orthologue. The gene is essential, and examination of clones of cells lacking Arl1 shows that it is required for recruitment of three of the four GRIP domain golgins to the Golgi, with Drosophila GCC185 being less dependent on Arl1. At a functional level, Arl1 is essential for formation of secretory granules in the larval salivary gland. When Arl1 is missing, Golgi are still present but there is a dispersal of adaptor protein 1 (AP-1), a clathrin adaptor that requires Arf1 for its membrane recruitment and which is known to be required for secretory granule biogenesis. Arl1 does not appear to be required for AP-1 recruitment in all tissues, suggesting that it is crucially required to enhance Arf1 activation at the trans-Golgi in particular tissues. PMID:24610947

  15. Codon usage affects the structure and function of the Drosophila circadian clock protein PERIOD

    PubMed Central

    Fu, Jingjing; Murphy, Katherine A.; Zhou, Mian; Li, Ying H.; Lam, Vu H.; Tabuloc, Christine A.; Chiu, Joanna C.; Liu, Yi

    2016-01-01

    Codon usage bias is a universal feature of all genomes, but its in vivo biological functions in animal systems are not clear. To investigate the in vivo role of codon usage in animals, we took advantage of the sensitivity and robustness of the Drosophila circadian system. By codon-optimizing parts of Drosophila period (dper), a core clock gene that encodes a critical component of the circadian oscillator, we showed that dper codon usage is important for circadian clock function. Codon optimization of dper resulted in conformational changes of the dPER protein, altered dPER phosphorylation profile and stability, and impaired dPER function in the circadian negative feedback loop, which manifests into changes in molecular rhythmicity and abnormal circadian behavioral output. This study provides an in vivo example that demonstrates the role of codon usage in determining protein structure and function in an animal system. These results suggest a universal mechanism in eukaryotes that uses a codon usage “code” within genetic codons to regulate cotranslational protein folding. PMID:27542830

  16. The Drosophila homologue of the amyloid precursor protein is a conserved modulator of Wnt PCP signaling.

    PubMed

    Soldano, Alessia; Okray, Zeynep; Janovska, Pavlina; Tmejová, Kateřina; Reynaud, Elodie; Claeys, Annelies; Yan, Jiekun; Atak, Zeynep Kalender; De Strooper, Bart; Dura, Jean-Maurice; Bryja, Vítězslav; Hassan, Bassem A

    2013-01-01

    Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. We therefore investigated the role of Drosophila APPL during brain development. We find that APPL is involved in the development of the Mushroom Body αβ neurons and, in particular, is required cell-autonomously for the β-axons and non-cell autonomously for the α-axons growth. Moreover, we find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover, we show that APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) by Abelson kinase (Abl). Taken together our data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth.

  17. Revisiting the protein-coding gene catalog of Drosophila melanogaster using 12 fly genomes

    PubMed Central

    Lin, Michael F.; Carlson, Joseph W.; Crosby, Madeline A.; Matthews, Beverley B.; Yu, Charles; Park, Soo; Wan, Kenneth H.; Schroeder, Andrew J.; Gramates, L. Sian; St. Pierre, Susan E.; Roark, Margaret; Wiley, Kenneth L.; Kulathinal, Rob J.; Zhang, Peili; Myrick, Kyl V.; Antone, Jerry V.; Celniker, Susan E.; Gelbart, William M.; Kellis, Manolis

    2007-01-01

    The availability of sequenced genomes from 12 Drosophila species has enabled the use of comparative genomics for the systematic discovery of functional elements conserved within this genus. We have developed quantitative metrics for the evolutionary signatures specific to protein-coding regions and applied them genome-wide, resulting in 1193 candidate new protein-coding exons in the D. melanogaster genome. We have reviewed these predictions by manual curation and validated a subset by directed cDNA screening and sequencing, revealing both new genes and new alternative splice forms of known genes. We also used these evolutionary signatures to evaluate existing gene annotations, resulting in the validation of 87% of genes lacking descriptive names and identifying 414 poorly conserved genes that are likely to be spurious predictions, noncoding, or species-specific genes. Furthermore, our methods suggest a variety of refinements to hundreds of existing gene models, such as modifications to translation start codons and exon splice boundaries. Finally, we performed directed genome-wide searches for unusual protein-coding structures, discovering 149 possible examples of stop codon readthrough, 125 new candidate ORFs of polycistronic mRNAs, and several candidate translational frameshifts. These results affect >10% of annotated fly genes and demonstrate the power of comparative genomics to enhance our understanding of genome organization, even in a model organism as intensively studied as Drosophila melanogaster. PMID:17989253

  18. Genes for Drosophila small heat shock proteins are regulated differently by ecdysterone

    SciTech Connect

    Amin, J.; Voellmy, R. ); Mestril, R. )

    1991-12-01

    Genes for small heat shock proteins (hsp27 to hsp22) are activated in late third-instar larvae of Drosophila melanogaster in the absence of heat stress. This regulation has been stimulated in cultured Drosophila cells in which the genes are activated by the addition of ecdysterone. Sequence elements (HERE) involved in ecdysterone regulation of the hsp27 and hsp23 genes have been defined by transfection studies and have recently been identified as binding sites for ecdysterone receptor. The authors report here that the shp27 and hsp23 genes are regulated differently by ecdysterone. The hsp27 gene is activated rapidly by ecdysterone, even in the absence of protein synthesis. In contrast, high-level expression of the hsp23 gene begins only after a lag of about 6 h, is dependent on the continuous presence of ecdysterone, and is sensitive to low concentrations of protein synthesis inhibitors. Transfection experiments with reported constructs show that this difference in regulation is at the transcriptional level. Synthetic hsp27 or hsp23 HERE sequences confer hsp27- or hsp23-type ecdysterone regulation on a basal promoter. These findings indicate that the hsp27 gene is primary, and the hsp23 gene is mainly a secondary, hormone-responsive gene. Ecdysterone receptor is implied to play a role in the regulation of both genes.

  19. Codon usage affects the structure and function of the Drosophila circadian clock protein PERIOD.

    PubMed

    Fu, Jingjing; Murphy, Katherine A; Zhou, Mian; Li, Ying H; Lam, Vu H; Tabuloc, Christine A; Chiu, Joanna C; Liu, Yi

    2016-08-01

    Codon usage bias is a universal feature of all genomes, but its in vivo biological functions in animal systems are not clear. To investigate the in vivo role of codon usage in animals, we took advantage of the sensitivity and robustness of the Drosophila circadian system. By codon-optimizing parts of Drosophila period (dper), a core clock gene that encodes a critical component of the circadian oscillator, we showed that dper codon usage is important for circadian clock function. Codon optimization of dper resulted in conformational changes of the dPER protein, altered dPER phosphorylation profile and stability, and impaired dPER function in the circadian negative feedback loop, which manifests into changes in molecular rhythmicity and abnormal circadian behavioral output. This study provides an in vivo example that demonstrates the role of codon usage in determining protein structure and function in an animal system. These results suggest a universal mechanism in eukaryotes that uses a codon usage "code" within genetic codons to regulate cotranslational protein folding. © 2016 Fu et al.; Published by Cold Spring Harbor Laboratory Press.

  20. Gudu, an Armadillo repeat-containing protein, is required for spermatogenesis in Drosophila.

    PubMed

    Cheng, Wei; Ip, Y Tony; Xu, Zuoshang

    2013-12-01

    The Drosophila annotated gene CG5155 encodes a protein that contains 10 Armadillo-repeats and has an unknown function. To fill this gap, we performed loss-of-function studies using RNAi. By analysis of four independent Drosophila RNAi lines targeting two non-overlapping regions of the CG5155 transcript, we demonstrate that this gene is required for male fertility. Therefore, we have named this gene Gudu. The transcript of Gudu is highly enriched in adult testes. Knockdown of Gudu by a ubiquitous driver leads to defects in the formation of the individualization complex that is required for spermatid maturation, thereby impairing spermatogenesis. Furthermore, testis-specific knockdown of Gudu by crossing the RNAi lines with the bam-Gal4 driver is sufficient to cause the infertility and defective spermatogenesis. Since Gudu is highly homologous to vertebrate ARMC4, also an Armadillo-repeat-containing protein enriched in testes, our results suggest that Gudu and ARMC4 are a subfamily of Armadillo-repeat containing proteins that may have an evolutionarily conserved function in spermatogenesis.

  1. Protein composition of interband regions in polytene and cell line chromosomes of Drosophila melanogaster

    PubMed Central

    2011-01-01

    Background Despite many efforts, little is known about distribution and interactions of chromatin proteins which contribute to the specificity of chromomeric organization of interphase chromosomes. To address this issue, we used publicly available datasets from several recent Drosophila genome-wide mapping and annotation projects, in particular, those from modENCODE project, and compared molecular organization of 13 interband regions which were accurately mapped previously. Results Here we demonstrate that in interphase chromosomes of Drosophila cell lines, the interband regions are enriched for a specific set of proteins generally characteristic of the "open" chromatin (RNA polymerase II, CHRIZ (CHRO), BEAF-32, BRE1, dMI-2, GAF, NURF301, WDS and TRX). These regions also display reduced nucleosome density, histone H1 depletion and pronounced enrichment for ORC2, a pre-replication complex component. Within the 13 interband regions analyzed, most were around 3-4 kb long, particularly those where many of said protein features were present. We estimate there are about 3500 regions with similar properties in chromosomes of D. melanogaster cell lines, which fits quite well the number of cytologically observed interbands in salivary gland polytene chromosomes. Conclusions Our observations suggest strikingly similar organization of interband chromatin in polytene chromosomes and in chromosomes from cell lines thereby reflecting the existence of a universal principle of interphase chromosome organization. PMID:22093916

  2. Developmental Roles of the Mi-2/NURD-Associated Protein p66 in Drosophila

    PubMed Central

    Kon, Charlene; Cadigan, Kenneth M.; da Silva, Sofia Lopes; Nusse, Roel

    2005-01-01

    The NURD and Sin3 histone deacetylase complexes are involved in transcriptional repression through global deacetylation of chromatin. Both complexes contain many different components that may control how histone deacetylase complexes are regulated and interact with other transcription factors. In a genetic screen for modifiers of wingless signaling in the Drosophila eye, we isolated mutations in the Drosophila homolog of p66, a protein previously purified as part of the Xenopus NURD/Mi-2 complex. p66 encodes a highly conserved nuclear zinc-finger protein that is required for development and we propose that the p66 protein acts as a regulatory component of the NURD complex. Animals homozygous mutant for p66 display defects during metamorphosis possibly caused by misregulation of ecdysone-regulated expression. Although heterozygosity for p66 enhances a wingless phenotype in the eye, loss-of-function clones in the wing and the eye discs do not have any detectable phenotype, possibly due to redundancy with the Sin3 complex. Overexpression of p66, on the other hand, can repress wingless-dependent phenotypes. Furthermore, p66 expression can repress multiple reporters in a cell culture assay, including a Wnt-responsive TCF reporter construct, implicating the NURD complex in repression of Wnt target genes. By co-immunoprecipitation, p66 associates with dMi-2, a known NURD complex member. PMID:15695365

  3. Characterization of the Drosophila BEAF-32A and BEAF-32B Insulator Proteins

    PubMed Central

    Avva, S. V. Satya Prakash

    2016-01-01

    Data implicate the Drosophila 32 kDa Boundary Element-Associated Factors BEAF-32A and BEAF-32B in both chromatin domain insulator element function and promoter function. They might also function as an epigenetic memory by remaining bound to mitotic chromosomes. Both proteins are made from the same gene. They differ in their N-terminal 80 amino acids, which contain single DNA-binding BED fingers. The remaining 200 amino acids are identical in the two proteins. The structure and function of the middle region of 120 amino acids is unknown, while the C-terminal region of 80 amino acids has a putative leucine zipper and a BESS domain and mediates BEAF-BEAF interactions. Here we report a further characterization of BEAF. We show that the BESS domain alone is sufficient to mediate BEAF-BEAF interactions, although the presence of the putative leucine zipper on at least one protein strengthens the interactions. BEAF-32B is sufficient to rescue a null BEAF mutation in flies. Using mutant BEAF-32B rescue transgenes, we show that the middle region and the BESS domain are essential. In contrast, the last 40 amino acids of the middle region, which is poorly conserved among Drosophila species, is dispensable. Deleting the putative leucine zipper results in a hypomorphic mutant BEAF-32B protein. Finally, we document the dynamics of BEAF-32A-EGFP and BEAF-32B-mRFP during mitosis in embryos. A subpopulation of both proteins appears to remain on mitotic chromosomes and also on the mitotic spindle, while much of the fluorescence is dispersed during mitosis. Differences in the dynamics of the two proteins are observed in syncytial embryos, and both proteins show differences between syncytial and later embryos. This characterization of BEAF lays a foundation for future studies into molecular mechanisms of BEAF function. PMID:27622635

  4. Protein Phosphatase 4 mediates localization of the Miranda complex during Drosophila neuroblast asymmetric divisions

    PubMed Central

    Sousa-Nunes, Rita; Chia, William; Somers, W. Greg

    2009-01-01

    Asymmetric localization of cell fate determinants is a crucial step in neuroblast asymmetric divisions. Whereas several protein kinases have been shown to mediate this process, no protein phosphatase has so far been implicated. In a clonal screen of larval neuroblasts we identified the evolutionarily conserved Protein Phosphatase 4 (PP4) regulatory subunit PP4R3/Falafel (Flfl) as a key mediator specific for the localization of Miranda (Mira) and associated cell fate determinants during both interphase and mitosis. Flfl is predominantly nuclear during interphase/prophase and cytoplasmic after nuclear envelope breakdown. Analyses of nuclear excluded as well as membrane targeted versions of the protein suggest that the asymmetric cortical localization of Mira and its associated proteins during mitosis depends on cytoplasmic/membrane-associated Flfl, whereas nuclear Flfl is required to exclude the cell fate determinant Prospero (Pros), and consequently Mira, from the nucleus during interphase/prophase. Attenuating the function of either the catalytic subunit of PP4 (PP4C; Pp4-19C in Drosophila) or of another regulatory subunit, PP4R2 (PPP4R2r in Drosophila), leads to similar defects in the localization of Mira and associated proteins. Flfl is capable of directly interacting with Mira, and genetic analyses indicate that flfl acts in parallel to or downstream from the tumor suppressor lethal (2) giant larvae (lgl). Our findings suggest that Flfl may target PP4 to the MIra protein complex to facilitate dephosphorylation step(s) crucial for its cortical association/asymmetric localization. PMID:19204120

  5. Actin capping protein alpha maintains vestigial-expressing cells within the Drosophila wing disc epithelium.

    PubMed

    Janody, Florence; Treisman, Jessica E

    2006-09-01

    Tissue patterning must be translated into morphogenesis through cell shape changes mediated by remodeling of the actin cytoskeleton. We have found that Capping protein alpha (Cpa) and Capping protein beta (Cpb), which prevent extension of the barbed ends of actin filaments, are specifically required in the wing blade primordium of the Drosophila wing disc. cpa or cpb mutant cells in this region, but not in the remainder of the wing disc, are extruded from the epithelium and undergo apoptosis. Excessive actin filament polymerization is not sufficient to explain this phenotype, as loss of Cofilin or Cyclase-associated protein does not cause cell extrusion or death. Misexpression of Vestigial, the transcription factor that specifies the wing blade, both increases cpa transcription and makes cells dependent on cpa for their maintenance in the epithelium. Our results suggest that Vestigial specifies the cytoskeletal changes that lead to morphogenesis of the adult wing.

  6. Characterization and gene cloning of neurotactin, a Drosophila transmembrane protein related to cholinesterases.

    PubMed Central

    de la Escalera, S; Bockamp, E O; Moya, F; Piovant, M; Jiménez, F

    1990-01-01

    Monoclonal antibodies have served to characterize neurotactin, a novel Drosophila protein for which a role in cell adhesion is postulated. Neurotactin is a transmembrane protein, as indicated by epitope mapping and amino acid sequence. Similarly to other cell adhesion molecules, neurotactin accumulates in parts of the membrane where neurotactin-expressing cells contact each other. The protein is only detected during cell proliferation and differentiation, and it is found mainly in neural tissue and also in mesoderm and imaginal discs. Neurotactin has a large cytoplasmic domain rich in charged residues and an extracellular domain similar to cholinesterase that lacks the active site serine required for esterase activity. The extracellular domain also contains three copies of the tripeptide leucine-arginine-glutamate, a motif that forms the primary sequence of the adhesive site of vertebrate s-laminin. Images Fig.1 Fig.2 Fig.3 Fig.4 Fig.5 Fig.6 PMID:2120047

  7. Characterization and gene cloning of neurotactin, a Drosophila transmembrane protein related to cholinesterases.

    PubMed

    de la Escalera, S; Bockamp, E O; Moya, F; Piovant, M; Jiménez, F

    1990-11-01

    Monoclonal antibodies have served to characterize neurotactin, a novel Drosophila protein for which a role in cell adhesion is postulated. Neurotactin is a transmembrane protein, as indicated by epitope mapping and amino acid sequence. Similarly to other cell adhesion molecules, neurotactin accumulates in parts of the membrane where neurotactin-expressing cells contact each other. The protein is only detected during cell proliferation and differentiation, and it is found mainly in neural tissue and also in mesoderm and imaginal discs. Neurotactin has a large cytoplasmic domain rich in charged residues and an extracellular domain similar to cholinesterase that lacks the active site serine required for esterase activity. The extracellular domain also contains three copies of the tripeptide leucine-arginine-glutamate, a motif that forms the primary sequence of the adhesive site of vertebrate s-laminin.

  8. Toucan protein is essential for the assembly of syncytial mitotic spindles in Drosophila melanogaster.

    PubMed

    Debec, A; Grammont, M; Berson, G; Dastugue, B; Sullivan, W; Couderc, J L

    2001-12-01

    The toc gene of Drosophila melanogaster encodes a 235-kD polypeptide with a coiled-coil domain, which is highly expressed during oogenesis (Grammont et al., 1997, 2000). We now report the localization of the Toucan protein during early embryonic development. The Toucan protein is present only during the syncytial stages and is associated with the nuclear envelope and the cytoskeletal structures of the syncytial embryo. In anaphase A, Toucan is concentrated at the spindle poles near the minus end of microtubules. This microtubule association is very dynamic during the nuclear cell cycle. Mutant embryos lacking the Toucan protein are blocked in a metaphase-like state. They display abnormal and nonfunctional spindles, characterized by broad poles, detachment of the centrosomes, and failure of migration of the chromosomes. These results strongly suggest that Toucan represents a factor essential for the assembly and the function of the syncytial mitotic spindles.

  9. Tafazzinsfrom Drosophila and Mammalian Cells Assemble in Large Protein Complexes with a Short Half-Life

    PubMed Central

    Xu, Yang; Malhotra, Ashim; Claypool, Steven M.; Ren, Mindong; Schlame, Michael

    2015-01-01

    Tafazzin is a transacylase that affects cardiolipin fatty acid composition and mitochondrial function. Mutations in human tafazzin cause Barth syndrome yet the enzyme has mostly been characterized in yeast. To study tafazzin in higher organisms, we isolated mitochondria from Drosophila and mammalian cell cultures. Our data indicate that tafazzin binds to multiple protein complexes in these organisms, and that the interactions of tafazzin lack strong specificity. Very large tafazzin complexes could only be detected in the presence of cardiolipin, but smaller complexes remained intact even upon treatment with phospholipase A2. In mammalian cells, tafazzin had a half-life of only 3–6 h, which was much shorter than the half-life of other mitochondrial proteins. The data suggest that tafazzin is a transient resident of multiple protein complexes. PMID:25598000

  10. Flagellar mitochondrial association of the male-specific Don Juan protein in Drosophila spermatozoa.

    PubMed

    Santel, A; Blümer, N; Kämpfer, M; Renkawitz-Pohl, R

    1998-11-01

    The Drosophila don juan gene encodes a basic protein (Don Juan protein), which is solely expressed postmeiotically during spermiogenesis in elongated spermatids and in mature sperm. Transgenic expression of a GFP-tagged Don Juan protein (DJ-GFP) in the male germ line showed an association of the fusion protein with the sperm tail. Detailed examination of DJ-GFP localization revealed novel insights into its distinct temporal and spatial distribution along the sperm tail during the last phase of spermatid maturation. Co-localization of DJ-GFP with actin-labeled cysts demonstrated its emergence in elongated spermatids during individualization. Additionally, the endogenous Don Juan protein was detected with epitope-specific antibodies in finally elongated nuclei of spermatids. After completion of nuclear shaping Don Juan is no longer detectable in the sperm heads with the onset of individualization. Mislocalization of the DJ-GFP protein in flagella of a mutant with defective mitochondrial differentiation provides evidence of mitochondrial association of the fusion protein with flagellar mitochondrial arrays. Ectopically expressed DJ-GFP in premeiotic germ cells as well as salivary gland cells confirmed the capability of the fusion protein to associate with mitochondria. Therefore we suppose that Don Juan is a nuclear-encoded, germ-cell specifically expressed mitochondrial protein, which might be involved in the final steps of mitochondrial differentiation within the flagellum.

  11. Involvement of a tissue-specific RNA recognition motif protein in Drosophila spermatogenesis.

    PubMed Central

    Haynes, S R; Cooper, M T; Pype, S; Stolow, D T

    1997-01-01

    RNA binding proteins mediate posttranscriptional regulation of gene expression via their roles in nuclear and cytoplasmic mRNA metabolism. Many of the proteins involved in these processes have a common RNA binding domain, the RNA recognition motif (RRM). We have characterized the Testis-specific RRM protein gene (Tsr), which plays an important role in spermatogenesis in Drosophila melanogaster. Disruption of Tsr led to a dramatic reduction in male fertility due to the production of spermatids with abnormalities in mitochondrial morphogenesis. Tsr is located on the third chromosome at 87F, adjacent to the nuclear pre-mRNA binding protein gene Hrb87F. A 1.7-kb Tsr transcript was expressed exclusively in the male germ line. It encoded a protein containing two RRMs similar to those found in HRB87F as well as a unique C-terminal domain. TSR protein was located in the cytoplasm of spermatocytes and young spermatids but was absent from mature sperm. The cellular proteins expressed in premeiotic primary spermatocytes from Tsr mutant and wild-type males were assessed by two-dimensional gel electrophoresis. Lack of TSR resulted in the premature expression of a few proteins prior to meiosis; this was abolished by a transgenic copy of Tsr. These data demonstrate that TSR negatively regulated the expression of some testis proteins and, in combination with its expression pattern and subcellular localization, suggest that TSR regulates the stability or translatability of some mRNAs during spermatogenesis. PMID:9111341

  12. Characterization of fragile X mental retardation protein recruitment and dynamics in Drosophila stress granules.

    PubMed

    Gareau, Cristina; Houssin, Elise; Martel, David; Coudert, Laetitia; Mellaoui, Samia; Huot, Marc-Etienne; Laprise, Patrick; Mazroui, Rachid

    2013-01-01

    The RNA-binding protein Fragile X Mental Retardation (FMRP) is an evolutionarily conserved protein that is particularly abundant in the brain due to its high expression in neurons. FMRP deficiency causes fragile X mental retardation syndrome. In neurons, FMRP controls the translation of target mRNAs in part by promoting dynamic transport in and out neuronal RNA granules. We and others have previously shown that upon stress, mammalian FMRP dissociates from translating polysomes to localize into neuronal-like granules termed stress granules (SG). This localization of FMRP in SG is conserved in Drosophila. Whether FMRP plays a key role in SG formation, how FMRP is recruited into SG, and whether its association with SG is dynamic are currently unknown. In contrast with mammalian FMRP, which has two paralog proteins, Drosophila FMR1 (dFMRP) is encoded by a single gene that has no paralog. Using this genetically simple model, we assessed the role of dFMRP in SG formation and defined the determinants required for its recruitment in SG as well as its dynamics in SG. We show that dFMRP is dispensable for SG formation in vitro and ex vivo. FRAP experiments showed that dFMRP shuttles in and out SG. The shuttling activity of dFMRP is mediated by a protein-protein interaction domain located at the N-terminus of the protein. This domain is, however, dispensable for the localization of dFMRP in SG. This localization of dFMRP in SG requires the KH and RGG motifs which are known to mediate RNA binding, as well as the C-terminal glutamine/asparagine rich domain. Our studies thus suggest that the mechanisms controlling the recruitment of FMRP into SG and those that promote its shuttling between granules and the cytosol are uncoupled. To our knowledge, this is the first demonstration of the regulated shuttling activity of a SG component between RNA granules and the cytosol.

  13. An Endocytic Scaffolding Protein together with Synapsin Regulates Synaptic Vesicle Clustering in the Drosophila Neuromuscular Junction.

    PubMed

    Winther, Åsa M E; Vorontsova, Olga; Rees, Kathryn A; Näreoja, Tuomas; Sopova, Elena; Jiao, Wei; Shupliakov, Oleg

    2015-11-04

    Many endocytic proteins accumulate in the reserve pool of synaptic vesicles (SVs) in synapses and relocalize to the endocytic periactive zone during neurotransmitter release. Currently little is known about their functions outside the periactive zone. Here we show that in the Drosophila neuromuscular junction (NMJ), the endocytic scaffolding protein Dap160 colocalizes during the SV cycle and forms a functional complex with the SV-associated phosphoprotein synapsin, previously implicated in SV clustering. This direct interaction is strongly enhanced under phosphorylation-promoting conditions and is essential for proper localization of synapsin at NMJs. In a dap160 rescue mutant lacking the interaction between Dap160 and synapsin, perturbed reclustering of SVs during synaptic activity is observed. Our data indicate that in addition to the function in endocytosis, Dap160 is a component of a network of protein-protein interactions that serves for clustering of SVs in conjunction with synapsin. During the SV cycle, Dap160 interacts with synapsin dispersed from SVs and helps direct synapsin back to vesicles. The proteins function in synergy to achieve efficient clustering of SVs in the reserve pool. We provide the first evidence for the function of the SH3 domain interaction in synaptic vesicle (SV) organization at the synaptic active zone. Using Drosophila neuromuscular junction as a model synapse, we describe the molecular mechanism that enables the protein implicated in SV clustering, synapsin, to return to the pool of vesicles during neurotransmitter release. We also identify the endocytic scaffolding complex that includes Dap160 as a regulator of the events linking exocytosis and endocytosis in synapses. Copyright © 2015 the authors 0270-6474/15/3514756-15$15.00/0.

  14. γCOP Is Required for Apical Protein Secretion and Epithelial Morphogenesis in Drosophila melanogaster

    PubMed Central

    Grieder, Nicole C.; Caussinus, Emmanuel; Parker, David S.; Cadigan, Kenneth; Affolter, Markus; Luschnig, Stefan

    2008-01-01

    Background There is increasing evidence that tissue-specific modifications of basic cellular functions play an important role in development and disease. To identify the functions of COPI coatomer-mediated membrane trafficking in Drosophila development, we were aiming to create loss-of-function mutations in the γCOP gene, which encodes a subunit of the COPI coatomer complex. Principal Findings We found that γCOP is essential for the viability of the Drosophila embryo. In the absence of zygotic γCOP activity, embryos die late in embryogenesis and display pronounced defects in morphogenesis of the embryonic epidermis and of tracheal tubes. The coordinated cell rearrangements and cell shape changes during tracheal tube morphogenesis critically depend on apical secretion of certain proteins. Investigation of tracheal morphogenesis in γCOP loss-of-function mutants revealed that several key proteins required for tracheal morphogenesis are not properly secreted into the apical lumen. As a consequence, γCOP mutants show defects in cell rearrangements during branch elongation, in tube dilation, as well as in tube fusion. We present genetic evidence that a specific subset of the tracheal defects in γCOP mutants is due to the reduced secretion of the Zona Pellucida protein Piopio. Thus, we identified a critical target protein of COPI-dependent secretion in epithelial tube morphogenesis. Conclusions/Significance These studies highlight the role of COPI coatomer-mediated vesicle trafficking in both general and tissue-specific secretion in a multicellular organism. Although COPI coatomer is generally required for protein secretion, we show that the phenotypic effect of γCOP mutations is surprisingly specific. Importantly, we attribute a distinct aspect of the γCOP phenotype to the effect on a specific key target protein. PMID:18802472

  15. Structure and expression of the Drosophila melanogaster gene for the U1 small nuclear ribonucleoprotein particle 70K protein.

    PubMed Central

    Mancebo, R; Lo, P C; Mount, S M

    1990-01-01

    A genomic clone encoding the Drosophila U1 small nuclear ribonucleoprotein particle 70K protein was isolated by hybridization with a human U1 small nuclear ribonucleoprotein particle 70K protein cDNA. Southern blot and in situ hybridizations showed that this U1 70K gene is unique in the Drosophila genome, residing at cytological position 27D1,2. Polyadenylated transcripts of 1.9 and 3.1 kilobases were observed. While the 1.9-kilobase mRNA is always more abundant, the ratio of these two transcripts is developmentally regulated. Analysis of cDNA and genomic sequences indicated that these two RNAs encode an identical protein with a predicted molecular weight of 52,879. Comparison of the U1 70K proteins predicted from Drosophila, human, and Xenopus cDNAs revealed 68% amino acid identity in the most amino-terminal 214 amino acids, which include a sequence motif common to many proteins which bind RNA. The carboxy-terminal half is less well conserved but is highly charged and contains distinctive arginine-rich regions in all three species. These arginine-rich regions contain stretches of arginine-serine dipeptides like those found in transformer, transformer-2, and suppressor-of-white-apricot proteins, all of which have been identified as regulators of mRNA splicing in Drosophila melanogaster. Images PMID:1692955

  16. Activity, expression and function of a second Drosophila protein kinase a catalytic subunit gene

    SciTech Connect

    Melendez, A.; Li, W.; Kalderon, D.

    1995-12-01

    The DC2 was isolated previously on the basis of sequence similarity to DC0, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development. 62 refs., 10 figs., 2 tabs.

  17. The role of polyamines in protein-dependent hypoxic tolerance of Drosophila

    PubMed Central

    Vigne, Paul; Frelin, Christian

    2008-01-01

    Background Chronic hypoxia is a major component of ischemic diseases such as stroke or myocardial infarction. Drosophila is more tolerant to hypoxia than most mammalian species. It is considered as a useful model organism to identify new mechanisms of hypoxic tolerance. The hypoxic tolerance of flies has previously been reported to be enhanced by low protein diets. This study analyses the mechanisms involved. Results Feeding adult Drosophila on a yeast diet dramatically reduced their longevities under chronic hypoxic conditions (5% O2). Mean and maximum longevities became close to the values observed for starving flies. The action of dietary yeast was mimicked by a whole casein hydrolysate and by anyone of the 20 natural amino acids that compose proteins. It was mimicked by amino acid intermediates of the urea cycle such as L-citrulline and L-ornithine, and by polyamines (putrescine, spermidine and spermine). α-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, partially protected hypoxic flies from amino acid toxicity but not from polyamine toxicity. N1-guanyl-1,7 diaminoheptane, a specific inhibitor of eIF5A hypusination, partially relieved the toxicities of both amino acids and polyamines. Conclusion Dietary amino acids reduced the longevity of chronically hypoxic flies fed on a sucrose diet. Pharmacological evidence suggests that the synthesis of polyamines and the hypusination of eIF5A contributed to the life-shortening effect of dietary amino acids. PMID:19055734

  18. The role of polyamines in protein-dependent hypoxic tolerance of Drosophila.

    PubMed

    Vigne, Paul; Frelin, Christian

    2008-12-02

    Chronic hypoxia is a major component of ischemic diseases such as stroke or myocardial infarction. Drosophila is more tolerant to hypoxia than most mammalian species. It is considered as a useful model organism to identify new mechanisms of hypoxic tolerance. The hypoxic tolerance of flies has previously been reported to be enhanced by low protein diets. This study analyses the mechanisms involved. Feeding adult Drosophila on a yeast diet dramatically reduced their longevities under chronic hypoxic conditions (5% O2). Mean and maximum longevities became close to the values observed for starving flies. The action of dietary yeast was mimicked by a whole casein hydrolysate and by anyone of the 20 natural amino acids that compose proteins. It was mimicked by amino acid intermediates of the urea cycle such as L-citrulline and L-ornithine, and by polyamines (putrescine, spermidine and spermine). alpha-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, partially protected hypoxic flies from amino acid toxicity but not from polyamine toxicity. N1-guanyl-1,7 diaminoheptane, a specific inhibitor of eIF5A hypusination, partially relieved the toxicities of both amino acids and polyamines. Dietary amino acids reduced the longevity of chronically hypoxic flies fed on a sucrose diet. Pharmacological evidence suggests that the synthesis of polyamines and the hypusination of eIF5A contributed to the life-shortening effect of dietary amino acids.

  19. Fatty-Acid Binding Proteins Modulate Sleep and Enhance Long-Term Memory Consolidation in Drosophila

    PubMed Central

    Gerstner, Jason R.; Vanderheyden, William M.; Shaw, Paul J.; Landry, Charles F.; Yin, Jerry C. P.

    2011-01-01

    Sleep is thought to be important for memory consolidation, since sleep deprivation has been shown to interfere with memory processing. However, the effects of augmenting sleep on memory formation are not well known, and testing the role of sleep in memory enhancement has been limited to pharmacological and behavioral approaches. Here we test the effect of overexpressing the brain-type fatty acid binding protein (Fabp7) on sleep and long-term memory (LTM) formation in Drosophila melanogaster. Transgenic flies carrying the murine Fabp7 or the Drosophila homologue dFabp had reduced baseline sleep but normal LTM, while Fabp induction produced increases in both net sleep and LTM. We also define a post-training consolidation “window” that is sufficient for the observed Fabp-mediated memory enhancement. Since Fabp overexpression increases consolidated daytime sleep bouts, these data support a role for longer naps in improving memory and provide a novel role for lipid-binding proteins in regulating memory consolidation concurrently with changes in behavioral state. PMID:21298037

  20. Quercetin ameliorates Aβ toxicity in Drosophila AD model by modulating cell cycle-related protein expression

    PubMed Central

    Kong, Yan; Li, Ke; Fu, Tingting; Wan, Chao; Zhang, Dongdong; Song, Hang; Zhang, Yao; Liu, Na; Gan, Zhenji; Yuan, Liudi

    2016-01-01

    Alzheimer's disease (AD) is a prevalent neurodegenerative disorder characterized by β amyloid (Aβ) deposition and neurofibril tangles. It has been reported that a bioflavonoid, quercetin, could ameliorate AD phenotypes in C. elegans and mice. However, the mechanism underlying the ameliorative effect of quercetin is not fully understood yet. Drosophila models could recapitulate AD-like phenotypes, such as shortened lifespan, impaired locomotive ability as well as defects in learning and memory. So in this study, we investigated the effects of quercetin on AD in Drosophila model and explored the underlying mechanisms. We found quercetin could effectively intervene in AD pathogenesis in vivo. Mechanism study showed quercetin could restore the expression of genes perturbed by Aβ accumulation, such as those involved in cell cycle and DNA replication. Cyclin B, an important cell cycle protein, was chosen to test whether it participated in the AD ameliorative effects of quercetin. We found that cyclin B RNAi in the brain could alleviate AD phenotypes. Taken together, the current study suggested that the neuroprotective effects of quercetin were mediated at least partially by targeting cell cycle-related proteins. PMID:27626494

  1. Quercetin ameliorates Aβ toxicity in Drosophila AD model by modulating cell cycle-related protein expression.

    PubMed

    Kong, Yan; Li, Ke; Fu, Tingting; Wan, Chao; Zhang, Dongdong; Song, Hang; Zhang, Yao; Liu, Na; Gan, Zhenji; Yuan, Liudi

    2016-10-18

    Alzheimer's disease (AD) is a prevalent neurodegenerative disorder characterized by β amyloid (Aβ) deposition and neurofibril tangles. It has been reported that a bioflavonoid, quercetin, could ameliorate AD phenotypes in C. elegans and mice. However, the mechanism underlying the ameliorative effect of quercetin is not fully understood yet. Drosophila models could recapitulate AD-like phenotypes, such as shortened lifespan, impaired locomotive ability as well as defects in learning and memory. So in this study, we investigated the effects of quercetin on AD in Drosophila model and explored the underlying mechanisms. We found quercetin could effectively intervene in AD pathogenesis in vivo. Mechanism study showed quercetin could restore the expression of genes perturbed by Aβ accumulation, such as those involved in cell cycle and DNA replication. Cyclin B, an important cell cycle protein, was chosen to test whether it participated in the AD ameliorative effects of quercetin. We found that cyclin B RNAi in the brain could alleviate AD phenotypes. Taken together, the current study suggested that the neuroprotective effects of quercetin were mediated at least partially by targeting cell cycle-related proteins.

  2. SLOB, a SLOWPOKE Channel Binding Protein, Regulates Insulin Pathway Signaling and Metabolism in Drosophila

    PubMed Central

    Sheldon, Amanda L.; Zhang, Jiaming; Fei, Hong; Levitan, Irwin B.

    2011-01-01

    There is ample evidence that ion channel modulation by accessory proteins within a macromolecular complex can regulate channel activity and thereby impact neuronal excitability. However, the downstream consequences of ion channel modulation remain largely undetermined. The Drosophila melanogaster large conductance calcium-activated potassium channel SLOWPOKE (SLO) undergoes modulation via its binding partner SLO-binding protein (SLOB). Regulation of SLO by SLOB influences the voltage dependence of SLO activation and modulates synaptic transmission. SLO and SLOB are expressed especially prominently in median neurosecretory cells (mNSCs) in the pars intercerebralis (PI) region of the brain; these cells also express and secrete Drosophila insulin like peptides (dILPs). Previously, we found that flies lacking SLOB exhibit increased resistance to starvation, and we reasoned that SLOB may regulate aspects of insulin signaling and metabolism. Here we investigate the role of SLOB in metabolism and find that slob null flies exhibit changes in energy storage and insulin pathway signaling. In addition, slob null flies have decreased levels of dilp3 and increased levels of takeout, a gene known to be involved in feeding and metabolism. Targeted expression of SLOB to mNSCs rescues these alterations in gene expression, as well as the metabolic phenotypes. Analysis of fly lines mutant for both slob and slo indicate that the effect of SLOB on metabolism and gene expression is via SLO. We propose that modulation of SLO by SLOB regulates neurotransmission in mNSCs, influencing downstream insulin pathway signaling and metabolism. PMID:21850269

  3. Activity, Expression and Function of a Second Drosophila Protein Kinase a Catalytic Subunit Gene

    PubMed Central

    Melendez, A.; Li, W.; Kalderon, D.

    1995-01-01

    The DC2 gene was isolated previously on the basis of sequence similarity to DCO, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 did not affect the viability or phenotype of imaginal disc cells lacking DC0 activity or embryonic hatching of animals with reduced DC0 activity. Furthermore, transgenes expressing DC2 from a DC0 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development. PMID:8601490

  4. The Drosophila fragile X mental retardation protein participates in the piRNA pathway.

    PubMed

    Bozzetti, Maria Pia; Specchia, Valeria; Cattenoz, Pierre B; Laneve, Pietro; Geusa, Annamaria; Sahin, H Bahar; Di Tommaso, Silvia; Friscini, Antonella; Massari, Serafina; Diebold, Celine; Giangrande, Angela

    2015-06-01

    RNA metabolism controls multiple biological processes, and a specific class of small RNAs, called piRNAs, act as genome guardians by silencing the expression of transposons and repetitive sequences in the gonads. Defects in the piRNA pathway affect genome integrity and fertility. The possible implications in physiopathological mechanisms of human diseases have made the piRNA pathway the object of intense investigation, and recent work suggests that there is a role for this pathway in somatic processes including synaptic plasticity. The RNA-binding fragile X mental retardation protein (FMRP, also known as FMR1) controls translation and its loss triggers the most frequent syndromic form of mental retardation as well as gonadal defects in humans. Here, we demonstrate for the first time that germline, as well as somatic expression, of Drosophila Fmr1 (denoted dFmr1), the Drosophila ortholog of FMRP, are necessary in a pathway mediated by piRNAs. Moreover, dFmr1 interacts genetically and biochemically with Aubergine, an Argonaute protein and a key player in this pathway. Our data provide novel perspectives for understanding the phenotypes observed in Fragile X patients and support the view that piRNAs might be at work in the nervous system.

  5. A genome-wide resource for the analysis of protein localisation in Drosophila

    PubMed Central

    Sarov, Mihail; Barz, Christiane; Jambor, Helena; Hein, Marco Y; Schmied, Christopher; Suchold, Dana; Stender, Bettina; Janosch, Stephan; KJ, Vinay Vikas; Krishnan, RT; Krishnamoorthy, Aishwarya; Ferreira, Irene RS; Ejsmont, Radoslaw K; Finkl, Katja; Hasse, Susanne; Kämpfer, Philipp; Plewka, Nicole; Vinis, Elisabeth; Schloissnig, Siegfried; Knust, Elisabeth; Hartenstein, Volker; Mann, Matthias; Ramaswami, Mani; VijayRaghavan, K; Tomancak, Pavel; Schnorrer, Frank

    2016-01-01

    The Drosophila genome contains >13000 protein-coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here, we present a genome-wide fosmid library of 10000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins, we created transgenic lines, and for a total of 207 lines, we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests, we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts. DOI: http://dx.doi.org/10.7554/eLife.12068.001 PMID:26896675

  6. A Common Suite of Coagulation Proteins Function in Drosophila Muscle Attachment.

    PubMed

    Green, Nicole; Odell, Nadia; Zych, Molly; Clark, Cheryl; Wang, Zong-Heng; Biersmith, Bridget; Bajzek, Clara; Cook, Kevin R; Dushay, Mitchell S; Geisbrecht, Erika R

    2016-11-01

    The organization and stability of higher order structures that form in the extracellular matrix (ECM) to mediate the attachment of muscles are poorly understood. We have made the surprising discovery that a subset of clotting factor proteins are also essential for muscle attachment in the model organism Drosophila melanogaster One such coagulation protein, Fondue (Fon), was identified as a novel muscle mutant in a pupal lethal genetic screen. Fon accumulates at muscle attachment sites and removal of this protein results in decreased locomotor behavior and detached larval muscles. A sensitized genetic background assay reveals that fon functions with the known muscle attachment genes Thrombospondin (Tsp) and Tiggrin (Tig). Interestingly, Tig is also a component of the hemolymph clot. We further demonstrate that an additional clotting protein, Larval serum protein 1γ (Lsp1γ), is also required for muscle attachment stability and accumulates where muscles attach to tendons. While the local biomechanical and organizational properties of the ECM vary greatly depending on the tissue microenvironment, we propose that shared extracellular protein-protein interactions influence the strength and elasticity of ECM proteins in both coagulation and muscle attachment. Copyright © 2016 by the Genetics Society of America.

  7. Retention of Ejaculate by Drosophila melanogaster Females Requires the Male-Derived Mating Plug Protein PEBme.

    PubMed

    Avila, Frank W; Cohen, Allie B; Ameerudeen, Fatima S; Duneau, David; Suresh, Shruthi; Mattei, Alexandra L; Wolfner, Mariana F

    2015-08-01

    Within the mated reproductive tracts of females of many taxa, seminal fluid proteins (SFPs) coagulate into a structure known as the mating plug (MP). MPs have diverse roles, including preventing female remating, altering female receptivity postmating, and being necessary for mated females to successfully store sperm. The Drosophila melanogaster MP, which is maintained in the mated female for several hours postmating, is comprised of a posterior MP (PMP) that forms quickly after mating begins and an anterior MP (AMP) that forms later. The PMP is composed of seminal proteins from the ejaculatory bulb (EB) of the male reproductive tract. To examine the role of the PMP protein PEBme in D. melanogaster reproduction, we identified an EB GAL4 driver and used it to target PEBme for RNA interference (RNAi) knockdown. PEBme knockdown in males compromised PMP coagulation in their mates and resulted in a significant reduction in female fertility, adversely affecting postmating uterine conformation, sperm storage, mating refractoriness, egg laying, and progeny generation. These defects resulted from the inability of females to retain the ejaculate in their reproductive tracts after mating. The uncoagulated MP impaired uncoupling by the knockdown male, and when he ultimately uncoupled, the ejaculate was often pulled out of the female. Thus, PEBme and MP coagulation are required for optimal fertility in D. melanogaster. Given the importance of the PMP for fertility, we identified additional MP proteins by mass spectrometry and found fertility functions for two of them. Our results highlight the importance of the MP and the proteins that comprise it in reproduction and suggest that in Drosophila the PMP is required to retain the ejaculate within the female reproductive tract, ensuring the storage of sperm by mated females. Copyright © 2015 by the Genetics Society of America.

  8. Retention of Ejaculate by Drosophila melanogaster Females Requires the Male-Derived Mating Plug Protein PEBme

    PubMed Central

    Avila, Frank W.; Cohen, Allie B.; Ameerudeen, Fatima S.; Duneau, David; Suresh, Shruthi; Mattei, Alexandra L.; Wolfner, Mariana F.

    2015-01-01

    Within the mated reproductive tracts of females of many taxa, seminal fluid proteins (SFPs) coagulate into a structure known as the mating plug (MP). MPs have diverse roles, including preventing female remating, altering female receptivity postmating, and being necessary for mated females to successfully store sperm. The Drosophila melanogaster MP, which is maintained in the mated female for several hours postmating, is comprised of a posterior MP (PMP) that forms quickly after mating begins and an anterior MP (AMP) that forms later. The PMP is composed of seminal proteins from the ejaculatory bulb (EB) of the male reproductive tract. To examine the role of the PMP protein PEBme in D. melanogaster reproduction, we identified an EB GAL4 driver and used it to target PEBme for RNA interference (RNAi) knockdown. PEBme knockdown in males compromised PMP coagulation in their mates and resulted in a significant reduction in female fertility, adversely affecting postmating uterine conformation, sperm storage, mating refractoriness, egg laying, and progeny generation. These defects resulted from the inability of females to retain the ejaculate in their reproductive tracts after mating. The uncoagulated MP impaired uncoupling by the knockdown male, and when he ultimately uncoupled, the ejaculate was often pulled out of the female. Thus, PEBme and MP coagulation are required for optimal fertility in D. melanogaster. Given the importance of the PMP for fertility, we identified additional MP proteins by mass spectrometry and found fertility functions for two of them. Our results highlight the importance of the MP and the proteins that comprise it in reproduction and suggest that in Drosophila the PMP is required to retain the ejaculate within the female reproductive tract, ensuring the storage of sperm by mated females. PMID:26058847

  9. Identification of N-glycosylated proteins from the central nervous system of Drosophila melanogaster.

    PubMed

    Koles, Kate; Lim, Jae-Min; Aoki, Kazuhiro; Porterfield, Mindy; Tiemeyer, Michael; Wells, Lance; Panin, Vlad

    2007-12-01

    Although the function of many glycoproteins in the nervous system of fruit flies is well understood, information about the glycosylation profile and glycan attachment sites for such proteins is scarce. In order to fill this gap and to facilitate the analysis of N-linked glycosylation in the nervous system, we have performed an extensive survey of membrane-associated glycoproteins and their N-glycosylation sites isolated from the adult Drosophila brain. Following subcellular fractionation and trypsin digestion, we used different lectin affinity chromatography steps to isolate N-glycosylated glycopeptides. We identified a total of 205 glycoproteins carrying N-linked glycans and revealed their 307 N-glycan attachment sites. The size of the resulting dataset furthermore allowed the statistical characterization of amino acid distribution around the N-linked glycosylation sites. Glycan profiles were analyzed separately for glycopeptides that were strongly and weakly bound to Concanavalin A (Con A), or that failed to bind Concanavalin A, but did bind to wheat germ agglutinin (WGA). High- or paucimannosidic glycans dominated each of the profiles, although the wheat germ agglutinin-bound glycan population was enriched in more extensively processed structures. A sialylated glycan structure was unambiguously detected in the wheat germ agglutinin-bound fraction. Despite the large amount of starting material, insufficient amount of glycopeptides was retained by the Wisteria floribunda (WFA) and Sambucus nigra columns to allow glycan or glycoprotein identification, providing further evidence that the vast majority of glycoproteins in the adult Drosophila brain carry primarily high-mannose, paucimannose, and hybrid glycans. The obtained results should facilitate future genetic and molecular approaches addressing the role of N-glycosylation in the central nervous system (CNS) of Drosophila.

  10. Time of day influences memory formation and dCREB2 proteins in Drosophila

    PubMed Central

    Fropf, Robin; Zhang, Jiabin; Tanenhaus, Anne K.; Fropf, Whitney J.; Siefkes, Ellen; Yin, Jerry C. P.

    2014-01-01

    Many biological phenomena oscillate under the control of the circadian system, exhibiting peaks and troughs of activity across the day/night cycle. In most animal models, memory formation also exhibits this property, but the underlying neuronal and molecular mechanisms remain unclear. The dCREB2 transcription factor shows circadian regulated oscillations in its activity, and has been shown to be important for both circadian biology and memory formation. We show that the time-of-day (TOD) of behavioral training affects Drosophila memory formation. dCREB2 exhibits complex changes in protein levels across the daytime and nighttime, and these changes in protein abundance are likely to contribute to oscillations in dCREB2 activity and TOD effects on memory formation. PMID:24744705

  11. Distinct protein domains and expression patterns confer divergent axon guidance functions for Drosophila Robo receptors.

    PubMed

    Spitzweck, Bettina; Brankatschk, Marko; Dickson, Barry J

    2010-02-05

    The orthogonal array of axon pathways in the Drosophila CNS is constructed in part under the control of three Robo family axon guidance receptors: Robo1, Robo2 and Robo3. Each of these receptors is responsible for a distinct set of guidance decisions. To determine the molecular basis for these functional specializations, we used homologous recombination to create a series of 9 "robo swap" alleles: expressing each of the three Robo receptors from each of the three robo loci. We demonstrate that the lateral positioning of longitudinal axon pathways relies primarily on differences in gene regulation, not distinct combinations of Robo proteins as previously thought. In contrast, specific features of the Robo1 and Robo2 proteins contribute to their distinct functions in commissure formation. These specializations allow Robo1 to prevent crossing and Robo2 to promote crossing. These data demonstrate how diversification of expression and structure within a single family of guidance receptors can shape complex patterns of neuronal wiring.

  12. A Chromatin-associated Kinesin-related Protein Required for Normal Mitotic Chromosome Segregation in Drosophila

    PubMed Central

    Molina, Isabel; Baars, Sigrid; Brill, Julie A.; Hales, Karen G.; Fuller, Margaret T.; Ripoll, Pedro

    1997-01-01

    The tiovivo (tio) gene of Drosophila encodes a kinesin-related protein, KLP38B, that colocalizes with condensed chromatin during cell division. Wild-type function of the tio gene product KLP38B is required for normal chromosome segregation during mitosis. Mitotic cells in tio larval brains displayed circular mitotic figures, increased ploidy, and abnormal anaphase figures. KLP38B mRNA is maternally provided and expressed in cells about to undergo division. We propose that KLP38B, perhaps redundantly with other chromosome-associated microtubule motor proteins, contributes to interactions between chromosome arms and microtubules important for establishing bipolar attachment of chromosomes and assembly of stable bipolar spindles. PMID:9396743

  13. The activity of the Drosophila Vestigial protein is modified by Scalloped-dependent phosphorylation.

    PubMed

    Pimmett, Virginia L; Deng, Hua; Haskins, Julie A; Mercier, Rebecca J; LaPointe, Paul; Simmonds, Andrew J

    2017-05-01

    The Drosophila vestigial gene is required for proliferation and differentiation of the adult wing and for differentiation of larval and adult muscle identity. Vestigial is part of a multi-protein transcription factor complex, which includes Scalloped, a TEAD-class DNA binding protein. Binding Scalloped is necessary for translocation of Vestigial into the nucleus. We show that Vestigial is extensively post-translationally modified and at least one of these modifications is required for proper function during development. We have shown that there is p38-dependent phosphorylation of Serine 215 in the carboxyl-terminal region of Vestigial. Phosphorylation of Serine 215 occurs in the nucleus and requires the presence of Scalloped. Comparison of a phosphomimetic and non-phosphorylatable mutant forms of Vestigial shows differences in the ability to rescue the wing and muscle phenotypes associated with a null vestigial allele. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. DNA regions that regulate the ovarian transcriptional specificity of Drosophila yolk protein genes.

    PubMed

    Logan, S K; Garabedian, M J; Wensink, P C

    1989-09-01

    Yolk protein genes 1 and 2 (yp1 and yp2) of Drosophila melanogaster are divergently transcribed neighboring genes. Both are transcribed in only two tissues, the ovarian follicle cells and the fat bodies of adult females. Previous work has identified a yolk protein enhancer between the genes that is sufficient to direct transcription in one of the tissues, female fat bodies. Using germ-line transformation methods, we identify two cis-acting regions with positive effects on transcription in ovaries. One, a 301-bp region located between the genes, influences both genes and is an enhancer determining the stage and cell type specificity of ovarian transcription. The other, a 105-bp region located in the first exon of yp2, acts across the yp2 promoter region to stimulate yp1 transcription in ovaries. Additional observations suggest how a single enhancer influences both promoters.

  15. Immunostaining of Drosophila polytene chromosomes to investigate recruitment of chromatin-binding proteins.

    PubMed

    Murawska, Magdalena; Brehm, Alexander

    2012-01-01

    Gene transcription is a complex process that involves a large number of proteins. These proteins can be brought to their target genes by a variety of different mechanisms: many transcription factors interact with specific DNA sequences in promoters or enhancers, several epigenetic regulators bind histones bearing specific modifications, elongation factors and some RNA processing factors bind to the transcribing RNA polymerase, and other factors interact directly with nascent transcripts or noncoding RNA. Immunostaining of Drosophila polytene chromosomes allows the genome-wide localization of factors involved at different stages of transcriptional regulation. In this chapter, we present protocols that adapt the general technique to probe different recruitment mechanisms employed by these factors, including specific interactions with phosphorylated RNA polymerase II and RNA-mediated chromatin associations.

  16. The kinesin-like protein KLP61F is essential for mitosis in Drosophila

    PubMed Central

    1993-01-01

    We report here that disruption of a recently discovered kinesin-like protein in Drosophila melanogaster, KLP61F, results in a mitotic mutation lethal to the organism. We show that in the absence of KLP61F function, spindle poles fail to separate, resulting in the formation of monopolar mitotic spindles. The resulting phenotype of metaphase arrest with polyploid cells is reminiscent of that seen in the fungal bimC and cut7 mutations, where it has also been shown that spindle pole bodies are not segregated. KLP61F is specifically expressed in proliferating tissues during embryonic and larval development, consistent with a primary role in cell division. The structural and functional homology of the KLP61F, bimC, cut7, and Eg5 kinesin-like proteins demonstrates the existence of a conserved family of kinesin-like molecules important for spindle pole separation and mitotic spindle dynamics. PMID:8227131

  17. Widespread Positive Selection Drives Differentiation of Centromeric Proteins in the Drosophila melanogaster subgroup

    PubMed Central

    Beck, Emily A.; Llopart, Ana

    2015-01-01

    Rapid evolution of centromeric satellite repeats is thought to cause compensatory amino acid evolution in interacting centromere-associated kinetochore proteins. Cid, a protein that mediates kinetochore/centromere interactions, displays particularly high amino acid turnover. Rapid evolution of both Cid and centromeric satellite repeats led us to hypothesize that the apparent compensatory evolution may extend to interacting partners in the Condensin I complex (i.e., SMC2, SMC4, Cap-H, Cap-D2, and Cap-G) and HP1s. Missense mutations in these proteins often result in improper centromere formation and aberrant chromosome segregation, thus selection for maintained function and coevolution among proteins of the complex is likely strong. Here, we report evidence of rapid evolution and recurrent positive selection in seven centromere-associated proteins in species of the Drosophila melanogaster subgroup, and further postulate that positive selection on these proteins could be a result of centromere drive and compensatory changes, with kinetochore proteins competing for optimal spindle attachment. PMID:26603658

  18. Widespread Positive Selection Drives Differentiation of Centromeric Proteins in the Drosophila melanogaster subgroup.

    PubMed

    Beck, Emily A; Llopart, Ana

    2015-11-25

    Rapid evolution of centromeric satellite repeats is thought to cause compensatory amino acid evolution in interacting centromere-associated kinetochore proteins. Cid, a protein that mediates kinetochore/centromere interactions, displays particularly high amino acid turnover. Rapid evolution of both Cid and centromeric satellite repeats led us to hypothesize that the apparent compensatory evolution may extend to interacting partners in the Condensin I complex (i.e., SMC2, SMC4, Cap-H, Cap-D2, and Cap-G) and HP1s. Missense mutations in these proteins often result in improper centromere formation and aberrant chromosome segregation, thus selection for maintained function and coevolution among proteins of the complex is likely strong. Here, we report evidence of rapid evolution and recurrent positive selection in seven centromere-associated proteins in species of the Drosophila melanogaster subgroup, and further postulate that positive selection on these proteins could be a result of centromere drive and compensatory changes, with kinetochore proteins competing for optimal spindle attachment.

  19. Reproductive hacking. A male seminal protein acts through intact reproductive pathways in female Drosophila.

    PubMed

    Rubinstein, C Dustin; Wolfner, Mariana F

    2014-01-01

    Seminal proteins are critical for reproductive success in all animals that have been studied. Although seminal proteins have been identified in many taxa, and female reproductive responses to receipt of these proteins have been documented in several, little is understood about the mechanisms by which seminal proteins affect female reproductive physiology. To explore this topic, we investigated how a Drosophila seminal protein, ovulin, increases ovulation rate in mated females. Ovulation is a relatively simple physiological process, with known female regulators: previous studies have shown that ovulation rate is promoted by the neuromodulator octopamine (OA) in D. melanogaster and other insects. We found that ovulin stimulates ovulation by increasing OA signaling in the female. This finding supports a model in which a male seminal protein acts through "hacking" a well-conserved, regulatory system females use to adjust reproductive output, rather than acting downstream of female mechanisms of control or in parallel pathways altogether. We also discuss similarities between 2 forms of intersexual control of behavior through chemical communication: seminal proteins and pheromones.

  20. Characterization of dRFX2, a novel RFX family protein in Drosophila.

    PubMed

    Otsuki, Kyoko; Hayashi, Yuko; Kato, Masaki; Yoshida, Hideki; Yamaguchi, Masamitsu

    2004-01-01

    A transcriptional regulatory element was identified in the region between URE (upstream regulatory element) and DRE (DNA replication-related element) in the Drosophila PCNA gene promoter. This element plays an important role in promoter activity in living flies. A yeast one-hybrid screening using this element as a bait allowed isolation of a cDNA encoding a protein which binds to the element in vitro. Nucleotide sequence analyses revealed that the cDNA encodes a novel protein containing a characteristic DNA-binding domain conserved among the regulatory factor X (RFX) family proteins. We termed this protein Drosophila RFX2 (dRFX2) and this element dRFX2 site. To investigate the function of dRFX2 in vivo, we took the strategy of analyzing the dominant negative effects against the endogenous dRFX2. Transgenic flies were established in which expression of HA-dRFX(202-480) carrying the amino acid sequences from 202 to 480 containing the RFX domain (DNA-binding domain) of dRFX2 was targeted to the cells in the eye imaginal discs. In the eye imaginal disc expressing the HA-dRFX(202-480), the G1-S transition and/or the progression of S phase were/was interrupted, and the ectopic apoptosis was induced, though photoreceptor cells differentiated normally. These results indicate that dRFX2 plays a role in G1-S transition and/or in progression of S phase.

  1. A functional heat shock protein 90 chaperone is essential for efficient flock house virus RNA polymerase synthesis in Drosophila cells.

    PubMed

    Castorena, Kathryn M; Weeks, Spencer A; Stapleford, Kenneth A; Cadwallader, Amy M; Miller, David J

    2007-08-01

    The molecular chaperone heat shock protein 90 (Hsp90) is involved in multiple cellular processes including protein maturation, complex assembly and disassembly, and intracellular transport. We have recently shown that a disruption of Hsp90 activity in cultured Drosophila melanogaster cells suppresses Flock House virus (FHV) replication and the accumulation of protein A, the FHV RNA-dependent RNA polymerase. In the present study, we investigated whether the defect in FHV RNA polymerase accumulation induced by Hsp90 suppression was secondary to an effect on protein A synthesis, degradation, or intracellular membrane association. Treatment with the Hsp90-specific inhibitor geldanamycin selectively reduced FHV RNA polymerase synthesis by 80% in Drosophila S2 cells stably transfected with an inducible protein A expression plasmid. The suppressive effect of geldanamycin on protein A synthesis was not attenuated by proteasome inhibition, nor was it sensitive to changes in either the mRNA untranslated regions or protein A intracellular membrane localization. Furthermore, geldanamycin did not promote premature protein A degradation, nor did it alter the extremely rapid kinetics of protein A membrane association. These results identify a novel role for Hsp90 in facilitating viral RNA polymerase synthesis in Drosophila cells and suggest that FHV subverts normal cellular pathways to assemble functional replication complexes.

  2. A Drosophila protein family implicated in pheromone perception is related to Tay-Sachs GM2-activator protein.

    PubMed

    Starostina, Elena; Xu, Aiguo; Lin, Heping; Pikielny, Claudio W

    2009-01-02

    Low volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons.

  3. A Drosophila Protein Family Implicated in Pheromone Perception Is Related to Tay-Sachs GM2-Activator Protein*

    PubMed Central

    Starostina, Elena; Xu, Aiguo; Lin, Heping; Pikielny, Claudio W.

    2009-01-01

    Low volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons. PMID:18952610

  4. Activity of cGMP-Dependent Protein Kinase (PKG) Affects Sucrose Responsiveness and Habituation in "Drosophila melanogaster"

    ERIC Educational Resources Information Center

    Scheiner, Ricarda; Sokolowski, Marla B.; Erber, Joachim

    2004-01-01

    The cGMP-dependent protein kinase (PKG) has many cellular functions in vertebrates and insects that affect complex behaviors such as locomotion and foraging. The "foraging" ("for") gene encodes a PKG in "Drosophila melanogaster." Here, we demonstrate a function for the "for" gene in sensory responsiveness and nonassociative learning. Larvae of the…

  5. Binding of Drosophila ORC proteins to anaphase chromosomes requires cessation of mitotic cyclin-dependent kinase activity.

    PubMed

    Baldinger, Tina; Gossen, Manfred

    2009-01-01

    The initial step in the acquisition of replication competence by eukaryotic chromosomes is the binding of the multisubunit origin recognition complex, ORC. We describe a transgenic Drosophila model which enables dynamic imaging of a green fluorescent protein (GFP)-tagged Drosophila melanogaster ORC subunit, DmOrc2-GFP. It is functional in genetic complementation, expressed at physiological levels, and participates quantitatively in complex formation. This fusion protein is therefore able to depict both the holocomplex DmOrc1-6 and the core complex DmOrc2-6 formed by the Drosophila initiator proteins. Its localization can be monitored in vivo along the cell cycle and development. DmOrc2-GFP is not detected on metaphase chromosomes but binds rapidly to anaphase chromatin in Drosophila embryos. Expression of either stable cyclin A, B, or B3 prevents this reassociation, suggesting that cessation of mitotic cyclin-dependent kinase activity is essential for binding of the DmOrc proteins to chromosomes.

  6. Overexpression of Drosophila juvenile hormone esterase binding protein results in anti-JH effects and reduced pheromone abundance

    USDA-ARS?s Scientific Manuscript database

    The titer of juvenile hormone (JH), which has wide ranging physiological effects in insects, is regulated in part by JH esterase (JHE). We show that overexpression in Drosophila melanogaster of the JHE binding protein, DmP29 results in a series of apparent anti-JH effects. We hypothesize that DmP29 ...

  7. Activity of cGMP-Dependent Protein Kinase (PKG) Affects Sucrose Responsiveness and Habituation in "Drosophila melanogaster"

    ERIC Educational Resources Information Center

    Scheiner, Ricarda; Sokolowski, Marla B.; Erber, Joachim

    2004-01-01

    The cGMP-dependent protein kinase (PKG) has many cellular functions in vertebrates and insects that affect complex behaviors such as locomotion and foraging. The "foraging" ("for") gene encodes a PKG in "Drosophila melanogaster." Here, we demonstrate a function for the "for" gene in sensory responsiveness and nonassociative learning. Larvae of the…

  8. M6 Membrane Protein Plays an Essential Role in Drosophila Oogenesis

    PubMed Central

    Zappia, María Paula; Brocco, Marcela Adriana; Billi, Silvia C.; Frasch, Alberto C.; Ceriani, María Fernanda

    2011-01-01

    We had previously shown that the transmembrane glycoprotein M6a, a member of the proteolipid protein (PLP) family, regulates neurite/filopodium outgrowth, hence, M6a might be involved in neuronal remodeling and differentiation. In this work we focused on M6, the only PLP family member present in Drosophila, and ortholog to M6a. Unexpectedly, we found that decreased expression of M6 leads to female sterility. M6 is expressed in the membrane of the follicular epithelium in ovarioles throughout oogenesis. Phenotypes triggered by M6 downregulation in hypomorphic mutants included egg collapse and egg permeability, thus suggesting M6 involvement in eggshell biosynthesis. In addition, RNAi-mediated M6 knockdown targeted specifically to follicle cells induced an arrest of egg chamber development, revealing that M6 is essential in oogenesis. Interestingly, M6-associated phenotypes evidenced abnormal changes of the follicle cell shape and disrupted follicular epithelium in mid- and late-stage egg chambers. Therefore, we propose that M6 plays a role in follicular epithelium maintenance involving membrane cell remodeling during oogenesis in Drosophila. PMID:21603606

  9. Piwi maintains germline stem cells and oogenesis in Drosophila through negative regulation of Polycomb group proteins.

    PubMed

    Peng, Jamy C; Valouev, Anton; Liu, Na; Lin, Haifan

    2016-03-01

    The Drosophila melanogaster Piwi protein regulates both niche and intrinsic mechanisms to maintain germline stem cells, but its underlying mechanism remains unclear. Here we report that Piwi interacts with Polycomb group complexes PRC1 and PRC2 in niche and germline cells to regulate ovarian germline stem cells and oogenesis. Piwi physically interacts with the PRC2 subunits Su(z)12 and Esc in the ovary and in vitro. Chromatin coimmunoprecipitation of Piwi, the PRC2 enzymatic subunit E(z), histone H3 trimethylated at lysine 27 (H3K27me3) and RNA polymerase II in wild-type and piwi mutant ovaries demonstrates that Piwi binds a conserved DNA motif at ∼ 72 genomic sites and inhibits PRC2 binding to many non-Piwi-binding genomic targets and H3K27 trimethylation. Moreover, Piwi influences RNA polymerase II activities in Drosophila ovaries, likely via inhibiting PRC2. We hypothesize that Piwi negatively regulates PRC2 binding by sequestering PRC2 in the nucleoplasm, thus reducing PRC2 binding to many targets and influencing transcription during oogenesis.

  10. Drosophila damaged DNA-binding protein 1 is an essential factor for development.

    PubMed

    Takata, Kei-ichi; Yoshida, Hideki; Yamaguchi, Masamitsu; Sakaguchi, Kengo

    2004-10-01

    The damaged DNA-binding protein (DDB) complex, thought to recognize (6-4) photoproducts and other lesions in DNA, has been implicated to have a role in global genomic nucleotide excision repair (NER) and E2F-1-mediated transcription. The complex consists of a heterodimer of p127 (DDB1) and p48 (DDB2), the latter also being known as XPE. We reported previously that in Drosophila expression of the DDB1 (D-DDB1) gene is controlled by the DRE/DREF system, and external injury to DNA is not essential for D-DDB1 function. In the present study of the function of D-DDB1 in a multicellular system, we prepared transgenic flies, which were knocked down for the D-DDB1 gene due to RNA interference (RNAi), and performed immunocytochemistry to ascertain the distribution of D-DDB1 in the eye imaginal disc. It was found to be abundant in the anterior of the morphogenetic furrow (MF). Whole-body overexpression of dsRNA of D-DDB1 in Drosophila using a GAL4-UAS targeted expression system induced melanotic tumors and caused complete lethality. When limited to the eye imaginal disc, a severe rough eye phenotype resulted. Correspondingly, all of the D-DDB1 gene knocked-out flies also died. D-DDB1 therefore appears to be an essential development-associated factor in a multicellular organism.

  11. Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknuellt protein

    SciTech Connect

    Yamaguchi, Masamitsu; Hirose, Fumiko; Nishida, Yasuyoshi; Matsukage, Akio )

    1991-10-01

    A 631-bp fragment containing the 5{prime}-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5{prime}-deletion derivatives revealed that the promoter function resided within a 192-bp region. Cotransfection with a zerknuellt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region fused with lacZ were established. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

  12. A novel Drosophila Girdin-like protein is involved in Akt pathway control of cell size

    SciTech Connect

    Puseenam, Aekkachai; Yoshioka, Yasuhide; Nagai, Rika; Hashimoto, Reina; Suyari, Osamu; Itoh, Masanobu; Enomoto, Atsushi; Takahashi, Masahide; Yamaguchi, Masamitsu

    2009-11-15

    The Akt signaling pathway is well known to regulate cell proliferation and growth. Girdin, a novel substrate of Akt, plays a crucial role in organization of the actin cytoskeleton and cell motility under the control of Akt. We here identified a novel Girdin-like protein in Drosophila (dGirdin), which has two isoforms, dGirdin PA and dGirdin PB. dGirdin shows high homology with human Girdin in the N-terminal and coiled-coil domains, while diverging at the C-terminal domain. On establishment of transgenic fly lines, featuring knockdown or overexpression of dGirdin in vivo, overexpression in the wing disc cells induced ectopic apoptosis, implying a role in directing apoptosis. Knockdown of dGirdin in the Drosophila wing imaginal disc cells resulted in reduction of cell size. Furthermore, this was enhanced by half reduction of the Akt gene dose, suggesting that Akt positively regulates dGirdin. In the wing disc, cells in which dGirdin was knocked down exhibited disruption of actin filaments. From these in vivo analyses, we conclude that dGirdin is required for actin organization and regulation of appropriate cell size under control of the Akt signaling pathway.

  13. Coordinate redeployment of PRC1 proteins suppresses tumor formation during Drosophila development.

    PubMed

    Loubière, Vincent; Delest, Anna; Thomas, Aubin; Bonev, Boyan; Schuettengruber, Bernd; Sati, Satish; Martinez, Anne-Marie; Cavalli, Giacomo

    2016-11-01

    Polycomb group proteins form two main complexes, PRC2 and PRC1, which generally coregulate their target genes. Here we show that PRC1 components act as neoplastic tumor suppressors independently of PRC2 function. By mapping the distribution of PRC1 components and trimethylation of histone H3 at Lys27 (H3K27me3) across the genome, we identify a large set of genes that acquire PRC1 in the absence of H3K27me3 in Drosophila larval tissues. These genes massively outnumber canonical targets and are mainly involved in the regulation of cell proliferation, signaling and polarity. Alterations in PRC1 components specifically deregulate this set of genes, whereas canonical targets are derepressed in both PRC1 and PRC2 mutants. In human embryonic stem cells, PRC1 components colocalize with H3K27me3 as in Drosophila embryos, whereas in differentiated cell types they are selectively recruited to a large set of proliferation and signaling-associated genes that lack H3K27me3, suggesting that the redeployment of PRC1 components during development is evolutionarily conserved.

  14. midlife crisis encodes a conserved zinc-finger protein required to maintain neuronal differentiation in Drosophila

    PubMed Central

    Carney, Travis D.; Struck, Adam J.; Doe, Chris Q.

    2013-01-01

    Stem cells generate progeny that undergo terminal differentiation. The initiation and maintenance of the differentiated status is crucial for tissue development, function and homeostasis. Drosophila neural stem cells (neuroblasts) are a model for stem cell self-renewal and differentiation; they divide asymmetrically to self-renew and generate the neurons and glia of the CNS. Here we report the identification of midlife crisis (mdlc; CG4973) as a gene required for the maintenance of neuronal differentiation and for neuroblast proliferation in Drosophila. mdlc encodes a ubiquitously expressed zinc-finger-containing protein with conserved orthologs from yeast to humans that are reported to have a role in RNA splicing. Using clonal analysis, we demonstrate that mdlc mutant neurons initiate but fail to complete differentiation, as judged by the loss of the pro-differentiation transcription factor Prospero, followed by derepression of the neuroblast factors Deadpan, Asense and Cyclin E. RNA-seq shows that loss of Mdlc decreases pros transcript levels and results in aberrant pros splicing. Importantly, misexpression of the full-length human ortholog, RNF113A, completely rescues all CNS defects in mdlc mutants. We conclude that Mdlc plays an essential role in maintaining neuronal differentiation, raising the possibility that RNF113A regulates neuronal differentiation in the human CNS. PMID:24026126

  15. midlife crisis encodes a conserved zinc-finger protein required to maintain neuronal differentiation in Drosophila.

    PubMed

    Carney, Travis D; Struck, Adam J; Doe, Chris Q

    2013-10-01

    Stem cells generate progeny that undergo terminal differentiation. The initiation and maintenance of the differentiated status is crucial for tissue development, function and homeostasis. Drosophila neural stem cells (neuroblasts) are a model for stem cell self-renewal and differentiation; they divide asymmetrically to self-renew and generate the neurons and glia of the CNS. Here we report the identification of midlife crisis (mdlc; CG4973) as a gene required for the maintenance of neuronal differentiation and for neuroblast proliferation in Drosophila. mdlc encodes a ubiquitously expressed zinc-finger-containing protein with conserved orthologs from yeast to humans that are reported to have a role in RNA splicing. Using clonal analysis, we demonstrate that mdlc mutant neurons initiate but fail to complete differentiation, as judged by the loss of the pro-differentiation transcription factor Prospero, followed by derepression of the neuroblast factors Deadpan, Asense and Cyclin E. RNA-seq shows that loss of Mdlc decreases pros transcript levels and results in aberrant pros splicing. Importantly, misexpression of the full-length human ortholog, RNF113A, completely rescues all CNS defects in mdlc mutants. We conclude that Mdlc plays an essential role in maintaining neuronal differentiation, raising the possibility that RNF113A regulates neuronal differentiation in the human CNS.

  16. Protein phosphatase 1ß limits ring canal constriction during Drosophila germline cyst formation.

    PubMed

    Yamamoto, Shinya; Bayat, Vafa; Bellen, Hugo J; Tan, Change

    2013-01-01

    Germline cyst formation is essential for the propagation of many organisms including humans and flies. The cytoplasm of germline cyst cells communicate with each other directly via large intercellular bridges called ring canals. Ring canals are often derived from arrested contractile rings during incomplete cytokinesis. However how ring canal formation, maintenance and growth are regulated remains unclear. To better understand this process, we carried out an unbiased genetic screen in Drosophila melanogaster germ cells and identified multiple alleles of flapwing (flw), a conserved serine/threonine-specific protein phosphatase. Flw had previously been reported to be unnecessary for early D. melanogaster oogenesis using a hypomorphic allele. We found that loss of Flw leads to over-constricted nascent ring canals and subsequently tiny mature ring canals, through which cytoplasmic transfer from nurse cells to the oocyte is impaired, resulting in small, non-functional eggs. Flw is expressed in germ cells undergoing incomplete cytokinesis, completely colocalized with the Drosophila myosin binding subunit of myosin phosphatase (DMYPT). This colocalization, together with genetic interaction studies, suggests that Flw functions together with DMYPT to negatively regulate myosin activity during ring canal formation. The identification of two subunits of the tripartite myosin phosphatase as the first two main players required for ring canal constriction indicates that tight regulation of myosin activity is essential for germline cyst formation and reproduction in D. melanogaster and probably other species as well.

  17. Drosophila DOCK family protein sponge regulates the JNK pathway during thorax development.

    PubMed

    Morishita, Kazushige; Ozasa, Fumito; Eguchi, Koichi; Yoshioka, Yasuhide; Yoshida, Hideki; Hiai, Hiroshi; Yamaguchi, Masamitsu

    2014-01-01

    The dedicator of cytokinesis (DOCK) family proteins that are conserved in a wide variety of species are known as DOCK1-DOCK11 in mammals. The Sponge (Spg) is a Drosophila counterpart to the mammalian DOCK3. Specific knockdown of spg by pannir-GAL4 or apterous-GAL4 driver in wing discs induced split thorax phenotype in adults. Reduction of the Drosophila c-Jun N-terminal kinase (JNK), basket (bsk) gene dose enhanced the spg knockdown-induced phenotype. Conversely, overexpression of bsk suppressed the split thorax phenotype. Monitoring JNK activity in the wing imaginal discs by immunostaining with anti-phosphorylated JNK (anti-pJNK) antibody together with examination of lacZ expression in a puckered-lacZ enhancer trap line revealed the strong reduction of the JNK activity in the spg knockdown clones. This was further confirmed by Western immunoblot analysis of extracts from wing discs of spg knockdown fly with anti-pJNK antibody. Furthermore, the Duolink in situ Proximity Ligation Assay method detected interaction signals between Spg and Rac1 in the wing discs. Taken together, these results indicate Spg positively regulates JNK pathway that is required for thorax development and the regulation is mediated by interaction with Rac1.

  18. Impaired fatty acid oxidation in a Drosophila model of mitochondrial trifunctional protein (MTP) deficiency.

    PubMed

    Kishita, Yoshihito; Tsuda, Manabu; Aigaki, Toshiro

    2012-03-09

    Mitochondrial trifunctional protein (MTP), which consists of the MTPα and MTPβ subunits, catalyzes long-chain fatty acid β-oxidation. MTP deficiency in humans results in Reye-like syndrome. Here, we generated Drosophila models of MTP deficiency by targeting two genes encoding Drosophila homologs of human MTPα and MTPβ, respectively. Both Mtpα(KO) and Mtpβ(KO) flies were viable, but demonstrated reduced lifespan, defective locomotor activity, and reduced fecundity represented by the number of eggs laid by the females. The phenotypes of Mtpα(KO) flies were generally more striking than those of Mtpβ(KO) flies. Mtpα(KO) flies were hypersensitive to fasting, and retained lipid droplets in their fat body cells as in non-fasting conditions. The amount of triglyceride was also unchanged upon fasting in Mtpα(KO) flies, suggesting that lipid mobilization was disrupted. Finally, we showed that both Mtpα(KO) and Mtpβ(KO) flies accumulated acylcarnitine and hydroxyacylcarnitine, diagnostic markers of MTP deficiencies in humans. Our results indicated that both Mtpα(KO) and Mtpβ(KO) flies were impaired in long-chain fatty acid β-oxidation. These flies should be useful as a model system to investigate the molecular pathogenesis of MTP deficiency.

  19. Cloning and functional analysis of TipE, a novel membrane protein that enhances Drosophila para sodium channel function.

    PubMed

    Feng, G; Deák, P; Chopra, M; Hall, L M

    1995-09-22

    Voltage-dependent sodium channels are involved in the initiation and propagation of action potentials in many excitable cells. Here we report that tipE, a gene defined by a temperature-sensitive paralytic mutation in Drosophila, encodes a novel integral membrane protein that dramatically stimulates functional expression in Xenopus oocytes of the Drosophila sodium channel alpha subunit encoded by the paralytic (para) locus. Using a heat shock promoter to control tipE+ gene expression in transgenic flies, we demonstrate that tipE+ gene expression is required during pupal development to rescue adult paralysis. In addition, we demonstrate a role for the tipE gene product in adults.

  20. Paraquat exposure and Sod2 knockdown have dissimilar impacts on the Drosophila melanogaster carbonylated protein proteome.

    PubMed

    Narayanasamy, Suresh K; Simpson, David C; Martin, Ian; Grotewiel, Mike; Gronert, Scott

    2014-11-01

    Exposure to Paraquat and RNA interference knockdown of mitochondrial superoxide dismutase (Sod2) are known to result in significant lifespan reduction, locomotor dysfunction, and mitochondrial degeneration in Drosophila melanogaster. Both perturbations increase the flux of the progenitor ROS, superoxide, but the molecular underpinnings of the resulting phenotypes are poorly understood. Improved understanding of such processes could lead to advances in the treatment of numerous age-related disorders. Superoxide toxicity can act through protein carbonylation. Analysis of carbonylated proteins is attractive since carbonyl groups are not present in the 20 canonical amino acids and are amenable to labeling and enrichment strategies. Here, carbonylated proteins were labeled with biotin hydrazide and enriched on streptavidin beads. On-bead digestion was used to release carbonylated protein peptides, with relative abundance ratios versus controls obtained using the iTRAQ MS-based proteomics approach. Western blotting and biotin quantitation assay approaches were also investigated. By both Western blotting and proteomics, Paraquat exposure, but not Sod2 knockdown, resulted in increased carbonylated protein relative abundance. For Paraquat exposure versus control, the median carbonylated protein relative abundance ratio (1.53) determined using MS-based proteomics was in good agreement with that obtained using a commercial biotin quantitation kit (1.36).

  1. Enabled and Capping protein play important roles in shaping cell behavior during Drosophila oogenesis

    PubMed Central

    Gates, Julie; Nowotarski, Stephanie H.; Yin, Hongyan; Mahaffey, James P.; Bridges, Tina; Herrera, Cristina; Homem, Catarina C. F.; Janody, Florence; Montell, Denise J.; Peifer, Mark

    2009-01-01

    During development, cells craft an impressive array of actin-based structures, mediating events as diverse as cytokinesis, apical constriction, and cell migration. One challenge is to determine how cells regulate actin assembly and disassembly to carry out these cell behaviors. During Drosophila oogenesis diverse cell behaviors are seen in the soma and germline. We used oogenesis to explore developmental roles of two important actin regulators: Enabled/VASP proteins and Capping protein. We found that Enabled plays an important role in cortical integrity of nurse cells, formation of robust bundled actin filaments in late nurse cells that facilitate nurse cell dumping, and migration of somatic border cells. During nurse cell dumping, Enabled localizes to barbed ends of the nurse cell actin filaments, suggesting its mechanism of action. We further pursued this mechanism using mutant Enabled proteins, each affecting one of its protein domains. These data suggest critical roles for the EVH2 domain and its tetramerization subdomain, while the EVH1 domain appears less critical. Enabled appears to be negatively regulated during oogenesis by Abelson kinase. We also explored the function of Capping protein. This revealed important roles in oocyte determination, nurse cell cortical integrity and nurse cell dumping, and support the idea that Capping protein and Enabled act antagonistically during dumping. Together these data reveal places these actin regulators shape oogenesis. PMID:19576200

  2. Paraquat exposure and Sod2 knockdown have dissimilar impacts on the Drosophila melanogaster carbonylated protein proteome

    PubMed Central

    Narayanasamy, Suresh K.; Simpson, David C.; Martin, Ian; Grotewiel, Mike; Gronert, Scott

    2014-01-01

    Exposure to Paraquat and RNA interference knockdown of Mn or mitochondrial superoxide dismutase (Sod2) are known to result in significant lifespan reduction, locomotor dysfunction, and mitochondrial degeneration in Drosophila melanogaster. Both perturbations increase the flux of the progenitor ROS, superoxide, but the molecular underpinnings of the resulting phenotypes are poorly understood. Improved understanding of such processes could lead to advances in the treatment of numerous age-related disorders. Superoxide toxicity can act through protein carbonylation. Analysis of carbonylated proteins is attractive since carbonyl groups are not present in the twenty canonical amino acids and are amenable to labeling and enrichment strategies. Here, carbonylated proteins were labeled with biotin hydrazide and enriched on streptavidin beads. On-bead digestion was used to release carbonylated protein peptides, with relative abundance ratios versus controls obtained using the iTRAQ MS-based proteomics approach. Western blotting and biotin quantitation assay approaches were also investigated. By both western blotting and proteomics, Paraquat exposure, but not Sod2 knockdown, resulted in increased carbonylated protein relative abundance. For Paraquat exposure versus control, the median carbonylated protein relative abundance ratio (1.53) determined using MS-based proteomics was in good agreement with that obtained using a commercial biotin quantitation kit (1.36). PMID:25091824

  3. Kebab: kinetochore and EB1 associated basic protein that dynamically changes its localisation during Drosophila mitosis.

    PubMed

    Meireles, Ana M; Dzhindzhev, Nikola S; Ohkura, Hiroyuki

    2011-01-01

    Microtubule plus ends are dynamic ends that interact with other cellular structures. Microtubule plus end tracking proteins are considered to play important roles in the regulation of microtubule plus ends. Recent studies revealed that EB1 is the central regulator for microtubule plus end tracking proteins by recruiting them to microtubule plus ends through direct interaction. Here we report the identification of a novel Drosophila protein, which we call Kebab (kinetochore and EB1 associated basic protein), through in vitro expression screening for EB1-interacting proteins. Kebab fused to GFP shows a novel pattern of dynamic localisation in mitosis. It localises to kinetochores weakly in metaphase and accumulates progressively during anaphase. In telophase, it associates with microtubules in central-spindle and centrosomal regions. The localisation to kinetochores depends on microtubules. The protein has a domain most similar to the atypical CH domain of Ndc80, and a coiled-coil domain. The interaction with EB1 is mediated by two SxIP motifs but is not required for the localisation. Depletion of Kebab in cultured cells by RNA interference did not show obvious defects in mitotic progression or microtubule organisation. Generation of mutants lacking the kebab gene indicated that Kebab is dispensable for viability and fertility.

  4. The Drosophila Fry protein interacts with Trc and is highly mobile in vivo

    PubMed Central

    2010-01-01

    Background Cell polarity is a common feature of eukaryotic cells. The NDR kinases have been found to regulate polarized growth in both animal cells and fungi. Drosophila Tricornered is an NDR kinase that is essential for the normal polarized growth of extensions of epidermal cells and for the tiling and branching of dendrites of da sensory neurons. Tricornered function requires interacting with the large Furry protein (3479 amino acid). Results We constructed a furry (fry) transgene and established that it rescued the lethality of fry null mutations. The encoded protein was tagged at both its amino and carboxy termini and this allowed us to demonstrate that the protein existed as an uncleaved protein in vivo. We used the C terminal GFP tag to follow the protein in vivo and found it to be highly mobile. Interestingly Fry accumulated at the distal tip of growing bristles. We established that Fry and Trc could be co-immunoprecipitated from wing discs. Conclusions The mobility of Fry in both bristles and dendrites suggests that it could function in directing/mediating the intracellular transport needed for polarized growth. Our observations that full length Fry and Trc show only partial co-localization in growing bristles while an amino terminal fragment of Fry shows close to complete co-localization with Trc suggests that the interaction between these proteins is transient and regulated. PMID:20406475

  5. Novel protein kinase signaling systems regulating lifespan identified by small molecule library screening using Drosophila.

    PubMed

    Spindler, Stephen R; Li, Rui; Dhahbi, Joseph M; Yamakawa, Amy; Sauer, Frank

    2012-01-01

    Protein kinase signaling cascades control most aspects of cellular function. The ATP binding domains of signaling protein kinases are the targets of most available inhibitors. These domains are highly conserved from mammals to flies. Herein we describe screening of a library of small molecule inhibitors of protein kinases for their ability to increase Drosophila lifespan. We developed an assay system which allowed screening using the small amounts of materials normally present in commercial chemical libraries. The studies identified 17 inhibitors, the majority of which targeted tyrosine kinases associated with the epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) receptors, G-protein coupled receptor (GPCR), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), the insulin and insulin-like growth factor (IGFI) receptors. Comparison of the protein kinase signaling effects of the inhibitors in vitro defined a consensus intracellular signaling profile which included decreased signaling by p38MAPK (p38), c-Jun N-terminal kinase (JNK) and protein kinase C (PKC). If confirmed, many of these kinases will be novel additions to the signaling cascades known to regulate metazoan longevity.

  6. Repression of hsp70 heat shock gene transcription by the suppressor of hairy-wing protein of Drosophila melanogaster

    SciTech Connect

    Holdridge, C.; Dorsett, D. )

    1991-04-01

    The suppressor of hairy-wing [su(Hw)] locus of Drosophila melanogaster encodes a zinc finger protein that binds a repeated motif in the gypsy retroposon. Mutations of su(Hw) suppress the phenotypes associated with mutations caused by gypsy insertions. To examine the mechanisms by which su(Hw) alters gene expression, a fragment of gypsy containing multiple su(Hw) protein-binding sites was inserted into various locations in the well-characterized Drosophila hsp70 heat shock gene promoter. The authors found no evidence for activation of basal hsp70 transcription by su(Hw) protein in cultured Drosophila cells but observed that it can repress heat shock-induced transcription. Repression occurred only when su(Hw) protein-binding sites were positioned between binding sites for proteins required for heat shock transcription. They propose that su(Hw) protein interferes nonspecifically with protein-protein interactions required for heat shock transcription, perhaps sterically, or by altering the ability of DNA to bend or twist.

  7. The PP2C Alphabet is a negative regulator of stress-activated protein kinase signaling in Drosophila.

    PubMed

    Baril, Caroline; Sahmi, Malha; Ashton-Beaucage, Dariel; Stronach, Beth; Therrien, Marc

    2009-02-01

    The Jun N-terminal kinase and p38 pathways, also known as stress-activated protein kinase (SAPK) pathways, are signaling conduits reiteratively used throughout the development and adult life of metazoans where they play central roles in the control of apoptosis, immune function, and environmental stress responses. We recently identified a Drosophila Ser/Thr phosphatase of the PP2C family, named Alphabet (Alph), which acts as a negative regulator of the Ras/ERK pathway. Here we show that Alph also plays an inhibitory role with respect to Drosophila SAPK signaling during development as well as under stress conditions such as oxidative or genotoxic stresses. Epistasis experiments suggest that Alph acts at a step upstream of the MAPKKs Hep and Lic. Consistent with this interpretation, biochemical experiments identify the upstream MAPKKKs Slpr, Tak1, and Wnd as putative substrates. Together with previous findings, this work identifies Alph as a general attenuator of MAPK signaling in Drosophila.

  8. Manipulations of Amyloid Precursor Protein Cleavage Disrupt the Circadian Clock in Aging Drosophila

    PubMed Central

    Blake, Matthew R.; Holbrook, Scott D.; Kotwica-Rolinska, Joanna; Chow, Eileen; Kretzschmar, Doris; Giebultowicz, Jadwiga M.

    2015-01-01

    Alzheimer’s disease (AD) is a neurodegenerative disease characterized by severe cognitive deterioration. While causes of AD pathology are debated, a large body of evidence suggests that increased cleavage of Amyloid Precursor Protein (APP) producing the neurotoxic Amyloid-β (Aβ) peptide plays a fundamental role in AD pathogenesis. One of the detrimental behavioral symptoms commonly associated with AD is the fragmentation of sleep-activity cycles with increased nighttime activity and daytime naps in humans. Sleep-activity cycles, as well as physiological and cellular rhythms, which may be important for neuronal homeostasis, are generated by a molecular system known as the circadian clock. Links between AD and the circadian system are increasingly evident but not well understood. Here we examined whether genetic manipulations of APP-like (APPL) protein cleavage in Drosophila melanogaster affect rest-activity rhythms and core circadian clock function in this model organism. We show that the increased β-cleavage of endogenous APPL by the β-secretase (dBACE) severely disrupts circadian behavior and leads to reduced expression of clock protein PER in central clock neurons of aging flies. Our data suggest that behavioral rhythm disruption is not a product of APPL-derived Aβ production but rather may be caused by a mechanism common to both α and β-cleavage pathways. Specifically, we show that increased production of the endogenous Drosophila Amyloid Intracellular Domain (dAICD) caused disruption of circadian rest-activity rhythms, while flies overexpressing endogenous APPL maintained stronger circadian rhythms during aging. In summary, our study offers a novel entry point toward understanding the mechanism of circadian rhythm disruption in Alzheimer’s disease. PMID:25766673

  9. Characterization of Fragile X Mental Retardation Protein granules formation and dynamics in Drosophila

    PubMed Central

    Gareau, Cristina; Martel, David; Coudert, Laetitia; Mellaoui, Samia; Mazroui, Rachid

    2013-01-01

    Summary FMRP is an evolutionarily conserved protein that is highly expressed in neurons and its deficiency causes fragile X mental retardation syndrome. FMRP controls the translation of target mRNAs in part by promoting their dynamic transport in neuronal RNA granules. We have previously shown that high expression of mammalian FMRP induces formation of granules termed FMRP granules. These RNA granules are reminiscent of neuronal granules, of stress granules, as well as of the recently described in vitro-assembled granules. In contrast with mammalian FMRP, which has two paralog proteins, Drosophila FMRP (dFMRP) is encoded by a single gene that has no paralog. Using this genetically simple organism, we investigated formation and dynamics of FMRP granules. We found that increased expression of dFMRP in Drosophila cells induces the formation of dynamic dFMRP RNA granules. Mutagenesis studies identified the N-terminal protein–protein domain of dFMRP as a key determinant for FMRP granules formation. The RGG RNA binding motif of dFMRP is dispensable for dFMRP granules formation since its deletion does not prevent formation of those granules. Deletion of the RGG motif reduced, however, dFMRP trafficking between FMRP granules and the cytosol. Similarly, deletion of a large part of the KH RNA binding motif of dFMRP had no effect on formation of dFMRP-granules, but diminished the shuttling activity of dFMRP. Our results thus suggest that the mechanisms controlling formation of RNA granules and those promoting their dynamics are uncoupled. This study opens new avenues to further elucidate the molecular mechanisms controlling FMRP trafficking with its associated mRNAs in and out of RNA granules. PMID:23336078

  10. Phylogenomic analysis reveals dynamic evolutionary history of the Drosophila heterochromatin protein 1 (HP1) gene family.

    PubMed

    Levine, Mia T; McCoy, Connor; Vermaak, Danielle; Lee, Yuh Chwen G; Hiatt, Mary Alice; Matsen, Frederick A; Malik, Harmit S

    2012-01-01

    Heterochromatin is the gene-poor, satellite-rich eukaryotic genome compartment that supports many essential cellular processes. The functional diversity of proteins that bind and often epigenetically define heterochromatic DNA sequence reflects the diverse functions supported by this enigmatic genome compartment. Moreover, heterogeneous signatures of selection at chromosomal proteins often mirror the heterogeneity of evolutionary forces that act on heterochromatic DNA. To identify new such surrogates for dissecting heterochromatin function and evolution, we conducted a comprehensive phylogenomic analysis of the Heterochromatin Protein 1 gene family across 40 million years of Drosophila evolution. Our study expands this gene family from 5 genes to at least 26 genes, including several uncharacterized genes in Drosophila melanogaster. The 21 newly defined HP1s introduce unprecedented structural diversity, lineage-restriction, and germline-biased expression patterns into the HP1 family. We find little evidence of positive selection at these HP1 genes in both population genetic and molecular evolution analyses. Instead, we find that dynamic evolution occurs via prolific gene gains and losses. Despite this dynamic gene turnover, the number of HP1 genes is relatively constant across species. We propose that karyotype evolution drives at least some HP1 gene turnover. For example, the loss of the male germline-restricted HP1E in the obscura group coincides with one episode of dramatic karyotypic evolution, including the gain of a neo-Y in this lineage. This expanded compendium of ovary- and testis-restricted HP1 genes revealed by our study, together with correlated gain/loss dynamics and chromosome fission/fusion events, will guide functional analyses of novel roles supported by germline chromatin.

  11. Identification of regions interacting with ovo{sup D} mutations: Potential new genes involved in germline sex determination or differentiation in Drosophila melanogaster

    SciTech Connect

    Pauli, D.; Oliver, B.; Mahowald, A.P.

    1995-02-01

    Only a few Drosophila melanogaster germline sex determination genes are known, and there have been no systematic screens to identify new genes involved in this important biological process. The ovarian phenotypes produced by females mutant for dominant alleles of the ovo gene are modified in flies with altered doses of other loci involved in germline sex determination in Drosophila (Sex-lethal{sup +}, snas fille{sup +} and ovarian tumor{sup +}). This observation constitutes the basis for a screen to identify additional genes required for proper establishment of germline sexual identity. We tested 300 deletions, which together cover {approximately}58% of the euchromatic portion of the genome, for genetic interactions with ovo{sup D}. Hemizygosity for more than a dozen small regions show interactions that either partially suppress or enhance the ovarian phenotypes of females mutant for one or more of the three dominant ovo mutations. These regions probably contain genes whose products act in developmental heirarchies that include ovo{sup +} protein. 40 refs, 7 figs., 5 tabs.

  12. 14-3-3 proteins regulate Tctp–Rheb interaction for organ growth in Drosophila

    PubMed Central

    Le, Thao Phuong; Vuong, Linh Thuong; Kim, Ah-Ram; Hsu, Ya-Chieh; Choi, Kwang-Wook

    2016-01-01

    14-3-3 family proteins regulate multiple signalling pathways. Understanding biological functions of 14-3-3 proteins has been limited by the functional redundancy of conserved isotypes. Here we provide evidence that 14-3-3 proteins regulate two interacting components of Tor signalling in Drosophila, translationally controlled tumour protein (Tctp) and Rheb GTPase. Single knockdown of 14-3-3ɛ or 14-3-3ζ isoform does not show obvious defects in organ development but causes synergistic genetic interaction with Tctp and Rheb to impair tissue growth. 14-3-3 proteins physically interact with Tctp and Rheb. Knockdown of both 14-3-3 isoforms abolishes the binding between Tctp and Rheb, disrupting organ development. Depletion of 14-3-3s also reduces the level of phosphorylated S6 kinase, phosphorylated Thor/4E-BP and cyclin E (CycE). Growth defects from knockdown of 14-3-3 and Tctp are suppressed by CycE overexpression. This study suggests a novel mechanism of Tor regulation mediated by 14-3-3 interaction with Tctp and Rheb. PMID:27151460

  13. Protein synthesis and degradation are essential to regulate germline stem cell homeostasis in Drosophila testes.

    PubMed

    Yu, Jun; Lan, Xiang; Chen, Xia; Yu, Chao; Xu, Yiwen; Liu, Yujuan; Xu, Lingna; Fan, Heng-Yu; Tong, Chao

    2016-08-15

    The homeostasis of self-renewal and differentiation in stem cells is controlled by intrinsic signals and their niche. We conducted a large-scale RNA interference (RNAi) screen in Drosophila testes and identified 221 genes required for germline stem cell (GSC) maintenance or differentiation. Knockdown of these genes in transit-amplifying spermatogonia and cyst cells further revealed various phenotypes. Complex analysis uncovered that many of the identified genes are involved in key steps of protein synthesis and degradation. A group of genes that are required for mRNA splicing and protein translation contributes to both GSC self-renewal and early germ cell differentiation. Loss of genes in the protein degradation pathway in cyst cells leads to testis tumors consisting of overproliferated germ cells. Importantly, in the Cullin 4-RING E3 ubiquitin ligase (CRL4) complex, we identified multiple proteins that are crucial to GSC self-renewal: pic/DDB1, a CRL4 linker protein, is not only required for GSC self-renewal in flies but also for maintenance of spermatogonial stem cells (SSCs) in mice.

  14. Gene and protein expression of Drosophila Starvin during cold stress and recovery from chill coma.

    PubMed

    Colinet, Hervé; Hoffmann, Ary

    2010-05-01

    In Drosophila melanogaster, the sole member of the Bcl-2-associated anthanogene (BAG)-family proteins, called Starvin (Stv), has only been recently described. BAG proteins regulate a large range of physiological processes including life/death cell balance and stress response. The role of Stv has been poorly studied in the context of abiotic stress and particularly during and after cold stress. In this study we investigated the temporal expression of Stv gene and protein in adult flies during both the cold stress (up to 9 h at 0 degrees C) and the subsequent recovery phase (up to 8 h at 25 degrees C). Because BAG proteins can regulate positively and negatively the function of Hsp70/Hsc70, we also checked whether Stv expression was related to Hsp70 and Hsc70. Stv mRNA and Stv protein both showed a similar expression pattern: no modulation during the cold period followed by a significant up-regulation during the recovery period. A coordinated response of Stv and Hsp70 mRNA was observed, but not for Hsc70. Our findings indicate that Stv expression is part of a stress-induced program in D. melanogaster. It probably acts as a co-chaperone modulating the activity of Hsp70 chaperone machinery during recovery from cold stress. Finally our results support the suggestion that Stv and human BAG3 may be functional homologs.

  15. [Drosophila melanogaster gene Merlin interacts with the clathrin adaptor protein gene lap].

    PubMed

    Kopyl, S A; Dorogova, N V; Akhmamet'eva, E M; Omel'ianchuk, L V; Chang, L -S

    2010-03-01

    The protein Merlin is involved in the regulation of cell proliferation and differentiation in the eyes and wings of Drosophila and is a homolog of the human protein encoded by the Neurofibromatosis 2 (NF2) gene whose mutations cause auricular nerve tumors. Recent studies show that Merlin and Expanded cooperatively regulate the recycling of membrane receptors, such as the epidermal growth factor receptor (EGFR). By performing a search for potential genetic interactions between Merlin (Mer) and the genes important for vesicular trafficking, we found that ectopic expression in the wing pouch of the clathrin adapter protein Lap involved in clathrin-mediated receptor endocytosis resulted in the formation of extra vein materials. On the one hand, coexpression of wild-type Merlin and lap in the wing pouch restored normal venation, while overexpression of a dominant-negative mutant Mer(DBB) together with lap enhanced ectopic vein formation. Using various constructs with Merlin truncated copies, we showed the C-terminal portion of the Merlin protein to be responsible for the Merlin-lap genetic interaction. Furthermore, we showed that the Merlin and Lap proteins colocalized at the cortex of the wing imaginal disc cells.

  16. Nature and function of insulator protein binding sites in the Drosophila genome.

    PubMed

    Schwartz, Yuri B; Linder-Basso, Daniela; Kharchenko, Peter V; Tolstorukov, Michael Y; Kim, Maria; Li, Hua-Bing; Gorchakov, Andrey A; Minoda, Aki; Shanower, Gregory; Alekseyenko, Artyom A; Riddle, Nicole C; Jung, Youngsook L; Gu, Tingting; Plachetka, Annette; Elgin, Sarah C R; Kuroda, Mitzi I; Park, Peter J; Savitsky, Mikhail; Karpen, Gary H; Pirrotta, Vincenzo

    2012-11-01

    Chromatin insulator elements and associated proteins have been proposed to partition eukaryotic genomes into sets of independently regulated domains. Here we test this hypothesis by quantitative genome-wide analysis of insulator protein binding to Drosophila chromatin. We find distinct combinatorial binding of insulator proteins to different classes of sites and uncover a novel type of insulator element that binds CP190 but not any other known insulator proteins. Functional characterization of different classes of binding sites indicates that only a small fraction act as robust insulators in standard enhancer-blocking assays. We show that insulators restrict the spreading of the H3K27me3 mark but only at a small number of Polycomb target regions and only to prevent repressive histone methylation within adjacent genes that are already transcriptionally inactive. RNAi knockdown of insulator proteins in cultured cells does not lead to major alterations in genome expression. Taken together, these observations argue against the concept of a genome partitioned by specialized boundary elements and suggest that insulators are reserved for specific regulation of selected genes.

  17. Nature and function of insulator protein binding sites in the Drosophila genome

    PubMed Central

    Schwartz, Yuri B.; Linder-Basso, Daniela; Kharchenko, Peter V.; Tolstorukov, Michael Y.; Kim, Maria; Li, Hua-Bing; Gorchakov, Andrey A.; Minoda, Aki; Shanower, Gregory; Alekseyenko, Artyom A.; Riddle, Nicole C.; Jung, Youngsook L.; Gu, Tingting; Plachetka, Annette; Elgin, Sarah C.R.; Kuroda, Mitzi I.; Park, Peter J.; Savitsky, Mikhail; Karpen, Gary H.; Pirrotta, Vincenzo

    2012-01-01

    Chromatin insulator elements and associated proteins have been proposed to partition eukaryotic genomes into sets of independently regulated domains. Here we test this hypothesis by quantitative genome-wide analysis of insulator protein binding to Drosophila chromatin. We find distinct combinatorial binding of insulator proteins to different classes of sites and uncover a novel type of insulator element that binds CP190 but not any other known insulator proteins. Functional characterization of different classes of binding sites indicates that only a small fraction act as robust insulators in standard enhancer-blocking assays. We show that insulators restrict the spreading of the H3K27me3 mark but only at a small number of Polycomb target regions and only to prevent repressive histone methylation within adjacent genes that are already transcriptionally inactive. RNAi knockdown of insulator proteins in cultured cells does not lead to major alterations in genome expression. Taken together, these observations argue against the concept of a genome partitioned by specialized boundary elements and suggest that insulators are reserved for specific regulation of selected genes. PMID:22767387

  18. Nuclear import of the Drosophila Rel protein Dorsal is regulated by phosphorylation.

    PubMed

    Drier, E A; Huang, L H; Steward, R

    1999-03-01

    In Drosophila, dorsal-ventral polarity is determined by a maternally encoded signal transduction pathway that culminates in the graded nuclear localization of the Rel protein, Dorsal. Dorsal is retained in the cytoplasm by the IkappaB protein, Cactus. Signal-dependent phosphorylation of Cactus results in the degradation of Cactus and the nuclear targeting of Dorsal. We present an in-depth study of the functional importance of Dorsal phosphorylation. We find that Dorsal is phosphorylated by the ventral signal while associated with Cactus, and that Dorsal phosphorylation is essential for its nuclear import. In vivo phospholabeling of Dorsal is limited to serine residues in both ovaries and early embryos. A protein bearing mutations in six conserved serines abolishes Dorsal activity, is constitutively cytoplasmic, and appears to eliminate Dorsal phosphorylation, but still interacts with Cactus. Two individual serine-to-alanine mutations produce unexpected results. In a wild-type signaling background, a mutation in the highly conserved PKA site (S312) produces only a weak loss-of-function; however, it completely destabilizes the protein in a cactus mutant background. Significantly, the phosphorylation of another completely conserved serine (S317) regulates the high level of nuclear import found in ventral cells. We conclude that the formation of a wild-type Dorsal nuclear gradient requires the phosphorylation of both Cactus and Dorsal. The strong conservation of the serines suggests that phosphorylation of other Rel proteins is essential for their proper nuclear targeting.

  19. Distinct functions of the Drosophila Nup153 and Nup214 FG domains in nuclear protein transport.

    PubMed

    Sabri, Nafiseh; Roth, Peggy; Xylourgidis, Nikos; Sadeghifar, Fatemeh; Adler, Jeremy; Samakovlis, Christos

    2007-08-13

    The phenylanine-glycine (FG)-rich regions of several nucleoporins both bind to nuclear transport receptors and collectively provide a diffusion barrier to the nuclear pores. However, the in vivo roles of FG nucleoporins in transport remain unclear. We have inactivated 30 putative nucleoporins in cultured Drosophila melanogaster S2 cells by RNA interference and analyzed the phenotypes on importin alpha/beta-mediated import and CRM1-dependent protein export. The fly homologues of FG nucleoporins Nup358, Nup153, and Nup54 are selectively required for import. The FG repeats of Nup153 are necessary for its function in transport, whereas the remainder of the protein maintains pore integrity. Inactivation of the CRM1 cofactor RanBP3 decreased the nuclear accumulation of CRM1 and protein export. We report a surprisingly antagonistic relationship between RanBP3 and the Nup214 FG region in determining CRM1 localization and its function in protein export. Our data suggest that peripheral metazoan FG nucleoporins have distinct functions in nuclear protein transport events.

  20. Drosophila IAP1-mediated ubiquitylation controls activation of the initiator caspase DRONC independent of protein degradation.

    PubMed

    Lee, Tom V; Fan, Yun; Wang, Shiuan; Srivastava, Mayank; Broemer, Meike; Meier, Pascal; Bergmann, Andreas

    2011-09-01

    Ubiquitylation targets proteins for proteasome-mediated degradation and plays important roles in many biological processes including apoptosis. However, non-proteolytic functions of ubiquitylation are also known. In Drosophila, the inhibitor of apoptosis protein 1 (DIAP1) is known to ubiquitylate the initiator caspase DRONC in vitro. Because DRONC protein accumulates in diap1 mutant cells that are kept alive by caspase inhibition ("undead" cells), it is thought that DIAP1-mediated ubiquitylation causes proteasomal degradation of DRONC, protecting cells from apoptosis. However, contrary to this model, we show here that DIAP1-mediated ubiquitylation does not trigger proteasomal degradation of full-length DRONC, but serves a non-proteolytic function. Our data suggest that DIAP1-mediated ubiquitylation blocks processing and activation of DRONC. Interestingly, while full-length DRONC is not subject to DIAP1-induced degradation, once it is processed and activated it has reduced protein stability. Finally, we show that DRONC protein accumulates in "undead" cells due to increased transcription of dronc in these cells. These data refine current models of caspase regulation by IAPs.

  1. Drosophila IAP1-Mediated Ubiquitylation Controls Activation of the Initiator Caspase DRONC Independent of Protein Degradation

    PubMed Central

    Wang, Shiuan; Srivastava, Mayank; Broemer, Meike; Meier, Pascal; Bergmann, Andreas

    2011-01-01

    Ubiquitylation targets proteins for proteasome-mediated degradation and plays important roles in many biological processes including apoptosis. However, non-proteolytic functions of ubiquitylation are also known. In Drosophila, the inhibitor of apoptosis protein 1 (DIAP1) is known to ubiquitylate the initiator caspase DRONC in vitro. Because DRONC protein accumulates in diap1 mutant cells that are kept alive by caspase inhibition (“undead” cells), it is thought that DIAP1-mediated ubiquitylation causes proteasomal degradation of DRONC, protecting cells from apoptosis. However, contrary to this model, we show here that DIAP1-mediated ubiquitylation does not trigger proteasomal degradation of full-length DRONC, but serves a non-proteolytic function. Our data suggest that DIAP1-mediated ubiquitylation blocks processing and activation of DRONC. Interestingly, while full-length DRONC is not subject to DIAP1-induced degradation, once it is processed and activated it has reduced protein stability. Finally, we show that DRONC protein accumulates in “undead” cells due to increased transcription of dronc in these cells. These data refine current models of caspase regulation by IAPs. PMID:21909282

  2. The spliceosome-associated protein Mfap1 binds to VCP in Drosophila

    PubMed Central

    Rode, Sandra; Ohm, Henrike; Zipfel, Jaqueline

    2017-01-01

    Posttranscriptional regulation of gene expression contributes to many developmental transitions. Previously, we found that the AAA chaperone Valosin-Containing Protein (VCP) regulates ecdysone-dependent dendrite pruning of Drosophila class IV dendritic arborization (c4da) neurons via an effect on RNA metabolism. In a search for RNA binding proteins associated with VCP, we identified the spliceosome-associated protein Mfap1, a component of the tri-snRNP complex. Mfap1 is a nucleolar protein in neurons and its levels are regulated by VCP. Mfap1 binds to VCP and TDP-43, a disease-associated RNA-binding protein. via distinct regions in its N- and C-terminal halfs. Similar to vcp mutations, Mfap1 overexpression causes c4da neuron dendrite pruning defects and mislocalization of TDP-43 in these cells, but genetic analyses show that Mfap1 is not a crucial VCP target during dendrite pruning. Finally, rescue experiments with a lethal mfap1 mutant show that the VCP binding region is not essential for Mfap1 function, but may act to increase its stability or activity. PMID:28837687

  3. The spliceosome-associated protein Mfap1 binds to VCP in Drosophila.

    PubMed

    Rode, Sandra; Ohm, Henrike; Zipfel, Jaqueline; Rumpf, Sebastian

    2017-01-01

    Posttranscriptional regulation of gene expression contributes to many developmental transitions. Previously, we found that the AAA chaperone Valosin-Containing Protein (VCP) regulates ecdysone-dependent dendrite pruning of Drosophila class IV dendritic arborization (c4da) neurons via an effect on RNA metabolism. In a search for RNA binding proteins associated with VCP, we identified the spliceosome-associated protein Mfap1, a component of the tri-snRNP complex. Mfap1 is a nucleolar protein in neurons and its levels are regulated by VCP. Mfap1 binds to VCP and TDP-43, a disease-associated RNA-binding protein. via distinct regions in its N- and C-terminal halfs. Similar to vcp mutations, Mfap1 overexpression causes c4da neuron dendrite pruning defects and mislocalization of TDP-43 in these cells, but genetic analyses show that Mfap1 is not a crucial VCP target during dendrite pruning. Finally, rescue experiments with a lethal mfap1 mutant show that the VCP binding region is not essential for Mfap1 function, but may act to increase its stability or activity.

  4. Proteomics approach to study the functions of Drosophila myosin VI through identification of multiple cargo-binding proteins

    PubMed Central

    Finan, Dina; Hartman, M. Amanda; Spudich, James A.

    2011-01-01

    Myosin VI is a molecular motor implicated in many processes, and it likely associates with a variety of cargoes that specify its functions. Although it is critical to Drosophila development, little is known about its cellular roles. To reveal its involvement in specific pathways, we sought to identify the binding partners of Drosophila myosin VI. We used affinity chromatography and mass spectrometry to discover interacting proteins, which we tested for direct binding. Using this approach, we found that the microtubule-associated protein Cornetto bound myosin VI, and we demonstrated a role for both in secretion of the lipidated morphogen Hedgehog. We also identified a number of other binding proteins, and further characterization of their interactions with myosin VI will advance our understanding of the roles of these complexes in cellular and developmental processes. Thus, our method has provided us the means to gain valuable insight into the multifaceted roles of a motor protein in vivo. PMID:21368190

  5. A visual screen for diet-regulated proteins in the Drosophila ovary using GFP protein trap lines.

    PubMed

    Hsu, Hwei-Jan; Drummond-Barbosa, Daniela

    2017-01-01

    The effect of diet on reproduction is well documented in a large number of organisms; however, much remains to be learned about the molecular mechanisms underlying this connection. The Drosophila ovary has a well described, fast and largely reversible response to diet. Ovarian stem cells and their progeny proliferate and grow faster on a yeast-rich diet than on a yeast-free (poor) diet, and death of early germline cysts, degeneration of early vitellogenic follicles and partial block in ovulation further contribute to the ∼60-fold decrease in egg laying observed on a poor diet. Multiple diet-dependent factors, including insulin-like peptides, the steroid ecdysone, the nutrient sensor Target of Rapamycin, AMP-dependent kinase, and adipocyte factors mediate this complex response. Here, we describe the results of a visual screen using a collection of green fluorescent protein (GFP) protein trap lines to identify additional factors potentially involved in this response. In each GFP protein trap line, an artificial GFP exon is fused in frame to an endogenous protein, such that the GFP fusion pattern parallels the levels and subcellular localization of the corresponding native protein. We identified 53 GFP-tagged proteins that exhibit changes in levels and/or subcellular localization in the ovary at 12-16 hours after switching females from rich to poor diets, suggesting them as potential candidates for future functional studies. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Drosophila enhancer-Gal4 lines show ectopic expression during development

    PubMed Central

    Arnés, Mercedes; Ferrús, Alberto

    2017-01-01

    In Drosophila melanogaster the most widely used technique to drive gene expression is the binary UAS/Gal4 system. We show here that a set of nervous system specific enhancers (elav, D42/Toll-6, OK6/RapGAP1) display ectopic activity in epithelial tissues during development, which is seldom considered in experimental studies. This ectopic activity is variable, unstable and influenced by the primary sequence of the enhancer and the insertion site in the chromosome. In addition, the ectopic activity is independent of the protein expressed, Gal4, as it is reproduced also with the expression of Gal80. Another enhancer, LN2 from the sex lethal (Sxl) gene, shows sex-dependent features in its ectopic expression. Feminization of LN2 expressing males does not alter the male specific pattern indicating that the sexual dimorphism of LN2 expression is an intrinsic feature of this enhancer. Other X chromosome enhancers corresponding to genes not related to sex determination do not show sexual dimorphism in their ectopic expressions. Although variable and unstable, the ectopic activation of enhancer-Gal4 lines seems to be regulated in terms of tissue and intensity. To characterize the full domain of expression of enhancer-Gal4 constructs is relevant for the design of transgenic animal models and biotechnology tools, as well as for the correct interpretation of developmental and behavioural studies in which Gal4 lines are used. PMID:28405401

  7. The Embryonically Active Gene, Unkempt, of Drosophila Encodes a Cys(3)his Finger Protein

    PubMed Central

    Mohler, J.; Weiss, N.; Murli, S.; Mohammadi, S.; Vani, K.; Vasilakis, G.; Song, C. H.; Epstein, A.; Kuang, T.; English, J.; Cherdak, D.

    1992-01-01

    The unkempt gene of Drosophila encodes a set of embryonic RNAs, which are abundant during early stages of embryogenesis and are present ubiquitously in most somatic tissues from the syncytial embryo through stage 15 of embryogenesis. Expression of unkempt RNAs becomes restricted predominantly to the central nervous system in stages 16 and early 17. Analysis of cDNAs from this locus reveals the presence of five Cys(3)His fingers in the protein product. Isolation and analysis of mutations affecting the unkempt gene, including complete deletions of this gene, indicate that there is no zygotic requirement for unkempt during embryogenesis, presumably due to the contribution of maternally supplied RNA, although the gene is essential during post-embryonic development. PMID:1339381

  8. Inducible protein expression in Drosophila Schneider 2 cells using the lac operator-repressor system.

    PubMed

    Wakiyama, Motoaki; Muramatsu, Reiko; Kaitsu, Yoko; Ikeda, Mariko; Yokoyama, Shigeyuki

    2011-12-01

    Schneider line 2 cells, derived from Drosophila melanogaster, can be used as a highly versatile gene expression system. Two powerful promoters derived from the actin5C (Ac5) and metallothionein (Mtn) genes are available. The Mtn promoter can be used for the inducible expression of heterologous proteins unsuitable for constitutive expression. However, to circumvent using CuSO(4) or CdCl(2) as inducers of the Mtn promoter, we created a modified Ac5 promoter, Ac5LacO, in which two short lac operator sequences are embedded. Expression from the Ac5LacO promoter was regulated with co-expression of the lac repressor and IPTG. More than 25-fold induction of firefly luciferase expression was achieved in transient transfection experiments. Furthermore, we demonstrated that the lac operator-repressor regulatory system functioned in chromosomally integrated cell lines.

  9. Neighboring genes for DNA-binding proteins rescue male sterility in Drosophila hybrids

    PubMed Central

    Liénard, Marjorie A.; Araripe, Luciana O.; Hartl, Daniel L.

    2016-01-01

    Crosses between closely related animal species often result in male hybrids that are sterile, and the molecular and functional basis of genetic factors for hybrid male sterility is of great interest. Here, we report a molecular and functional analysis of HMS1, a region of 9.2 kb in chromosome 3 of Drosophila mauritiana, which results in virtually complete hybrid male sterility when homozygous in the genetic background of sibling species Drosophila simulans. The HMS1 region contains two strong candidate genes for the genetic incompatibility, agt and Taf1. Both encode unrelated DNA-binding proteins, agt for an alkyl-cysteine-S-alkyltransferase and Taf1 for a subunit of transcription factor TFIID that serves as a multifunctional transcriptional regulator. The contribution of each gene to hybrid male sterility was assessed by means of germ-line transformation, with constructs containing complete agt and Taf1 genomic sequences as well as various chimeric constructs. Both agt and Taf1 contribute about equally to HMS1 hybrid male sterility. Transgenes containing either locus rescue sterility in about one-half of the males, and among fertile males the number of offspring is in the normal range. This finding suggests compensatory proliferation of the rescued, nondysfunctional germ cells. Results with chimeric transgenes imply that the hybrid incompatibilities result from interactions among nucleotide differences residing along both agt and Taf1. Our results challenge a number of preliminary generalizations about the molecular and functional basis of hybrid male sterility, and strongly reinforce the role of DNA-binding proteins as a class of genes contributing to the maintenance of postzygotic reproductive isolation. PMID:27357670

  10. Crumbs affects protein dynamics in anterior regions of the developing Drosophila embryo.

    PubMed

    Firmino, João; Tinevez, Jean-Yves; Knust, Elisabeth

    2013-01-01

    Maintenance of apico-basal polarity is essential for epithelial integrity and requires particular reinforcement during tissue morphogenesis, when cells are reorganised, undergo shape changes and remodel their junctions. It is well established that epithelial integrity during morphogenetic processes depends on the dynamic exchange of adherens junction components, but our knowledge on the dynamics of other proteins and their dynamics during these processes is still limited. The early Drosophila embryo is an ideal system to study membrane dynamics during morphogenesis. Here, morphogenetic activities differ along the anterior-posterior axis, with the extending germband showing a high degree of epithelial remodelling. We developed a Fluorescence Recovery After Photobleaching (FRAP) assay with a higher temporal resolution, which allowed the distinction between a fast and a slow component of recovery of membrane proteins during the germband extension stage. We show for the first time that the recovery kinetics of a general membrane marker, SpiderGFP, differs in the anterior and posterior parts of the embryo, which correlates well with the different morphogenetic activities of the respective embryonic regions. Interestingly, absence of crumbs, a polarity regulator essential for epithelial integrity in the Drosophila embryo, decreases the fast component of SpiderGFP and of the apical marker Stranded at Second-Venus specifically in the anterior region. We suggest that the defects in kinetics observed in crumbs mutant embryos are the first signs of tissue instability in this region, explaining the earlier breakdown of the head epidermis in comparison to that of the trunk, and that diffusion in the plasma membrane is affected by the absence of Crumbs.

  11. Fragile X mental retardation protein regulates trans-synaptic signaling in Drosophila.

    PubMed

    Friedman, Samuel H; Dani, Neil; Rushton, Emma; Broadie, Kendal

    2013-11-01

    Fragile X syndrome (FXS), the most common inherited determinant of intellectual disability and autism spectrum disorders, is caused by loss of the fragile X mental retardation 1 (FMR1) gene product (FMRP), an mRNA-binding translational repressor. A number of conserved FMRP targets have been identified in the well-characterized Drosophila FXS disease model, but FMRP is highly pleiotropic in function and the full spectrum of FMRP targets has yet to be revealed. In this study, screens for upregulated neural proteins in Drosophila fmr1 (dfmr1) null mutants reveal strong elevation of two synaptic heparan sulfate proteoglycans (HSPGs): GPI-anchored glypican Dally-like protein (Dlp) and transmembrane Syndecan (Sdc). Our recent work has shown that Dlp and Sdc act as co-receptors regulating extracellular ligands upstream of intracellular signal transduction in multiple trans-synaptic pathways that drive synaptogenesis. Consistently, dfmr1 null synapses exhibit altered WNT signaling, with changes in both Wingless (Wg) ligand abundance and downstream Frizzled-2 (Fz2) receptor C-terminal nuclear import. Similarly, a parallel anterograde signaling ligand, Jelly belly (Jeb), and downstream ERK phosphorylation (dpERK) are depressed at dfmr1 null synapses. In contrast, the retrograde BMP ligand Glass bottom boat (Gbb) and downstream signaling via phosphorylation of the transcription factor MAD (pMAD) seem not to be affected. To determine whether HSPG upregulation is causative for synaptogenic defects, HSPGs were genetically reduced to control levels in the dfmr1 null background. HSPG correction restored both (1) Wg and Jeb trans-synaptic signaling, and (2) synaptic architecture and transmission strength back to wild-type levels. Taken together, these data suggest that FMRP negatively regulates HSPG co-receptors controlling trans-synaptic signaling during synaptogenesis, and that loss of this regulation causes synaptic structure and function defects characterizing the FXS disease

  12. Drosophila DOCK Family Protein Zizimin Involves in Pigment Cell Differentiation in Pupal Retinae.

    PubMed

    Ozasa, Fumito; Morishita, Kazushige; Dang, Ngoc Anh Suong; Miyata, Seiji; Yoshida, Hideki; Yamaguchi, Masamitsu

    2017-08-26

    The dedicator of cytokinesis (DOCK) family proteins are known as one of guanine nucleotide exchange factors (GEFs), that contribute to cellular signaling processes by activating small G proteins. Although mammalian Zizimin is known to be a GEF for Cdc42 of Rho family small GTPase, its role in vivo is not well understood. Here we studied in vivo function of Drosophila Zizimin (Ziz). Knockdown of Ziz in eye imaginal discs induced the rough eye phenotype accompanied with fusion of ommatidia, loss of bristles and loss of pigments. Immunostaining analyses revealed that Ziz mainly localizes in the secondary pigment cells (SPCs) and tertiary pigment cells (TPCs) in pupal retinae. Ziz-knockdown induced SPC- and TPC-like cells with aberrant morphology in the pupal retina. Delta (Dl), a downstream target of EGFR signaling is known to regulate pigment cell differentiation. Loss-of-function mutation of Dl suppressed the rough eye phenotype and the defect in differentiation of SPCs and TPCs in Ziz-knockdown flies. Moreover, Ziz-knockdown increased Dl expression level especially in SPCs and TPCs. In addition, mutations of rhomboid-1 and roughoid that are activators of EGFR signaling pathway also suppressed both the rough eye phenotype and the defect in differentiation of SPCs and TPCs in Ziz-knockdown flies. Activation of EGFR signaling in Ziz-knockdown flies were further confirmed by immunostaining with anti-diphospho ERK IgG. These results indicate that Ziz negatively regulates the Dl expression in SPCs and TPCs to control differentiation of pigment cells and this regulation is mediated by EGFR signaling pathway.Key words: Zizimin, DOCK, EGFR signaling pathway, pigment cell, Drosophila.

  13. Localization and Function of Pals1-associated Tight Junction Protein in Drosophila Is Regulated by Two Distinct Apical Complexes.

    PubMed

    Sen, Arnab; Sun, Rui; Krahn, Michael P

    2015-05-22

    The transmembrane protein Crumbs (Crb) and its intracellular adaptor protein Pals1 (Stardust, Sdt in Drosophila) play a crucial role in the establishment and maintenance of apical-basal polarity in epithelial cells in various organisms. In contrast, the multiple PDZ domain-containing protein Pals1-associated tight junction protein (PATJ), which has been described to form a complex with Crb/Sdt, is not essential for apical basal polarity or for the stability of the Crb/Sdt complex in the Drosophila epidermis. Here we show that, in the embryonic epidermis, Sdt is essential for the correct subcellular localization of PATJ in differentiated epithelial cells but not during cellularization. Consistently, the L27 domain of PATJ is crucial for the correct localization and function of the protein. Our data further indicate that the four PDZ domains of PATJ function, to a large extent, in redundancy, regulating the function of the protein. Interestingly, the PATJ-Sdt heterodimer is not only recruited to the apical cell-cell contacts by binding to Crb but depends on functional Bazooka (Baz). However, biochemical experiments show that PATJ associates with both complexes, the Baz-Sdt and the Crb-Sdt complex, in the mature epithelium of the embryonic epidermis, suggesting a role of these two complexes for the function of PATJ during the development of Drosophila.

  14. The Distribution of GYR- and YLP-Like Motifs in Drosophila Suggests a General Role in Cuticle Assembly and Other Protein-Protein Interactions

    PubMed Central

    Cornman, R. Scott

    2010-01-01

    Background Arthropod cuticle is composed predominantly of a self-assembling matrix of chitin and protein. Genes encoding structural cuticular proteins are remarkably abundant in arthropod genomes, yet there has been no systematic survey of conserved motifs across cuticular protein families. Methodology/Principal Findings Two short sequence motifs with conserved tyrosines were identified in Drosophila cuticular proteins that were similar to the GYR and YLP Interpro domains. These motifs were found in members of the CPR, Tweedle, CPF/CPFL, and (in Anopheles gambiae) CPLCG cuticular protein families, and the Dusky/Miniature family of cuticle-associated proteins. Tweedle proteins have a characteristic motif architecture that is shared with the Drosophila protein GCR1 and its orthologs in other species, suggesting that GCR1 is also cuticular. A resilin repeat, which has been shown to confer elasticity, matched one of the motifs; a number of other Drosophila proteins of unknown function exhibit a motif architecture similar to that of resilin. The motifs were also present in some proteins of the peritrophic matrix and the eggshell, suggesting molecular convergence among distinct extracellular matrices. More surprisingly, gene regulation, development, and proteolysis were statistically over-represented ontology terms for all non-cuticular matches in Drosophila. Searches against other arthropod genomes indicate that the motifs are taxonomically widespread. Conclusions This survey suggests a more general definition for GYR and YLP motifs and reveals their contribution to several types of extracellular matrix. They may define sites of protein interaction with DNA or other proteins, based on ontology analysis. These results can help guide experimental studies on the biochemistry of cuticle assembly. PMID:20824096

  15. Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins in Drosophila Oocytes

    PubMed Central

    Das, Arunika; Shah, Shital J.; Fan, Bensen; Paik, Daniel; DiSanto, Daniel J.; Hinman, Anna Maria; Cesario, Jeffry M.; Battaglia, Rachel A.; Demos, Nicole; McKim, Kim S.

    2016-01-01

    Oocytes segregate chromosomes in the absence of centrosomes. In this situation, the chromosomes direct spindle assembly. It is still unclear in this system which factors are required for homologous chromosome bi-orientation and spindle assembly. The Drosophila kinesin-6 protein Subito, although nonessential for mitotic spindle assembly, is required to organize a bipolar meiotic spindle and chromosome bi-orientation in oocytes. Along with the chromosomal passenger complex (CPC), Subito is an important part of the metaphase I central spindle. In this study we have conducted genetic screens to identify genes that interact with subito or the CPC component Incenp. In addition, the meiotic mutant phenotype for some of the genes identified in these screens were characterized. We show, in part through the use of a heat-shock-inducible system, that the Centralspindlin component RacGAP50C and downstream regulators of cytokinesis Rho1, Sticky, and RhoGEF2 are required for homologous chromosome bi-orientation in metaphase I oocytes. This suggests a novel function for proteins normally involved in mitotic cell division in the regulation of microtubule–chromosome interactions. We also show that the kinetochore protein, Polo kinase, is required for maintaining chromosome alignment and spindle organization in metaphase I oocytes. In combination our results support a model where the meiotic central spindle and associated proteins are essential for acentrosomal chromosome segregation. PMID:26564158

  16. The SCF ubiquitin ligase protein slimb regulates centrosome duplication in Drosophila.

    PubMed

    Wojcik, E J; Glover, D M; Hays, T S

    2000-09-21

    The duplication of the centrosome is a key event in the cell-division cycle. Although defects in centrosome duplication are thought to contribute to genomic instability [1-3] and are a hallmark of certain transformed cells and human cancer [4-6], the mechanism responsible for centrosome duplication is not understood. Recent experiments have established that centrosome duplication requires the activity of cyclin-dependent kinase 2 (Cdk2) and cyclins E and A [7-9]. The stability of cyclin E is regulated by the ubiquitin ligase SCF, which is a protein complex composed of Skp1, Cdc53 (Cullin) and F-box proteins [10-12]. The Skp1 and Cullin components have been detected on mammalian centrosomes, and shown to be essential for centrosome duplication and separation in Xenopus [13]. Here, we report that Slimb, an F-box protein that targets proteins to the SCFcomplex [14,15], plays a role in limiting centrosome replication. We found that, in the fruit fly Drosophila, the hypomorphic mutation slimb(crd) causes the appearance of additional centrosomes and mitotic defects in mutant larval neuroblasts.

  17. Distinct Roles of Chromatin Insulator Proteins in Control of the Drosophila Bithorax Complex

    PubMed Central

    Savitsky, Mikhail; Kim, Maria; Kravchuk, Oksana; Schwartz, Yuri B.

    2016-01-01

    Chromatin insulators are remarkable regulatory elements that can bring distant genomic sites together and block unscheduled enhancer–promoter communications. Insulators act via associated insulator proteins of two classes: sequence-specific DNA binding factors and “bridging” proteins. The latter are required to mediate interactions between distant insulator elements. Chromatin insulators are critical for correct expression of complex loci; however, their mode of action is poorly understood. Here, we use the Drosophila bithorax complex as a model to investigate the roles of the bridging proteins Cp190 and Mod(mdg4). The bithorax complex consists of three evolutionarily conserved homeotic genes Ubx, abd-A, and Abd-B, which specify anterior–posterior identity of the last thoracic and all abdominal segments of the fly. Looking at effects of CTCF, mod(mdg4), and Cp190 mutations on expression of the bithorax complex genes, we provide the first functional evidence that Mod(mdg4) acts in concert with the DNA binding insulator protein CTCF. We find that Mod(mdg4) and Cp190 are not redundant and may have distinct functional properties. We, for the first time, demonstrate that Cp190 is critical for correct regulation of the bithorax complex and show that Cp190 is required at an exceptionally strong Fub insulator to partition the bithorax complex into two topological domains. PMID:26715665

  18. Lava lamp, a novel peripheral golgi protein, is required for Drosophila melanogaster cellularization.

    PubMed

    Sisson, J C; Field, C; Ventura, R; Royou, A; Sullivan, W

    2000-11-13

    Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, we have conducted a biochemical screen for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. We now show that a novel MMAP, Lava Lamp (Lva), is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Our functional analysis shows that cellularization is dramatically inhibited upon injecting anti-Lva antibodies (IgG and Fab) into embryos. In addition, we show that brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. Our results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion.

  19. Lava Lamp, a Novel Peripheral Golgi Protein, Is Required for Drosophila melanogaster Cellularization

    PubMed Central

    Sisson, John C.; Field, Christine; Ventura, Richard; Royou, Anne; Sullivan, William

    2000-01-01

    Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, we have conducted a biochemical screen for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. We now show that a novel MMAP, Lava Lamp (Lva), is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Our functional analysis shows that cellularization is dramatically inhibited upon injecting anti–Lva antibodies (IgG and Fab) into embryos. In addition, we show that brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. Our results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion. PMID:11076973

  20. Aging results in an unusual expression of Drosophila heat shock proteins

    SciTech Connect

    Fleming, J.E.; Walton, J.K.; Dubitsky, R.; Bensch, K.G. )

    1988-06-01

    The authors used high-resolution two-dimensional polyacrylamide gel electrophoresis to evaluate the effect of aging on the heat shock response in Drosophila melanogaster. Although the aging process is not well understood at the molecular level, recent observations suggest that quantitative changes in gene expression occur as these fruit flies approach senescence. Such genetic alterations are in accord with our present data, which clearly show marked differences in the synthesis of heat shock proteins between young and old fruit flies. In 10-day-old flies, a heat shock of 20 min results in the expression of 14 new proteins as detectable by two-dimensional electrophoresis of ({sup 35}S)methionine-labeled polypeptides, whereas identical treatment of 45-day-old flies leads to the expression of at least 50 new or highly up-regulated proteins. In addition, there is also a concomitant increase in the rate of synthesis of a number of the normal proteins in the older animals. Microdensitometric determinations of the low molecular weight heat shock polypeptides on autoradiographs of five age groups revealed that their maximum expression occurs at 47 days for a population of flies with a mean life span of 33.7 days. Moreover, a heat shock effect similar to that observed in senescent flies occurs in young flies fed canavanine, an arginine analogue, before heat shock.

  1. The Drosophila nuclear lamina protein otefin is required for germline stem cell survival.

    PubMed

    Barton, Lacy J; Pinto, Belinda S; Wallrath, Lori L; Geyer, Pamela K

    2013-06-24

    LEM domain (LEM-D) proteins are components of an extensive protein network that assembles beneath the inner nuclear envelope. Defects in LEM-D proteins cause tissue-restricted human diseases associated with altered stem cell homeostasis. Otefin (Ote) is a Drosophila LEM-D protein that is intrinsically required for female germline stem cell (GSC) maintenance. Previous studies linked Ote loss with transcriptional activation of the key differentiation gene bag-of-marbles (bam), leading to the model in which Ote tethers the bam gene to the nuclear periphery for gene silencing. Using genetic and phenotypic analyses of multiple ote(-/-) backgrounds, we obtained evidence that is inconsistent with this model. We show that bam repression is maintained in ote(-/-) GSCs and that germ cell loss persists in ote(-/-), bam(-/-) mutants, together demonstrating that GSC loss is independent of bam transcription. We show that the primary defect in ote(-/-) GSCs is a block of differentiation, which ultimately leads to germ cell death.

  2. Distinct Roles of Chromatin Insulator Proteins in Control of the Drosophila Bithorax Complex.

    PubMed

    Savitsky, Mikhail; Kim, Maria; Kravchuk, Oksana; Schwartz, Yuri B

    2016-02-01

    Chromatin insulators are remarkable regulatory elements that can bring distant genomic sites together and block unscheduled enhancer-promoter communications. Insulators act via associated insulator proteins of two classes: sequence-specific DNA binding factors and "bridging" proteins. The latter are required to mediate interactions between distant insulator elements. Chromatin insulators are critical for correct expression of complex loci; however, their mode of action is poorly understood. Here, we use the Drosophila bithorax complex as a model to investigate the roles of the bridging proteins Cp190 and Mod(mdg4). The bithorax complex consists of three evolutionarily conserved homeotic genes Ubx, abd-A, and Abd-B, which specify anterior-posterior identity of the last thoracic and all abdominal segments of the fly. Looking at effects of CTCF, mod(mdg4), and Cp190 mutations on expression of the bithorax complex genes, we provide the first functional evidence that Mod(mdg4) acts in concert with the DNA binding insulator protein CTCF. We find that Mod(mdg4) and Cp190 are not redundant and may have distinct functional properties. We, for the first time, demonstrate that Cp190 is critical for correct regulation of the bithorax complex and show that Cp190 is required at an exceptionally strong Fub insulator to partition the bithorax complex into two topological domains. Copyright © 2016 by the Genetics Society of America.

  3. Select Neuropeptides and their G-Protein Coupled Receptors in Caenorhabditis Elegans and Drosophila Melanogaster

    PubMed Central

    Bendena, William G.; Campbell, Jason; Zara, Lian; Tobe, Stephen S.; Chin-Sang, Ian D.

    2012-01-01

    The G-protein coupled receptor (GPCR) family is comprised of seven transmembrane domain proteins and play important roles in nerve transmission, locomotion, proliferation and development, sensory perception, metabolism, and neuromodulation. GPCR research has been targeted by drug developers as a consequence of the wide variety of critical physiological functions regulated by this protein family. Neuropeptide GPCRs are the least characterized of the GPCR family as genetic systems to characterize their functions have lagged behind GPCR gene discovery. Drosophila melanogaster and Caenorhabditis elegans are genetic model organisms that have proved useful in characterizing neuropeptide GPCRs. The strength of a genetic approach leads to an appreciation of the behavioral plasticity that can result from subtle alterations in GPCRs or regulatory proteins in the pathways that GPCRs control. Many of these invertebrate neuropeptides, GPCRs, and signaling pathway components serve as models for mammalian counterparts as they have conserved sequences and function. This review provides an overview of the methods to match neuropeptides to their cognate receptor and a state of the art account of neuropeptide GPCRs that have been characterized in D. melanogaster and C. elegans and the behaviors that have been uncovered through genetic manipulation. PMID:22908006

  4. Proteomic analysis reveals CCT is a target of Fragile X mental retardation protein regulation in Drosophila.

    PubMed

    Monzo, Kate; Dowd, Susan R; Minden, Jonathan S; Sisson, John C

    2010-04-15

    Fragile X mental retardation protein (FMRP) is an RNA-binding protein that is required for the translational regulation of specific target mRNAs. Loss of FMRP causes Fragile X syndrome (FXS), the most common form of inherited mental retardation in humans. Understanding the basis for FXS has been limited because few in vivo targets of FMRP have been identified and mechanisms for how FMRP regulates physiological targets are unclear. We have previously demonstrated that Drosophila FMRP (dFMRP) is required in early embryos for cleavage furrow formation. In an effort to identify new targets of dFMRP-dependent regulation and new effectors of cleavage furrow formation, we used two-dimensional difference gel electrophoresis and mass spectrometry to identify proteins that are misexpressed in dfmr1 mutant embryos. Of the 28 proteins identified, we have identified three subunits of the Chaperonin containing TCP-1 (CCT) complex as new direct targets of dFMRP-dependent regulation. Furthermore, we found that the septin Peanut, a known effector of cleavage, is a likely conserved substrate of fly CCT and is mislocalized in both cct and in dfmr1 mutant embryos. Based on these results we propose that dFMRP-dependent regulation of CCT subunits is required for cleavage furrow formation and that at least one of its substrates is affected in dfmr1- embryos suggesting that dFMRP-dependent regulation of CCT contributes to the cleavage furrow formation phenotype.

  5. Dietary live yeast alters metabolic profiles, protein biosynthesis and thermal stress tolerance of Drosophila melanogaster.

    PubMed

    Colinet, Hervé; Renault, David

    2014-04-01

    The impact of nutritional factors on insect's life-history traits such as reproduction and lifespan has been excessively examined; however, nutritional determinant of insect's thermal tolerance has not received a lot of attention. Dietary live yeast represents a prominent source of proteins and amino acids for laboratory-reared drosophilids. In this study, Drosophila melanogaster adults were fed on diets supplemented or not with live yeast. We hypothesized that manipulating nutritional conditions through live yeast supplementation would translate into altered physiology and stress tolerance. We verified how live yeast supplementation affected body mass characteristics, total lipids and proteins, metabolic profiles and cold tolerance (acute and chronic stress). Females fed with live yeast had increased body mass and contained more lipids and proteins. Using GC/MS profiling, we found distinct metabolic fingerprints according to nutritional conditions. Metabolite pathway enrichment analysis corroborated that live yeast supplementation was associated with amino acid and protein biosyntheses. The cold assays revealed that the presence of dietary live yeast greatly promoted cold tolerance. Hence, this study conclusively demonstrates a significant interaction between nutritional conditions and thermal tolerance.

  6. Suppression of PERIOD protein abundance and circadian cycling by the Drosophila clock mutation timeless.

    PubMed Central

    Price, J L; Dembinska, M E; Young, M W; Rosbash, M

    1995-01-01

    The timeless mutation (tim) leads to loss of circadian behavioral rhythms in Drosophila melanogaster. The effects of tim on rhythmicity involve interactions with period (per), a second essential clock gene, as the tim mutation suppresses circadian oscillations of per transcription and blocks nuclear localization of a PER reporter protein. In the present study it was found that the tim mutant constitutively produces a low level of PER protein that is comparable with that produced late in the day by wild-type flies. In addition, it was shown that tim suppresses circadian cycling of PER protein abundance and circadian regulation of PER phosphorylation. Transfer of wild-type flies to constant light also suppressed cycling of PER abundance and phosphorylation and produced constitutively low levels of PER. In the tim mutant there was no additional effect of constant light on PER. These results suggest that constant light and the tim mutation produce related changes in the underlying biological clock. We further suggest that the multiple effects of tim are due to a primary effect on per expression at the posttranscriptional level. The effects of tim on behavioral rhythms and per RNA cycling are therefore likely to involve effects on PER protein through previously proposed feedback mechanisms. Images PMID:7664743

  7. Characterization and localization of the even-skipped protein of Drosophila.

    PubMed Central

    Frasch, M; Hoey, T; Rushlow, C; Doyle, H; Levine, M

    1987-01-01

    On the basis of homeo box cross-homology we have isolated the pair-rule gene even-skipped (eve) of Drosophila. The eve transcription unit appears to be less than 1.5 kb in length, and encodes a single mRNA of approximately 1.4 kb. The nucleotide sequence of genomic and cDNA clones indicates that the eve protein is composed of 376 amino acid residues, and that its homeo domain shares only approximately 50% amino acid identity with the homeo domains of previously characterized genes. Using antibodies raised against a beta-galactosidase fusion protein we show that the eve protein is distributed in a series of seven transverse stripes at the cellular blastoderm stage, and is localized primarily within the nuclear regions of those embryonic cells that express the gene. After gastrulation, seven weakly stained stripes of eve expression appear, resulting in a transient pattern that consists of a total of 14 evenly spaced stripes. Both the original and new stripes gradually disappear during germ band elongation. A second expression pattern emerges during neurogenesis, whereby eve protein is detected in discrete subsets of neurons in each of the ventral ganglia. Images Fig. 2. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:2884106

  8. [Use of immunogold labelling technique for immunoelectron microscope localization of proteins in Drosophila polytene chromosomes].

    PubMed

    Semeshin, V F; Shloma, V V; Andreeva, E N; Saumweber, H; Zhimulev, I F

    2003-01-01

    Using gold labeled antibodies, we developed and tested an immunoelectron microscope (IEM) method for detection of protein localization in Drosophila melanogaster polytene chromosomes. This method is based on procedures widely used for indirect immunofluorescent (IF) staining of salivary gland polytene chromosome squashes. The application of IEM was evaluated by using specific antibodies against proteins earlier localized in both decondensed (interbands and puffs) and compact (bands) regions of polytene chromosomes. In all the experiments, IEM and IF images for homologous chromosome regions were compared. When applied to regions of loose structures, IEM enabled us to localize, with high precision, signals in fine bands, interbands and puffs. There was a good correspondence between immunogold EM and IF data. However, there was no correspondence for dense bands: gold particles were distributed at their boundaries, while the entire bands showed bright fluorescence. This discrepancy probably resulted from a poor penetration of antibodies conjugated to gold particles in the tightly packaged structures. From the results obtained it may by concluded that the IEM method is advantageous for studying the fine protein topography of loose decompacted regions of polytene chromosomes. And this must be taken into consideration when protein localization in polytene chromosomes is performed.

  9. Drosophila domino Exhibits Genetic Interactions with a Wide Spectrum of Chromatin Protein-Encoding Loci.

    PubMed

    Ellis, Kaitlyn; Friedman, Chloe; Yedvobnick, Barry

    2015-01-01

    The Drosophila domino gene encodes protein of the SWI2/SNF2 family that has widespread roles in transcription, replication, recombination and DNA repair. Here, the potential relationship of Domino protein to other chromatin-associated proteins has been investigated through a genetic interaction analysis. We scored for genetic modification of a domino wing margin phenotype through coexpression of RNAi directed against a set of previously characterized and more newly characterized chromatin-encoding loci. A set of other SWI2/SNF2 loci were also assayed for interaction with domino. Our results show that the majority of tested loci exhibit synergistic enhancement or suppression of the domino wing phenotype. Therefore, depression in domino function sensitizes the wing margin to alterations in the activity of numerous chromatin components. In several cases the genetic interactions are associated with changes in the level of cell death measured across the dorsal-ventral margin of the wing imaginal disc. These results highlight the broad realms of action of many chromatin proteins and suggest significant overlap with Domino function in fundamental cell processes, including cell proliferation, cell death and cell signaling.

  10. Drosophila DPP2C1, a novel member of the protein phosphatase 2C (PP2C) family.

    PubMed

    Dick, T; Bahri, S M; Chia, W

    1997-10-15

    We report the molecular cloning, chromosome mapping and developmental transcription pattern of a putative serine/threonine protein phosphatase 2C (PP2C), DPP2C1, from Drosophila melanogaster. The 6-kb transcript of this first Drosophila PP2C gene encodes a 1428-aa deduced protein. The DPP2C1 protein contains a approximately 330-aa PP2C-like catalytic domain flanked by extensive N- and C-terminal sequences showing no similarities to other PP2Cs. The dpp2c1 gene maps to 4E1-2 on the X chromosome, 1.5 kb upstream of the ddlc1 gene. Northern blot analyses showed that dpp2c1 transcription is developmentally regulated, accumulating maximally during early (0-6 h) and late (12-24 h) embryogensis. The presented molecular characterisation provides the basis for a genetic dissection of DPP2C1 function.

  11. Identification and Functional Characterization of N-Terminally Acetylated Proteins in Drosophila melanogaster

    PubMed Central

    Gerrits, Bertran; Roschitzki, Bernd; Mohanty, Sonali; Niederer, Eva M.; Laczko, Endre; Timmerman, Evy; Lange, Vinzenz; Hafen, Ernst; Aebersold, Ruedi; Vandekerckhove, Joël; Basler, Konrad; Ahrens, Christian H.; Gevaert, Kris; Brunner, Erich

    2009-01-01

    Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species. PMID:19885390

  12. Drosophila male and female germline stem cell niches require the nuclear lamina protein Otefin.

    PubMed

    Barton, Lacy J; Lovander, Kaylee E; Pinto, Belinda S; Geyer, Pamela K

    2016-07-01

    The nuclear lamina is an extensive protein network that underlies the inner nuclear envelope. This network includes the LAP2-emerin-MAN1-domain (LEM-D) protein family, proteins that share an association with the chromatin binding protein Barrier-to-autointegration factor (BAF). Loss of individual LEM-D proteins causes progressive, tissue-restricted diseases, known as laminopathies. Mechanisms associated with laminopathies are not yet understood. Here we present our studies of one of the Drosophila nuclear lamina LEM-D proteins, Otefin (Ote), a homologue of emerin. Previous studies have shown that Ote is autonomously required for the survival of female germline stem cells (GSCs). We demonstrate that Ote is also required for survival of somatic cells in the ovarian niche, with loss of Ote causing a decrease in cap cell number and altered signal transduction. We show germ cell-restricted expression of Ote rescues these defects, revealing a non-autonomous function for Ote in niche maintenance and emphasizing that GSCs contribute to the maintenance of their own niches. Further, we investigate the requirement of Ote in the male fertility. We show that ote mutant males become prematurely sterile as they age. Parallel to observations in females, this sterility is associated with GSC loss and changes in somatic cells of the niche, phenotypes that are largely rescued by germ cell-restricted Ote expression. Taken together, our studies demonstrate that Ote is required autonomously for survival of two stem cell populations, as well as non-autonomously for maintenance of two somatic niches. Finally, our data add to growing evidence that LEM-D proteins have critical roles in stem cell survival and tissue homeostasis.

  13. Inducible Protein Traps with Dominant Phenotypes for Functional Analysis of the Drosophila Genome

    PubMed Central

    Singari, Swetha; Javeed, Naureen; Tardi, Nicholas J.; Marada, Suresh; Carlson, Jeff C.; Kirk, Steven; Thorn, Judith M.; Edwards, Kevin A.

    2014-01-01

    The Drosophila melanogaster genome has been extensively characterized, but there remains a pressing need to associate gene products with phenotypes, subcellular localizations, and interaction partners. A multifunctional, Minos transposon-based protein trapping system called Hostile takeover (Hto) was developed to facilitate in vivo analyses of endogenous genes, including live imaging, purification of protein complexes, and mutagenesis. The Hto transposon features a UAS enhancer with a basal promoter, followed by an artificial exon 1 and a standard 5′ splice site. Upon GAL4 induction, exon 1 can splice to the next exon downstream in the flanking genomic DNA, belonging to a random target gene. Exon 1 encodes a dual tag (FLAG epitope and mCherry red fluorescent protein), which becomes fused to the target protein. Hto was mobilized throughout the genome and then activated by eye-specific GAL4; an F1 screen for abnormal eye phenotypes was used to identify inserts that express disruptive fusion proteins. Approximately 1.7% of new inserts cause eye phenotypes. Of the first 23 verified target genes, 21 can be described as regulators of cell biology and development. Most are transcription factor genes, including AP-2, CG17181, cut, klu, mamo, Sox102F, and sv. Other target genes [l(1)G0232, nuf, pum, and Syt4] make cytoplasmic proteins, and these lines produce diverse fluorescence localization patterns. Hto permits the expression of stable carboxy-terminal subfragments of proteins, which are rarely tested in conventional genetic screens. Some of these may disrupt specific cell pathways, as exemplified by truncated forms of Mastermind and Nuf. PMID:24172131

  14. Inverse regulation of two classic Hippo pathway target genes in Drosophila by the dimerization hub protein Ctp

    PubMed Central

    Barron, Daniel A.; Moberg, Kenneth

    2016-01-01

    The LC8 family of small ~8 kD proteins are highly conserved and interact with multiple protein partners in eukaryotic cells. LC8-binding modulates target protein activity, often through induced dimerization via LC8:LC8 homodimers. Although many LC8-interactors have roles in signaling cascades, LC8’s role in developing epithelia is poorly understood. Using the Drosophila wing as a developmental model, we find that the LC8 family member Cut up (Ctp) is primarily required to promote epithelial growth, which correlates with effects on the pro-growth factor dMyc and two genes, diap1 and bantam, that are classic targets of the Hippo pathway coactivator Yorkie. Genetic tests confirm that Ctp supports Yorkie-driven tissue overgrowth and indicate that Ctp acts through Yorkie to control bantam (ban) and diap1 transcription. Quite unexpectedly however, Ctp loss has inverse effects on ban and diap1: it elevates ban expression but reduces diap1 expression. In both cases these transcriptional changes map to small segments of these promoters that recruit Yorkie. Although LC8 complexes with Yap1, a Yorkie homolog, in human cells, an orthologous interaction was not detected in Drosophila cells. Collectively these findings reveal that that Drosophila Ctp is a required regulator of Yorkie-target genes in vivo and suggest that Ctp may interact with a Hippo pathway protein(s) to exert inverse transcriptional effects on Yorkie-target genes. PMID:26972460

  15. Neurotoxic protein expression reveals connections between the circadian clock and mating behavior in Drosophila

    PubMed Central

    Kadener, Sebastian; Villella, Adriana; Kula, Elzbieta; Palm, Kristyna; Pyza, Elzbieta; Botas, Juan; Hall, Jeffrey C.; Rosbash, Michael

    2006-01-01

    To investigate the functions of circadian neurons, we added two strategies to the standard Drosophila behavioral genetics repertoire. The first was to express a polyglutamine-expanded neurotoxic protein (MJDtr78Q; MJD, Machado–Joseph disease) in the major timeless (tim)-expressing cells of the adult brain. These Tim-MJD flies were viable, in contrast to the use of cell-death gene expression for tim neuron inactivation. Moreover, they were more arrhythmic than flies expressing other neurotoxins and had low but detectable tim mRNA levels. The second extended standard microarray technology from fly heads to dissected fly brains. By combining the two approaches, we identified a population of Tim-MJD-affected mRNAs. Some had been previously identified as sex-specific and relevant to courtship, including mRNAs localized to brain-proximal fat-body tissue and brain courtship centers. Finally, we found a decrease in the number of neurons that expressed male-specific forms of the fruitless protein in the laterodorsal region of the brain. The decrease was not a consequence of toxic protein expression within these specialized cells but a likely effect of communication with neighboring TIM-expressing neurons. The data suggest a functional interaction between adjacent circadian and mating circuits within the fly brain, as well as an interaction between circadian circuits and brain-proximal fat body. PMID:16938865

  16. reduced ocelli encodes the leucine rich repeat protein Pray For Elves in Drosophila melanogaster.

    PubMed

    Caldwell, Jason C; Fineberg, Sarah K; Eberl, Daniel F

    2007-01-01

    The ocelli are three simple photoreceptors on the vertex of the fruit fly head. We sought to identify the gene encoded by the classical ocellar mutant, reduced ocelli (rdo). Deficiency and inversion breakpoint mapping and P-element induced male recombination analyses were performed and Pray For Elves (PFE; CG15151; Fbgn0032661) emerged as a promising candidate for the rdo phenotype. The PFE locus maps to polytene region 36E on chromosome 2L between elfless (Fbgn0032660) and Arrestin 1 (Fbgn0000120). FlyBase annotation predicts that PFE encodes a serine/threonine kinase, yet protein prediction programs revealed no kinase domain. These analyses suggest that PFE simply encodes a leucine rich repeat molecule of unknown function, but presumably functions in nervous system protein-protein interaction. Two classical spontaneous alleles of rdo, rdo(1) and rdo(2), were characterized and the underlying mutations result from a small deletion spanning exon 1/intron 1 and a B104/roo insertion into the 3'UTR of PFE, respectively. Transposase-mediated excisions of several P-elements inserted into the PFE locus revert the rdo phenotype and a full-length PFE cDNA is sufficient to rescue rdo. A Gal4 enhancer trap reveals a broad adult neural expression pattern for PFE. Our identification and initial characterization of the rdo locus will contribute to the understanding of neurogenesis and neural development in the simple photoreceptors of the Drosophila visual system.

  17. Septate Junction Proteins Play Essential Roles in Morphogenesis Throughout Embryonic Development in Drosophila

    PubMed Central

    Hall, Sonia; Ward, Robert E.

    2016-01-01

    The septate junction (SJ) is the occluding junction found in the ectodermal epithelia of invertebrate organisms, and is essential to maintain chemically distinct compartments in epithelial organs, to provide the blood–brain barrier in the nervous system, and to provide an important line of defense against invading pathogens. More than 20 genes have been identified to function in the establishment or maintenance of SJs in Drosophila melanogaster. Numerous studies have demonstrated the cell biological function of these proteins in establishing the occluding junction, whereas very few studies have examined further developmental roles for them. Here we examined embryos with mutations in nine different core SJ genes and found that all nine result in defects in embryonic development as early as germ band retraction, with the most penetrant defect observed in head involution. SJ genes are also required for cell shape changes and cell rearrangements that drive the elongation of the salivary gland during midembryogenesis. Interestingly, these developmental events occur at a time prior to the formation of the occluding junction, when SJ proteins localize along the lateral membrane and have not yet coalesced into the region of the SJ. Together, these observations reveal an underappreciated role for a large group of SJ genes in essential developmental events during embryogenesis, and suggest that the function of these proteins in facilitating cell shape changes and rearrangements is independent of their role in the occluding junction. PMID:27261004

  18. Drosophila amyloid precursor protein-like is required for long-term memory.

    PubMed

    Goguel, Valérie; Belair, Anne-Laure; Ayaz, Derya; Lampin-Saint-Amaux, Aurélie; Scaplehorn, Niki; Hassan, Bassem A; Preat, Thomas

    2011-01-19

    The amyloid precursor protein (APP) plays an important role in Alzheimer's disease (AD), a progressive neurodegenerative pathology that first manifests as a decline of memory. While the main hypothesis for AD pathology centers on the proteolytic processing of APP, very little is known about the physiological function of the APP protein in the adult brain. Likewise, whether APP loss of function contributes to AD remains unclear. Drosophila has been used extensively as a model organism to study neuronal function and pathology. In addition, many of the molecular mechanisms underlying memory are thought to be conserved from flies to mammals, prompting us to study the function of APPL, the fly APP ortholog, during associative memory. It was previously shown that APPL expression is highly enriched in the mushroom bodies (MBs), a specialized brain structure involved in olfactory memory. We analyzed memory in flies in which APPL expression has been silenced specifically and transiently in the adult MBs. Our results show that in adult flies, APPL is not required for learning but is specifically involved in long-term memory, a long lasting memory whose formation requires de novo protein synthesis and is thought to require synaptic structural plasticity. These data support the hypothesis that disruption of normal APP function may contribute to early AD cognitive impairment.

  19. A role for the membrane protein M6 in the Drosophila visual system

    PubMed Central

    2012-01-01

    Background Members of the proteolipid protein family, including the four-transmembrane glycoprotein M6a, are involved in neuronal plasticity in mammals. Results from our group previously demonstrated that M6, the only proteolipid protein expressed in Drosophila, localizes to the cell membrane in follicle cells. M6 loss triggers female sterility, which suggests a role for M6 in follicular cell remodeling. These results were the basis of the present study, which focused on the function and requirements of M6 in the fly nervous system. Results The present study identified two novel, tissue-regulated M6 isoforms with variable N- and C- termini, and showed that M6 is the functional fly ortholog of Gpm6a. In the adult brain, the protein was localized to several neuropils, such as the optic lobe, the central complex, and the mushroom bodies. Interestingly, although reduced M6 levels triggered a mild rough-eye phenotype, hypomorphic M6 mutants exhibited a defective response to light. Conclusions Based on its ability to induce filopodium formation we propose that M6 is key in cell remodeling processes underlying visual system function. These results bring further insight into the role of M6/M6a in biological processes involving neuronal plasticity and behavior in flies and mammals. PMID:22762289

  20. Abberant protein synthesis in G2019S LRRK2 Drosophila Parkinson disease-related phenotypes

    PubMed Central

    Martin, Ian; Abalde-Atristain, Leire; Kim, Jungwoo Wren; Dawson, Ted M; Dawson, Valina L

    2014-01-01

    LRRK2 mutations are a frequent cause of familial Parkinson disease (PD) and are also found in a number of sporadic PD cases. PD-linked G2019S and I2020T mutations in the kinase domain of LRRK2 result in elevated kinase activity, which is required for the toxicity of these pathogenic variants in cell and animal models of PD. We recently reported that LRRK2 interacts with and phosphorylates a number of mammalian ribosomal proteins, several of which exhibit increased phosphorylation via both G2019S and I2020T LRRK2. Blocking the phosphorylation of ribosomal protein s15 through expression of phospho-deficient T136A s15 prevents age-associated locomotor deficits and dopamine neuron loss caused by G2019S LRRK2 expression in Drosophila indicating that s15 is a pathogenic LRRK2 substrate. We previously described that G2019S LRRK2 causes an induction of bulk mRNA translation that is blocked by T136A s15 or the protein synthesis inhibitor anisomycin. Here, we report the protective effects of the eIF4E/eIF4G interaction inhibitor 4EGI-1, in preventing neurodegenerative phenotypes in G2019S LRRK2 flies, and discuss how our findings and those of other groups provide a framework to begin investigating the mechanistic impact of LRRK2 on translation. PMID:25483009

  1. A GFP trap study uncovers the functions of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis.

    PubMed

    Nerusheva, O O; Dorogova, N V; Gubanova, N V; Yudina, O S; Omelyanchuk, L V

    2009-05-01

    The function of the gene gilgamesh (89B9-12) encoding a casein kinase in Drosophila spermatogenesis was studied. The chimeric Gilgamesh-GFP protein in spermatocytes is cortically located. In the polar and apolar spermatocytes, it concentrates at the terminal ends of the fusome, the organelle that passes through the system of ring canals of the spermatocyte cyst. At the stage of spermatid elongation, the protein associates with the nucleus. A spot of the highest Gilgamesh-GFP concentration in the nucleus co-localizes with gamma-tubulin in the basal body. At later stages, Gilgamesh is localized to the individualization complex (IC), leaving the nuclei somewhat before the IC investment cones, as detected by actin binding. The sterile mutation due to the gilgamesh gene leads to the phenotype of scattered nuclei and altered structure of actin cones in the individualizing spermatid cyst. Ultrastructural evidence confirmed defective spermatid individualization due to the mutation. The phylogenetic origin of the protein, and the connection between vesicular trafficking and spermatid individualization, are discussed.

  2. The Drosophila DOCK family protein Sponge is required for development of the air sac primordium.

    PubMed

    Morishita, Kazushge; Anh Suong, Dang Ngoc; Yoshida, Hideki; Yamaguchi, Masamitsu

    2017-05-15

    Dedicator of cytokinesis (DOCK) family genes are known as DOCK1-DOCK11 in mammals. DOCK family proteins mainly regulate actin filament polymerization and/or depolymerization and are GEF proteins, which contribute to cellular signaling events by activating small G proteins. Sponge (Spg) is a Drosophila counterpart to mammalian DOCK3/DOCK4, and plays a role in embryonic central nervous system development, R7 photoreceptor cell differentiation, and adult thorax development. In order to conduct further functional analyses on Spg in vivo, we examined its localization in third instar larval wing imaginal discs. Immunostaining with purified anti-Spg IgG revealed that Spg mainly localized in the air sac primordium (ASP) in wing imaginal discs. Spg is therefore predicted to play an important role in the ASP. The specific knockdown of Spg by the breathless-GAL4 driver in tracheal cells induced lethality accompanied with a defect in ASP development and the induction of apoptosis. The monitoring of ERK signaling activity in wing imaginal discs by immunostaining with anti-diphospho-ERK IgG revealed reductions in the ERK signal cascade in Spg knockdown clones. Furthermore, the overexpression of D-raf suppressed defects in survival and the proliferation of cells in the ASP induced by the knockdown of Spg. Collectively, these results indicate that Spg plays a critical role in ASP development and tracheal cell viability that is mediated by the ERK signaling pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Multimerization of Drosophila sperm protein Mst77F causes a unique condensed chromatin structure

    PubMed Central

    Kost, Nils; Kaiser, Sophie; Ostwal, Yogesh; Riedel, Dietmar; Stützer, Alexandra; Nikolov, Miroslav; Rathke, Christina; Renkawitz-Pohl, Renate; Fischle, Wolfgang

    2015-01-01

    Despite insights on the cellular level, the molecular details of chromatin reorganization in sperm development, which involves replacement of histone proteins by specialized factors to allow ultra most condensation of the genome, are not well understood. Protamines are dispensable for DNA condensation during Drosophila post-meiotic spermatogenesis. Therefore, we analyzed the interaction of Mst77F, another very basic testis-specific protein with chromatin and DNA as well as studied the molecular consequences of such binding. We show that Mst77F on its own causes severe chromatin and DNA aggregation. An intrinsically unstructured domain in the C-terminus of Mst77F binds DNA via electrostatic interaction. This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein. Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation. In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm. Based on these findings we postulate that Mst77F is crucial for sperm development by giving rise to a unique condensed chromatin structure. PMID:25735749

  4. Protein synthesis elongation factor EF-1 alpha expression and longevity in Drosophila melanogaster.

    PubMed Central

    Shikama, N; Ackermann, R; Brack, C

    1994-01-01

    It has been proposed that the decline in protein synthesis observed in aging organisms may result from a decrease in elongation factor EF-1 alpha. Transgenic Drosophila melanogaster flies carrying an additional copy of the EF-1 alpha gene under control of a heat-inducible promoter have an extended lifespan, further indicating that the EF-1 alpha gene may play an important role in determining longevity. To test this hypothesis, we have quantitated EF-1 alpha mRNA, EF-1 alpha protein, and the EF-1 alpha complex-formation activity in these transgenic flies. Furthermore, we have tested whether the transgene construct is functional--i.e., whether transgenic mRNA is induced when flies are grown at higher temperature. The results show that although there is a clear difference in mean lifespan between the EF-1 alpha transgenic (E) flies and the control transgenic (C) flies, E flies do not express more EF-1 alpha protein or mRNA than C flies kept at the same experimental conditions. Although the transgene can be induced when E flies are heat-shocked at 37 degrees C, transgenic mRNA is not detectable in E flies aged at 29 degrees C. In both lines, the loss in catalytic activity with age is the same. We conclude that the E flies examined here do not live longer because of overexpressing the EF-1 alpha gene. Images PMID:8183891

  5. Requirement for sex comb on midleg protein interactions in Drosophila polycomb group repression.

    PubMed

    Peterson, Aidan J; Mallin, Daniel R; Francis, Nicole J; Ketel, Carrie S; Stamm, Joyce; Voeller, Rochus K; Kingston, Robert E; Simon, Jeffrey A

    2004-07-01

    The Drosophila Sex Comb on Midleg (SCM) protein is a transcriptional repressor of the Polycomb group (PcG). Although genetic studies establish SCM as a crucial PcG member, its molecular role is not known. To investigate how SCM might link to PcG complexes, we analyzed the in vivo role of a conserved protein interaction module, the SPM domain. This domain is found in SCM and in another PcG protein, Polyhomeotic (PH), which is a core component of Polycomb repressive complex 1 (PRC1). SCM-PH interactions in vitro are mediated by their respective SPM domains. Yeast two-hybrid and in vitro binding assays were used to isolate and characterize >30 missense mutations in the SPM domain of SCM. Genetic rescue assays showed that SCM repressor function in vivo is disrupted by mutations that impair SPM domain interactions in vitro. Furthermore, overexpression of an isolated, wild-type SPM domain produced PcG loss-of-function phenotypes in flies. Coassembly of SCM with a reconstituted PRC1 core complex shows that SCM can partner with PRC1. However, gel filtration chromatography showed that the bulk of SCM is biochemically separable from PH in embryo nuclear extracts. These results suggest that SCM, although not a core component of PRC1, interacts and functions with PRC1 in gene silencing.

  6. Flightin, a novel myofibrillar protein of Drosophila stretch-activated muscles

    PubMed Central

    1993-01-01

    The indirect flight muscles of Drosophila are adapted for rapid oscillatory movements which depend on properties of the contractile apparatus itself. Flight muscles are stretch activated and the frequency of contraction in these muscles is independent of the rate of nerve impulses. Little is known about the molecular basis of these adaptations. We now report a novel protein that is found only in flight muscles and has, therefore, been named flightin. Although we detect only one gene (in polytene region 76D) for flightin, this protein has several isoforms (relative gel mobilities, 27-30 kD; pIs, 4.6-6.0). These isoforms appear to be created by posttranslational modifications. A subset of these isoforms is absent in newly emerged adults but appears when the adult develops the ability to fly. In intact muscles flightin is associated with the A band of the sarcomere, where evidence suggests it interacts with the myosin filaments. Computer database searches do not reveal extensive similarity to any known protein. However, the NH2-terminal 12 residues show similarity to the NH2- terminal sequence of actin, a region that interacts with myosin. These features suggest a role for flightin in the regulation of contraction, possibly by modulating actin-myosin interaction. PMID:8486738

  7. reduced ocelli Encodes the Leucine Rich Repeat Protein Pray For Elves in Drosophila melanogaster

    PubMed Central

    Caldwell, Jason C.; Fineberg, Sarah K.; Eberl, Daniel F.

    2009-01-01

    The ocelli are three simple photoreceptors on the vertex of the fruit fly head. We sought to identify the gene encoded by the classical ocellar mutant, reduced ocelli (rdo). Deficiency and inversion breakpoint mapping and P-element induced male recombination analyses were performed and Pray For Elves (PFE; CG15151; Fbgn0032661) emerged as a promising candidate for the rdo phenotype. The PFE locus maps to polytene region 36E on chromosome 2L between elfless (Fbgn0032660) and Arrestin 1 (Fbgn0000120). FlyBase annotation predicts that PFE encodes a serine/threonine kinase, yet protein prediction programs revealed no kinase domain. These analyses suggest that PFE simply encodes a leucine rich repeat molecule of unknown function, but presumably functions in nervous system protein-protein interaction. Two classical spontaneous alleles of rdo, rdo1 and rdo2, were characterized and the underlying mutations result from a small deletion spanning exon 1/intron 1 and a B104/roo insertion into the 3′UTR of PFE, respectively. Transposase-mediated excisions of several P-elements inserted into the PFE locus revert the rdo phenotype and a full-length PFE cDNA is sufficient to rescue rdo. A Gal4 enhancer trap reveals a broad adult neural expression pattern for PFE. Our identification and initial characterization of the rdo locus will contribute to the understanding of neurogenesis and neural development in the simple photoreceptors of the Drosophila visual system. PMID:18820435

  8. Requirement for sex comb on midleg protein interactions in Drosophila polycomb group repression.

    PubMed Central

    Peterson, Aidan J; Mallin, Daniel R; Francis, Nicole J; Ketel, Carrie S; Stamm, Joyce; Voeller, Rochus K; Kingston, Robert E; Simon, Jeffrey A

    2004-01-01

    The Drosophila Sex Comb on Midleg (SCM) protein is a transcriptional repressor of the Polycomb group (PcG). Although genetic studies establish SCM as a crucial PcG member, its molecular role is not known. To investigate how SCM might link to PcG complexes, we analyzed the in vivo role of a conserved protein interaction module, the SPM domain. This domain is found in SCM and in another PcG protein, Polyhomeotic (PH), which is a core component of Polycomb repressive complex 1 (PRC1). SCM-PH interactions in vitro are mediated by their respective SPM domains. Yeast two-hybrid and in vitro binding assays were used to isolate and characterize >30 missense mutations in the SPM domain of SCM. Genetic rescue assays showed that SCM repressor function in vivo is disrupted by mutations that impair SPM domain interactions in vitro. Furthermore, overexpression of an isolated, wild-type SPM domain produced PcG loss-of-function phenotypes in flies. Coassembly of SCM with a reconstituted PRC1 core complex shows that SCM can partner with PRC1. However, gel filtration chromatography showed that the bulk of SCM is biochemically separable from PH in embryo nuclear extracts. These results suggest that SCM, although not a core component of PRC1, interacts and functions with PRC1 in gene silencing. PMID:15280237

  9. Variation in sperm displacement and its association with accessory gland protein loci in Drosophila melanogaster

    SciTech Connect

    Clark, A.G.; Prout, T.; Harshman, L.G.

    1995-01-01

    Genes that influence mating and/or fertilization success may be targets for strong natural selection. If females remate frequently relative to the duration of sperm storage and rate of sperm use, sperm displacement may be an important component of male reproductive success. Although it has long been known that mutant laboratory stocks of Drosophila differ in sperm displacement, the magnitude of the naturally occurring genetic variation in this character has not been systematically quantified. Here we report the results of a screen for variation in sperm displacement among 152 lines of Drosophila melanogaster that were made homozygous for second and/or third chromosomes recovered from natural populations. Sperm displacement was assayed by scoring the progeny of cn;bw females that had been mated sequentially to cn;bw and tested males in either order. Highly significant differences were seen in both the ability to displace sperm that is resident in the female`s reproductive tract and in the ability to resist displacement by subsequent sperm. Most lines exhibited nearly complete displacement, having nearly all progeny sired by the second male, but several lines had as few as half the progeny fathered by the second male. Lines that were identified in the screen for naturally occurring variation in sperm displacement were also characterized for single-strand conformation polymorphisms (SSCP) at seven accessory gland protein (Acp) genes. Significant associations were found between particular Acp alleles at four different loci (Acp26Aa/Ab, Acp29B, Acp36DE and Acp53E) and the ability of males to resist displacement by subsequent sperm. There was no correlation between the ability to displace resident sperm and the ability to resist being displaced by subsequent sperm. This lack of correlation, and the association of Acp alleles with resisting subsequent sperm only, suggests that different mechanisms mediate the two components of sperm displacement. 36 refs., 4 figs., 7 tabs.

  10. Mushroom body miscellanea: transgenic Drosophila strains expressing anatomical and physiological sensor proteins in Kenyon cells

    PubMed Central

    Pech, Ulrike; Dipt, Shubham; Barth, Jonas; Singh, Priyanka; Jauch, Mandy; Thum, Andreas S.; Fiala, André; Riemensperger, Thomas

    2013-01-01

    The fruit fly Drosophila melanogaster represents a key model organism for analyzing how neuronal circuits regulate behavior. The mushroom body in the central brain is a particularly prominent brain region that has been intensely studied in several insect species and been implicated in a variety of behaviors, e.g., associative learning, locomotor activity, and sleep. Drosophila melanogaster offers the advantage that transgenes can be easily expressed in neuronal subpopulations, e.g., in intrinsic mushroom body neurons (Kenyon cells). A number of transgenes has been described and engineered to visualize the anatomy of neurons, to monitor physiological parameters of neuronal activity, and to manipulate neuronal function artificially. To target the expression of these transgenes selectively to specific neurons several sophisticated bi- or even multipartite transcription systems have been invented. However, the number of transgenes that can be combined in the genome of an individual fly is limited in practice. To facilitate the analysis of the mushroom body we provide a compilation of transgenic fruit flies that express transgenes under direct control of the Kenyon-cell specific promoter, mb247. The transgenes expressed are fluorescence reporters to analyze neuroanatomical aspects of the mushroom body, proteins to restrict ectopic gene expression to mushroom bodies, or fluorescent sensors to monitor physiological parameters of neuronal activity of Kenyon cells. Some of the transgenic animals compiled here have been published already, whereas others are novel and characterized here for the first time. Overall, the collection of transgenic flies expressing sensor and reporter genes in Kenyon cells facilitates combinations with binary transcription systems and might, ultimately, advance the physiological analysis of mushroom body function. PMID:24065891

  11. Cooperation of the BTB-Zinc finger protein, Abrupt, with cytoskeletal regulators in Drosophila epithelial tumorigenesis

    PubMed Central

    Turkel, Nezaket; Portela, Marta; Poon, Carole; Li, Jason; Brumby, Anthony M.; Richardson, Helena E.

    2015-01-01

    ABSTRACT The deregulation of cell polarity or cytoskeletal regulators is a common occurrence in human epithelial cancers. Moreover, there is accumulating evidence in human epithelial cancer that BTB-ZF genes, such as Bcl6 and ZBTB7A, are oncogenic. From our previous studies in the vinegar fly, Drosophila melanogaster, we have identified a cooperative interaction between a mutation in the apico-basal cell polarity regulator Scribble (Scrib) and overexpression of the BTB-ZF protein Abrupt (Ab). Herein, we show that co-expression of ab with actin cytoskeletal regulators, RhoGEF2 or Src64B, in the developing eye-antennal epithelial tissue results in the formation of overgrown amorphous tumours, whereas ab and DRac1 co-expression leads to non-cell autonomous overgrowth. Together with ab, these genes affect the expression of differentiation genes, resulting in tumours locked in a progenitor cell fate. Finally, we show that the expression of two mammalian genes related to ab, Bcl6 and ZBTB7A, which are oncogenes in mammalian epithelial cancers, significantly correlate with the upregulation of cytoskeletal genes or downregulation of apico-basal cell polarity neoplastic tumour suppressor genes in colorectal, lung and other human epithelial cancers. Altogether, this analysis has revealed that upregulation of cytoskeletal regulators cooperate with Abrupt in Drosophila epithelial tumorigenesis, and that high expression of human BTB-ZF genes, Bcl6 and ZBTB7A, shows significant correlations with cytoskeletal and cell polarity gene expression in specific epithelial tumour types. This highlights the need for further investigation of the cooperation between these genes in mammalian systems. PMID:26187947

  12. The Circadian Clock Protein Timeless Regulates Phagocytosis of Bacteria in Drosophila

    PubMed Central

    Ayres, Janelle S.; Pham, Linh N.; Ziauddin, Junaid; Shirasu-Hiza, Mimi M.

    2012-01-01

    Survival of bacterial infection is the result of complex host-pathogen interactions. An often-overlooked aspect of these interactions is the circadian state of the host. Previously, we demonstrated that Drosophila mutants lacking the circadian regulatory proteins Timeless (Tim) and Period (Per) are sensitive to infection by S. pneumoniae. Sensitivity to infection can be mediated either by changes in resistance (control of microbial load) or tolerance (endurance of the pathogenic effects of infection). Here we show that Tim regulates resistance against both S. pneumoniae and S. marcescens. We set out to characterize and identify the underlying mechanism of resistance that is circadian-regulated. Using S. pneumoniae, we found that resistance oscillates daily in adult wild-type flies and that these oscillations are absent in Tim mutants. Drosophila have at least three main resistance mechanisms to kill high levels of bacteria in their hemolymph: melanization, antimicrobial peptides, and phagocytosis. We found that melanization is not circadian-regulated. We further found that basal levels of AMP gene expression exhibit time-of-day oscillations but that these are Tim-independent; moreover, infection-induced AMP gene expression is not circadian-regulated. We then show that phagocytosis is circadian-regulated. Wild-type flies exhibit up-regulated phagocytic activity at night; Tim mutants have normal phagocytic activity during the day but lack this night-time peak. Tim appears to regulate an upstream event in phagocytosis, such as bacterial recognition or activation of phagocytic hemocytes. Interestingly, inhibition of phagocytosis in wild type flies results in survival kinetics similar to Tim mutants after infection with S. pneumoniae. Taken together, these results suggest that loss of circadian oscillation of a specific immune function (phagocytosis) can have significant effects on long-term survival of infection. PMID:22253593

  13. AMP-activated protein kinase has diet-dependent and -independent roles in Drosophila oogenesis.

    PubMed

    Laws, Kaitlin M; Drummond-Barbosa, Daniela

    2016-12-01

    Multiple aspects of organismal physiology influence the number and activity of stem cells and their progeny, including nutritional status. Previous studies demonstrated that Drosophila germline stem cells (GSCs), follicle stem cells (FSCs), and their progeny sense and respond to diet via complex mechanisms involving many systemic and local signals. AMP-activated protein kinase, or AMPK, is a highly conserved regulator of energy homeostasis known to be activated under low cellular energy conditions; however, its role in the ovarian response to diet has not been investigated. Here, we describe nutrient-dependent and -independent requirements for AMPK in Drosophila oogenesis. We found that AMPK is cell autonomously required for the slow down in GSC and follicle cell proliferation that occurs on a poor diet. Similarly, AMPK activity is necessary in the germline for the degeneration of vitellogenic stages in response to nutrient deprivation. In contrast, AMPK activity is not required within the germline to modulate its growth. Instead, AMPK acts in follicle cells to negatively regulate their growth and proliferation, thereby indirectly limiting the size of the underlying germline cyst within developing follicles. Paradoxically, AMPK is required for GSC maintenance in well-fed flies (when AMPK activity is presumably at its lowest), suggesting potentially important roles for basal AMPK activity in specific cell types. Finally, we identified a nutrient-independent, developmental role for AMPK in cyst encapsulation by follicle cells. These results uncover specific AMPK requirements in multiple cell types in the ovary and suggest that AMPK can function outside of its canonical nutrient-sensing role in specific developmental contexts.

  14. Wolbachia Endosymbionts Modify Drosophila Ovary Protein Levels in a Context-Dependent Manner

    PubMed Central

    Christensen, Steen; Pérez Dulzaides, Ricardo; Hedrick, Victoria E.; Momtaz, A. J. M. Zehadee; Nakayasu, Ernesto S.; Paul, Lake N.

    2016-01-01

    ABSTRACT Endosymbiosis is a unique form of interaction between organisms, with one organism dwelling inside the other. One of the most widespread endosymbionts is Wolbachia pipientis, a maternally transmitted bacterium carried by insects, crustaceans, mites, and filarial nematodes. Although candidate proteins that contribute to maternal transmission have been identified, the molecular basis for maternal Wolbachia transmission remains largely unknown. To investigate transmission-related processes in response to Wolbachia infection, ovarian proteomes were analyzed from Wolbachia-infected Drosophila melanogaster and D. simulans. Endogenous and variant host-strain combinations were investigated. Significant and differentially abundant ovarian proteins were detected, indicating substantial regulatory changes in response to Wolbachia. Variant Wolbachia strains were associated with a broader impact on the ovary proteome than endogenous Wolbachia strains. The D. melanogaster ovarian environment also exhibited a higher level of diversity of proteomic responses to Wolbachia than D. simulans. Overall, many Wolbachia-responsive ovarian proteins detected in this study were consistent with expectations from the experimental literature. This suggests that context-specific changes in protein abundance contribute to Wolbachia manipulation of transmission-related mechanisms in oogenesis. IMPORTANCE Millions of insect species naturally carry bacterial endosymbionts called Wolbachia. Wolbachia bacteria are transmitted by females to their offspring through a robust egg-loading mechanism. The molecular basis for Wolbachia transmission remains poorly understood at this time, however. This proteomic study identified specific fruit fly ovarian proteins as being upregulated or downregulated in response to Wolbachia infection. The majority of these protein responses correlated specifically with the type of host and Wolbachia strain involved. This work corroborates previously identified

  15. The role of the Suppressor of Hairy-wing insulator protein in Drosophila oogenesis

    PubMed Central

    Baxley, Ryan M.; Soshnev, Alexey A.; Koryakov, Dmitry E.; Zhimulev, Igor F.; Geyer, Pamela K.

    2011-01-01

    The Drosophila Suppressor of Hairy wing [Su(Hw)] insulator protein has an essential role in the development of the female germline. Here we investigate the function of Su(Hw) in the ovary. We show that Su(Hw) is universally expressed in somatic cells, while germ cell expression is dynamic. Robust levels accumulate in post-mitotic germ cells, where Su(Hw) localization is limited to chromosomes within nurse cells, the specialized cells that support oocyte growth. Although loss of Su(Hw) causes global defects in nurse cell chromosome structure, we demonstrate that these architectural changes are not responsible for the block in oogenesis. Connections between the fertility and insulator functions of Su(Hw) were investigated through studies of the two gypsy insulator proteins, Modifier of (mdg4)67.2 (Mod67.2) and Centrosomal Protein of 190 kD (CP190). Accumulation of these proteins is distinct from Su(Hw), with Mod67.2 and CP190 showing uniform expression in all cells during early stages of oogenesis that diminishes in later stages. Although Mod67.2 and CP190 extensively co-localize with Su(Hw) on nurse cell chromosomes, neither protein is required for nurse cell chromosome development or oocyte production. These data indicate that while the gypsy insulator function requires both Mod67.2 and CP190, these proteins are not essential for oogenesis. These studies represent the first molecular investigations of Su(Hw) function in the germline, which uncover distinct requirements for Su(Hw) insulator and ovary functions. PMID:21651900

  16. Drosophila DJ-1 Decreases Neural Sensitivity to Stress by Negatively Regulating Daxx-Like Protein through dFOXO

    PubMed Central

    Choi, Gahee; Suh, Yoon Seok; Han, Seung Yeop; Lee, Minjung; Park, Seung Hwan; Lee, Jang Ho; Lee, Soojin; Bang, Se Min; Jeong, Yuji; Chung, Won-Ju; Lee, Im-Soon; Jeong, Gilsang; Chung, Jongkyeong; Cho, Kyoung Sang

    2013-01-01

    DJ-1, a Parkinson's disease (PD)–associated gene, has been shown to protect against oxidative stress in Drosophila. However, the molecular mechanism underlying oxidative stress-induced phenotypes, including apoptosis, locomotive defects, and lethality, in DJ-1-deficient flies is not fully understood. Here we showed that Daxx-like protein (DLP), a Drosophila homologue of the mammalian Death domain-associated protein (Daxx), was upregulated under oxidative stress conditions in the loss-of-function mutants of Drosophila DJ-1β, a Drosophila homologue of DJ-1. DLP overexpression induced apoptosis via the c-Jun N-terminal kinase (JNK)/Drosophila forkhead box subgroup O (dFOXO) pathway, whereas loss of DLP increased resistance to oxidative stress and UV irradiation. Moreover, the oxidative stress-induced phenotypes of DJ-1β mutants were dramatically rescued by DLP deficiency, suggesting that enhanced expression of DLP contributes to the DJ-1β mutant phenotypes. Interestingly, we found that dFOXO was required for the increase in DLP expression in DJ-1β mutants and that dFOXO activity was increased in the heads of DJ-1β mutants. In addition, subcellular localization of DLP appeared to be influenced by DJ-1 expression so that cytosolic DLP was increased in DJ-1β mutants. Similarly, in mammalian cells, Daxx translocation from the nucleus to the cytosol was suppressed by overexpressed DJ-1β under oxidative stress conditions; and, furthermore, targeted expression of DJ-1β to mitochondria efficiently inhibited the Daxx translocation. Taken together, our findings demonstrate that DJ-1β protects flies against oxidative stress- and UV-induced apoptosis by regulating the subcellular localization and gene expression of DLP, thus implying that Daxx-induced apoptosis is involved in the pathogenesis of DJ-1-associated PD. PMID:23593018

  17. Sxl-Dependent, tra/tra2-Independent Alternative Splicing of the Drosophila melanogaster X-Linked Gene found in neurons.

    PubMed

    Sun, Xia; Yang, Haiwang; Sturgill, David; Oliver, Brian; Rabinow, Leonard; Samson, Marie-Laure

    2015-10-28

    Somatic sexual determination and behavior in Drosophila melanogaster are under the control of a genetic cascade initiated by Sex lethal (Sxl). In the female soma, SXL RNA-binding protein regulates the splicing of transformer (tra) transcripts into a female-specific form. The RNA-binding protein TRA and its cofactor TRA2 function in concert in females, whereas SXL, TRA, and TRA2 are thought to not function in males. To better understand sex-specific regulation of gene expression, we analyzed male and female head transcriptome datasets for expression levels and splicing, quantifying sex-biased gene expression via RNA-Seq and qPCR. Our data uncouple the effects of Sxl and tra/tra2 in females in the-sex-biased alternative splicing of head transcripts from the X-linked locus found in neurons (fne), encoding a pan-neuronal RNA-binding protein of the ELAV family. We show that FNE protein levels are downregulated by Sxl in female heads, also independently of tra/tra2. We argue that this regulation may have important sexually dimorphic consequences for the regulation of nervous system development or function. Copyright © 2015 Sun et al.

  18. Sxl-Dependent, tra/tra2-Independent Alternative Splicing of the Drosophila melanogaster X-Linked Gene found in neurons

    PubMed Central

    Sun, Xia; Yang, Haiwang; Sturgill, David; Oliver, Brian; Rabinow, Leonard; Samson, Marie-Laure

    2015-01-01

    Somatic sexual determination and behavior in Drosophila melanogaster are under the control of a genetic cascade initiated by Sex lethal (Sxl). In the female soma, SXL RNA-binding protein regulates the splicing of transformer (tra) transcripts into a female-specific form. The RNA-binding protein TRA and its cofactor TRA2 function in concert in females, whereas SXL, TRA, and TRA2 are thought to not function in males. To better understand sex-specific regulation of gene expression, we analyzed male and female head transcriptome datasets for expression levels and splicing, quantifying sex-biased gene expression via RNA-Seq and qPCR. Our data uncouple the effects of Sxl and tra/tra2 in females in the-sex-biased alternative splicing of head transcripts from the X-linked locus found in neurons (fne), encoding a pan-neuronal RNA-binding protein of the ELAV family. We show that FNE protein levels are downregulated by Sxl in female heads, also independently of tra/tra2. We argue that this regulation may have important sexually dimorphic consequences for the regulation of nervous system development or function. PMID:26511498

  19. Transcriptional responses of ecologically diverse Drosophila species to larval diets differing in relative sugar and protein ratios.

    PubMed

    Nazario-Yepiz, Nestor O; Loustalot-Laclette, Mariana Ramirez; Carpinteyro-Ponce, Javier; Abreu-Goodger, Cei; Markow, Therese Ann

    2017-01-01

    We utilized three ecologically diverse Drosophila species to explore the influence of ecological adaptation on transcriptomic responses to isocaloric diets differing in their relative proportions of protein to sugar. Drosophila melanogaster, a cosmopolitan species that breeds in decaying fruit, exemplifies individuals long exposed to a Western diet higher in sugar, while the natural diet of the cactophilic D. mojavensis, is much lower in carbohydrates. Drosophila arizonae, the sister species of D. mojavensis, is largely cactophilic, but also utilizes rotting fruits that are higher in sugars than cacti. We exposed third instar larvae for 24 hours to diets either (1) high in protein relative to sugar, (2) diets with equal amounts of protein and sugar, and (3) diets low in protein but high in sugar. As we predicted, based upon earlier interspecific studies of development and metabolism, the most extreme differences in gene expression under different dietary conditions were found in D. mojavensis followed by D. arizonae. No differential expression among diets was observed for D. melanogaster, a species that survives well under all three conditions, with little impact on its metabolism. We suggest that these three species together provide a model to examine individual and population differences in vulnerability to lifestyle-associated health problems such as metabolic syndrome and diabetes.

  20. Drosophila heterochromatin protein 1 (HP1)/origin recognition complex (ORC) protein is associated with HP1 and ORC and functions in heterochromatin-induced silencing.

    PubMed

    Shareef, M M; King, C; Damaj, M; Badagu, R; Huang, D W; Kellum, R

    2001-06-01

    Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.

  1. Drosophila Heterochromatin Protein 1 (HP1)/Origin Recognition Complex (ORC) Protein Is Associated with HP1 and ORC and Functions in Heterochromatin-induced Silencing

    PubMed Central

    Shareef, Mohammed Momin; King, Chadwick; Damaj, Mona; Badagu, RamaKrishna; Huang, Da Wei; Kellum, Rebecca

    2001-01-01

    Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit. PMID:11408576

  2. The Drosophila BTB Domain Protein Jim Lovell Has Roles in Multiple Larval and Adult Behaviors

    PubMed Central

    Bjorum, Sonia M.; Simonette, Rebecca A.; Alanis, Raul; Wang, Jennifer E.; Lewis, Benjamin M.; Trejo, Michael H.; Hanson, Keith A.; Beckingham, Kathleen M.

    2013-01-01

    Innate behaviors have their origins in the specification of neural fates during development. Within Drosophila, BTB (Bric-a-brac,Tramtrack, Broad) domain proteins such as Fruitless are known to play key roles in the neural differentiation underlying such responses. We previously identified a gene, which we have termed jim lovell (lov), encoding a BTB protein with a role in gravity responses. To understand more fully the behavioral roles of this gene we have investigated its function through several approaches. Transcript and protein expression patterns have been examined and behavioral phenotypes of new lov mutations have been characterized. Lov is a nuclear protein, suggesting a role as a transcriptional regulator, as for other BTB proteins. In late embryogenesis, Lov is expressed in many CNS and PNS neurons. An examination of the PNS expression indicates that lov functions in the late specification of several classes of sensory neurons. In particular, only two of the five abdominal lateral chordotonal neurons express Lov, predicting functional variation within this highly similar group. Surprisingly, Lov is also expressed very early in embryogenesis in ways that suggests roles in morphogenetic movements, amnioserosa function and head neurogenesis. The phenotypes of two new lov mutations that delete adjacent non-coding DNA regions are strikingly different suggesting removal of different regulatory elements. In lov47, Lov expression is lost in many embryonic neurons including the two lateral chordotonal neurons. lov47 mutant larvae show feeding and locomotor defects including spontaneous backward movement. Adult lov47 males perform aberrant courtship behavior distinguished by courtship displays that are not directed at the female. lov47 adults also show more defective negative gravitaxis than the previously isolated lov91Y mutant. In contrast, lov66 produces largely normal behavior but severe female sterility associated with ectopic lov expression in the ovary. We

  3. The Drosophila protein palmitoylome: Characterizing palmitoyl-thioesterases and DHHC palmitoyl-transferases

    PubMed Central

    Bannan, Barbra A.; Van Etten, Jamie; Kohler, John A.; Tsoi, Yui; Hansen, Nicole M.; Sigmon, Stacey; Fowler, Elizabeth; Buff, Haley; Williams, Tiffany S.; Ault, Jeffrey G.; Glaser, Robert L.; Korey, Christopher A.

    2010-01-01

    Palmitoylation is the post-translational addition of a palmitate moiety to a cysteine residue through a covalent thioester bond. The addition and removal of this modification is controlled by both palmitoyl acyl-transferases and thioesterases. Using bioinformatic analysis, we identified 22 DHHC family palmitoyl acyl-transferase homologs in the Drosophila genome. We used in situ hybridization, RT-PCR, and published FlyAtlas microarray data to characterize the expression patterns of all 22 fly homologs. Our results indicate that all are expressed genes, but several, including CG1407, CG4676, CG5620, CG6017/dHIP14, CG6618, CG6627, and CG17257 appear to be enriched in neural tissues suggesting that they are important for neural function. Furthermore, we have found that several may be expressed in a sex-specific manner with adult male-specific expression of CG4483 and CG17195. Using tagged versions of the DHHC genes, we demonstrate that fly DHHC proteins are primarily located in either the Golgi Apparatus or Endoplasmic Reticulum in S2 cells, except for CG1407, which was found on the plasma membrane. We also characterized the subcellular localization and expression of the three known thioesterases: Palmitoyl-protein Thioesterase 1 (Ppt1), Palmitoyl-protein Thioesterase 2 (Ppt2), and Acyl-protein Thioesterase 1 (APT1). Our results indicate that Ppt1 and Ppt2 are the major lysosomal thioesterases while APT1 is the likely cytoplasmic thioesterase. Finally, in vivo rescue experiments show that Ppt2 expression cannot rescue the neural inclusion phenotypes associated with loss of Ppt1, further supporting distinct functions and substrates for these two thioesterases. These results will serve as the basis for a more complete understanding of the protein palmitoylome's normal cellular functions in the fly and will lead to further insights into the molecular etiology of diseases associated with the mis-regulation of palmitoylation. PMID:18719403

  4. Drosophila Ack targets its substrate, the sorting nexin DSH3PX1, to a protein complex involved in axonal guidance.

    PubMed

    Worby, Carolyn A; Simonson-Leff, Nancy; Clemens, James C; Huddler, Donald; Muda, Marco; Dixon, Jack E

    2002-03-15

    Dock, the Drosophila orthologue of Nck, is an adaptor protein that is known to function in axonal guidance paradigms in the fly including proper development of neuronal connections in photoreceptor cells and axonal tracking in Bolwig's organ. To develop a better understanding of axonal guidance at the molecular level, we purified proteins in a complex with the SH2 domain of Dock from fly Schneider 2 cells. A protein designated p145 was identified and shown to be a tyrosine kinase with sequence similarity to mammalian Cdc-42-associated tyrosine kinases. We demonstrate that Drosophila Ack (DAck) can be co-immunoprecipitated with Dock and DSH3PX1 from fly cell extracts. The domains responsible for the in vitro interaction between Drosophila Ack and Dock were identified, and direct protein-protein interactions between complex members were established. We conclude that DSH3PX1 is a substrate for DAck in vivo and in vitro and define one of the major in vitro sites of DSH3PX1 phosphorylation to be Tyr-56. Tyr-56 is located within the SH3 domain of DSH3PX1, placing it in an important position for regulating the binding of proline-rich targets. We demonstrate that Tyr-56 phosphorylation by DAck diminishes the DSH3PX1 SH3 domain interaction with the Wiskott-Aldrich Syndrome protein while enabling DSH3PX1 to associate with Dock. Furthermore, when Tyr-56 is mutated to aspartate or glutamate, the binding to Wiskott-Aldrich Syndrome protein is abrogated. These results suggest that the phosphorylation of DSH3PX1 by DAck targets this sorting nexin to a protein complex that includes Dock, an adaptor protein important for axonal guidance.

  5. The Drosophila lingerer protein cooperates with Orb2 in long-term memory formation.

    PubMed

    Kimura, Shingo; Sakakibara, Yasufumi; Sato, Kosei; Ote, Manabu; Ito, Hiroki; Koganezawa, Masayuki; Yamamoto, Daisuke

    2015-03-01

    Recently mated Drosophila females were shown to be reluctant to copulate and to exhibit rejecting behavior when courted by a male. Males that experience mate refusal by a mated female subsequently attenuate their courtship effort toward not only mated females but also virgin females. This courtship suppression persists for more than a day, and thus represents long-term memory. The courtship long-term memory has been shown to be impaired in heterozygotes as well as homozygotes of mutants in orb2, a locus encoding a set of CPEB RNA-binding proteins. We show that the impaired courtship long-term memory in orb2-mutant heterozygotes is restored by reducing the activity of lig, another putative RNA-binding protein gene, yet on its own the loss-of-function lig mutation is without effect. We further show that Lig forms a complex with Orb2. We infer that a reduction in the Lig levels compensates the Orb2 deficiency by mitigating the negative feedback for Orb2 expression and thereby alleviating defects in long-term memory.

  6. Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein.

    PubMed

    Bhogal, Balpreet; Jepson, James E; Savva, Yiannis A; Pepper, Anita S-R; Reenan, Robert A; Jongens, Thomas A

    2011-10-30

    Loss of FMR1 gene function results in fragile X syndrome, the most common heritable form of intellectual disability. The protein encoded by this locus (FMRP) is an RNA-binding protein that is thought to primarily act as a translational regulator; however, recent studies have implicated FMRP in other mechanisms of gene regulation. We found that the Drosophila fragile X homolog (dFMR1) biochemically interacted with the adenosine-to-inosine RNA-editing enzyme dADAR. Adar and Fmr1 mutant larvae exhibited distinct morphological neuromuscular junction (NMJ) defects. Epistasis experiments based on these phenotypic differences revealed that Adar acts downstream of Fmr1 and that dFMR1 modulates dADAR activity. Furthermore, sequence analyses revealed that a loss or overexpression of dFMR1 affects editing efficiency on certain dADAR targets with defined roles in synaptic transmission. These results link dFMR1 with the RNA-editing pathway and suggest that proper NMJ synaptic architecture requires modulation of dADAR activity by dFMR1.

  7. Australin: a chromosomal passenger protein required specifically for Drosophila melanogaster male meiosis

    PubMed Central

    Gao, Shan; Giansanti, Maria Grazia; Buttrick, Graham J.; Ramasubramanyan, Sharada; Auton, Adam; Gatti, Maurizio; Wakefield, James G.

    2008-01-01

    The chromosomal passenger complex (CPC), which is composed of conserved proteins aurora B, inner centromere protein (INCENP), survivin, and Borealin/DASRA, localizes to chromatin, kinetochores, microtubules, and the cell cortex in a cell cycle–dependent manner. The CPC is required for multiple aspects of cell division. Here we find that Drosophila melanogaster encodes two Borealin paralogues, Borealin-related (Borr) and Australin (Aust). Although Borr is a passenger in all mitotic tissues studied, it is specifically replaced by Aust for the two male meiotic divisions. We analyzed aust mutant spermatocytes to assess the effects of fully inactivating the Aust-dependent functions of the CPC. Our results indicate that Aust is required for sister chromatid cohesion, recruitment of the CPC to kinetochores, and chromosome alignment and segregation but not for meiotic histone phosphorylation or spindle formation. Furthermore, we show that the CPC is required earlier in cytokinesis than previously thought; cells lacking Aust do not initiate central spindle formation, accumulate anillin or actin at the cell equator, or undergo equatorial constriction. PMID:18268101

  8. Materials composed of the Drosophila Hox protein Ultrabithorax are biocompatible and nonimmunogenic.

    PubMed

    Patterson, Jan L; Arenas-Gamboa, Angela M; Wang, Ting-Yi; Hsiao, Hao-Ching; Howell, David W; Pellois, Jean-Philippe; Rice-Ficht, Allison; Bondos, Sarah E

    2015-04-01

    Although the in vivo function of the Drosophila melanogaster Hox protein Ultrabithorax (Ubx) is to regulate transcription, in vitro Ubx hierarchically self-assembles to form nanoscale to macroscale materials. The morphology, mechanical properties, and functionality (via protein chimeras) of Ubx materials are all easily engineered. Ubx materials are also compatible with cells in culture. These properties make Ubx attractive as a potential tissue engineering scaffold, but to be used as such they must be biocompatible and nonimmunogenic. In this study, we assess whether Ubx materials are suitable for in vivo applications. When implanted into mice, Ubx fibers attracted few immune cells to the implant area. Sera from mice implanted with Ubx contain little to no antibodies capable of recognizing Ubx. Furthermore, Ubx fibers cultured with macrophages in vitro did not lyse or activate the macrophages, as measured by TNF-α and NO secretion. Finally, Ubx fibers do not cause hemolysis when incubated with human red blood cells. The minimal effects observed are comparable with those induced by biomaterials used successfully in vivo. We conclude Ubx materials are biocompatible and nonimmunogenic.

  9. Tropomyosin is an interaction partner of the Drosophila coiled coil protein yuri gagarin.

    PubMed

    Texada, Michael J; Simonette, Rebecca A; Deery, William J; Beckingham, Kathleen M

    2011-02-15

    The Drosophila gene yuri gagarin is a complex locus encoding three protein isoform classes that are ubiquitously expressed in the organism. Mutations to the gene affect processes as diverse as gravitactic behavior and spermatogenesis. The larger Yuri isoforms contain extensive coiled-coil regions. Our previous studies indicate that one of the large isoform classes (Yuri-65) is required for formation of specialized F-actin-containing structures generated during spermatogenesis, including the so-called actin "cones" that mediate spermatid individualization. We used the tandem affinity purification of a tagged version of Yuri-65 (the TAP-tagging technique) to identify proteins associated with Yuri-65 in the intact organism. Tropomyosin, primarily as the 284-residue isoform derived from the ubiquitously expressed Tropomyosin 1 gene was thus identified as a major Yuri interaction partner. Co-immunoprecipitation experiments confirmed this interaction. We have established that the stable F-actin cones of spermatogenesis contain Tropomyosin 1 (Tm1) and that in mutant yuri(F64), failure of F-actin cone formation is associated with failure of Tm1 to accumulate at the cone initiation sites. In investigating possible interactions of Tm1 and Yuri in other tissues, we discovered that Tm1 and Yuri frequently colocalize with the endoplasmic reticulum. Tropomyosin has been implicated in actin-mediated membrane trafficking activity in other systems. Our findings suggest that Yuri-Tm1 complexes participate in related functions.

  10. TROPOMYOSIN IS AN INTERACTION PARTNER OF THE DROSOPHILA COILED COIL PROTEIN YURI GAGARIN

    PubMed Central

    Texada, Michael J.; Simonette, Rebecca A.; Deery, William J.; Beckingham, Kathleen M.

    2011-01-01

    The Drosophila gene yuri gagarin is a complex locus encoding three protein isoform classes that are ubiquitously expressed in the organism. Mutations to the gene affect processes as diverse as gravitactic behavior and spermatogenesis. The larger Yuri isoforms contain extensive coiled-coil regions. Our previous studies indicate that one of the large isoform classes (Yuri-65) is required for formation of specialized F-actin-containing structures generated during spermatogenesis, including the so-called actin “cones” that mediate spermatid individualization. We used tandem affinity purification of a tagged version of Yuri-65 (the TAP-tagging technique) to identify proteins associated with Yuri-65 in the intact organism. Tropomyosin, primarily as the 284-residue isoform derived from the ubiquitously expressed Tropomyosin 1 gene was thus identified as a major Yuri interaction partner. Co-immunoprecipitation experiments confirmed this interaction. We have established that the stable F-actin cones of spermatogenesis contain Tropomyosin 1 (Tm1) and that in mutant yuriF64, failure of F-actin cone formation is associated with failure of Tm1 to accumulate at the cone initiation sites. In investigating possible interactions of Tm1 and Yuri in other tissues, we discovered that Tm1 and Yuri frequently colocalize with the endoplasmic reticulum. Tropomyosin has been implicated in actin-mediated membrane trafficking activity in other systems. Our findings suggest that Yuri-Tm1 complexes participate in related functions. PMID:21126519

  11. The Drosophila melanogaster seminal fluid protein Acp62F is a protease inhibitor that is toxic upon ectopic expression.

    PubMed Central

    Lung, Oliver; Tram, Uyen; Finnerty, Casey M; Eipper-Mains, Marcie A; Kalb, John M; Wolfner, Mariana F

    2002-01-01

    Drosophila melanogaster seminal fluid proteins stimulate sperm storage and egg laying in the mated female but also cause a reduction in her life span. We report here that of eight Drosophila seminal fluid proteins (Acps) and one non-Acp tested, only Acp62F is toxic when ectopically expressed. Toxicity to preadult male or female Drosophila occurs upon one exposure, whereas multiple exposures are needed for toxicity to adult female flies. Of the Acp62F received by females during mating, approximately 10% enters the circulatory system while approximately 90% remains in the reproductive tract. We show that in the reproductive tract, Acp62F localizes to the lumen of the uterus and the female's sperm storage organs. Analysis of Acp62F's sequence, and biochemical assays, reveals that it encodes a trypsin inhibitor with sequence and structural similarities to extracellular serine protease inhibitors from the nematode Ascaris. In light of previous results demonstrating entry of Acp62F into the mated female's hemolymph, we propose that Acp62F is a candidate for a molecule to contribute to the Acp-dependent decrease in female life span. We propose that Acp62F's protease inhibitor activity exerts positive protective functions in the mated female's reproductive tract but that entry of a small amount of this protein into the female's hemolymph could contribute to the cost of mating. PMID:11805057

  12. The kinesin-associated protein UNC-76 is required for axonal transport in the Drosophila nervous system.

    PubMed

    Gindhart, Joseph G; Chen, Jinyun; Faulkner, Melissa; Gandhi, Rita; Doerner, Karl; Wisniewski, Tiffany; Nandlestadt, Aline

    2003-08-01

    Kinesin-I is essential for the transport of membrane-bound organelles in neural and nonneural cells. However, the means by which kinesin interacts with its intracellular cargoes, and the means by which kinesin-cargo interactions are regulated in response to cellular transport requirements are not fully understood. The C terminus of the Drosophila kinesin heavy chain (KHC) was used in a two-hybrid screen of a Drosophila cDNA library to identify proteins that bind specifically to the kinesin tail domain. UNC-76 is an evolutionarily conserved cytosolic protein that binds to the tail domain of KHC in two-hybrid and copurification assays, indicating that kinesin and UNC-76 form a stable complex in vivo. Loss of Drosophila Unc-76 function results in locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants, suggesting that UNC-76 is required for kinesin-dependent axonal transport. Unc-76 exhibits dosage-sensitive genetic relationships with Khc and Kinesin light chain mutations, further supporting the hypothesis that UNC-76 and kinesin-I work in a common transport pathway. Given the interaction of FEZ1, the mammalian homolog of UNC-76, with protein kinase Czeta, and the role of FEZ1 in axon outgrowth, we propose that UNC-76 helps integrate kinesin activity in response to transport requirements in axons.

  13. Nutrient-Dependent Requirement for SOD1 in Lifespan Extension by Protein Restriction in Drosophila melanogaster

    PubMed Central

    Sun, Xiaoping; Komatsu, Toshimitsu; Lim, Jinhwan; Laslo, Mara; Yolitz, Jason; Wang, Cecilia; Poirier, Luc; Alberico, Thomas; Zou, Sige

    2012-01-01

    Summary Reactive oxygen species (ROS) modulate aging and aging-related diseases. Dietary composition is critical in modulating lifespan. However, how ROS modulate dietary effects on lifespan remains poorly understood. Superoxide dismutase 1 (SOD1) is a major cytosolic enzyme responsible for scavenging superoxides. Here we investigated the role of SOD1 in lifespan modulation by diet in Drosophila. We found that a high sugar-low protein (HS-LP) diet or low-calorie diet with low-sugar content, representing protein restriction, increased lifespan but not resistance to acute oxidative stress in wild-type flies, relative to a standard base diet. A low sugar-high protein diet had an opposite effect. Our genetic analysis indicated that SOD1 overexpression or dfoxo deletion did not alter lifespan patterns of flies responding to diets. However, sod1 reduction blunted lifespan extension by the HS-LP diet but not the low-calorie diet. HS-LP and low-calorie diets both reduced target-of-rapamycin (TOR) signaling and only the HS-LP diet increased oxidative damage. sod1 knockdown did not affect phosphorylation of S6 kinase, suggesting that SOD1 acts in parallel with or downstream of TOR signaling. Surprisingly rapamycin decreased lifespan in sod1 mutant but not wild-type males fed the standard, HS-LP and low calorie diets, whereas antioxidant N-acetylcysteine only increased lifespan in sod1 mutant males fed the HS-LP diet, when compared to diet-matched controls. Our findings suggest that SOD1 is required for lifespan extension by protein restriction only when dietary sugar is high, and support the context-dependent role of ROS in aging and caution the use of rapamycin and antioxidants in aging interventions. PMID:22672579

  14. Glucocerebrosidase Deficiency in Drosophila Results in α-Synuclein-Independent Protein Aggregation and Neurodegeneration.

    PubMed

    Davis, Marie Y; Trinh, Kien; Thomas, Ruth E; Yu, Selina; Germanos, Alexandre A; Whitley, Brittany N; Sardi, Sergio Pablo; Montine, Thomas J; Pallanck, Leo J

    2016-03-01

    Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher's disease, and are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB) excluding variants of low penetrance. Because α-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing α-synuclein toxicity. To explore this hypothesis, we deleted the Drosophila GBA1 homolog, dGBA1b, and compared the phenotypes of dGBA1b mutants in the presence and absence of α-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human α-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased α-synuclein aggregation relative to controls. However, α-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. Our findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of α-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity.

  15. Glucocerebrosidase Deficiency in Drosophila Results in α-Synuclein-Independent Protein Aggregation and Neurodegeneration

    PubMed Central

    Thomas, Ruth E.; Yu, Selina; Germanos, Alexandre A.; Whitley, Brittany N.; Sardi, Sergio Pablo; Montine, Thomas J.; Pallanck, Leo J.

    2016-01-01

    Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher’s disease, and are the most common genetic risk factor for Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) excluding variants of low penetrance. Because α-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing α-synuclein toxicity. To explore this hypothesis, we deleted the Drosophila GBA1 homolog, dGBA1b, and compared the phenotypes of dGBA1b mutants in the presence and absence of α-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human α-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased α-synuclein aggregation relative to controls. However, α-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. Our findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of α-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity. PMID:27019408

  16. Tubulin domains for the interaction of microtubule associated protein DMAP-85 from Drosophila melanogaster.

    PubMed

    Henríquez, J P; Cambiazo, V; Maccioni, R B

    1996-05-24

    The interaction of microtubule associated proteins (MAPs) with the microtubule system has been characterized in depth in neuronal cells from various mammalian species. These proteins interact with well-defined domains within the acidic tubulin carboxyl-terminal regulatory region. However, there is little information on the mechanisms of MAPs-tubulin interactions in nonmammalian systems. Recently, a novel tau-like protein designated as DMAP-85 has been identified in Drosophila melanogaster, and the regulation of its interactions with cytoskeletal elements was analyzed throughout different developmental stages of this organism. In this report, the topographic domains involved in the binding of DMAP-85 with tubulin heterodimer were investigated. Affinity chromatography of DMAP-85 in matrixes of taxol-stabilized microtubules showed the reversible interaction of DMAP-85 with domains on the microtubular surface. Co-sedimentation studies using the subtilisin-treated tubulin (S-tubulin) indicated the lack of association of DMAP-85 to this tubulin moiety. Moreover, studies on affinity chromatography of the purified 4 kDa C-terminal tubulin peptide bound to an affinity column, confirmed that DMAP-85 interacts directly with this regulatory domain on tubulin subunits. Further studies on sequential affinity chromatography using a calmodulin affinity column followed by the microtubule column confirmed the similarities in the interaction behaviour of DMAP-85 with that of tau. DMAP-85 associated to both calmodulin and the microtubular polymer. These studies support the idea that the carboxyl-terminal region on tubulin constitutes a common binding domain for most microtubule-interacting proteins.

  17. Bonus, a Drosophila Homolog of TIF1 Proteins, Interacts with Nuclear Receptors and Can Inhibit βFTZ-F1-Dependent Transcription

    PubMed Central

    Beckstead, Robert; Ortiz, José A; Sanchez, Cecilia; Prokopenko, Sergei N; Chambon, Pierre; Losson, Régine; Bellen, Hugo J

    2013-01-01

    The Drosophila bonus (bon) gene encodes a homolog of the vertebrate TIF1 transcriptional cofactors. bon is required for male viability, molting, and numerous events in metamorphosis including leg elongation, bristle development, and pigmentation. Most of these processes are associated with genes that have been implicated in the ecdysone pathway, a nuclear hormone receptor pathway required throughout Drosophila development. Bon is associated with sites on the polytene chromosomes and can interact with numerous Drosophila nuclear receptor proteins. Bon binds via an LxxLL motif to the AF-2 activation domain present in the ligand binding domain of βFTZ-F1 and behaves as a transcriptional inhibitor in vivo. PMID:11336699

  18. Structural analysis of the Drosophila rpA1 gene, a member of the eucaryotic 'A' type ribosomal protein family.

    PubMed Central

    Qian, S; Zhang, J Y; Kay, M A; Jacobs-Lorena, M

    1987-01-01

    The expression of ribosomal protein (r-protein) genes is uniquely regulated at the translational level during early development of Drosophila. Here we report results of a detailed analysis of the r-protein rpA1 gene. A cloned DNA sequence coding for rpA1 has been identified by hybrid-selected translation and amino acid composition analysis. The rpA1 gene was localized to polytene chromosome band 53CD. The nucleotide sequence of the rpA1 gene and its cDNA have been determined. rpA1 is a single copy gene and sequence comparison between the gene and its cDNA indicates that this r-protein gene is intronless. Allelic restriction site polymorphisms outside of the gene were observed, while the coding sequence is well conserved between two Drosophila strains. The protein has unusual domains rich in Ala and charged residues. The rpA1 is homologous to the "A" family of eucaryotic acidic r-proteins which are known to play a key role in the initiation and elongation steps of protein synthesis. Images PMID:3103101

  19. The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span

    PubMed Central

    Wang, Ji-Wu; Brent, Jonathan R.; Tomlinson, Andrew; Shneider, Neil A.; McCabe, Brian D.

    2011-01-01

    The fatal adult motor neuron disease amyotrophic lateral sclerosis (ALS) shares some clinical and pathological overlap with frontotemporal dementia (FTD), an early-onset neurodegenerative disorder. The RNA/DNA-binding proteins fused in sarcoma (FUS; also known as TLS) and TAR DNA binding protein-43 (TDP-43) have recently been shown to be genetically and pathologically associated with familial forms of ALS and FTD. It is currently unknown whether perturbation of these proteins results in disease through mechanisms that are independent of normal protein function or via the pathophysiological disruption of molecular processes in which they are both critical. Here, we report that Drosophila mutants in which the homolog of FUS is disrupted exhibit decreased adult viability, diminished locomotor speed, and reduced life span compared with controls. These phenotypes were fully rescued by wild-type human FUS, but not ALS-associated mutant FUS proteins. A mutant of the Drosophila homolog of TDP-43 had similar, but more severe, deficits. Through cross-rescue analysis, we demonstrated that FUS acted together with and downstream of TDP-43 in a common genetic pathway in neurons. Furthermore, we found that these proteins associated with each other in an RNA-dependent complex. Our results establish that FUS and TDP-43 function together in vivo and suggest that molecular pathways requiring the combined activities of both of these proteins may be disrupted in ALS and FTD. PMID:21881207

  20. Plasticity in patterns of histone modifications and chromosomal proteins in Drosophila heterochromatin

    PubMed Central

    Riddle, Nicole C.; Minoda, Aki; Kharchenko, Peter V.; Alekseyenko, Artyom A.; Schwartz, Yuri B.; Tolstorukov, Michael Y.; Gorchakov, Andrey A.; Jaffe, Jacob D.; Kennedy, Cameron; Linder-Basso, Daniela; Peach, Sally E.; Shanower, Gregory; Zheng, Haiyan; Kuroda, Mitzi I.; Pirrotta, Vincenzo; Park, Peter J.; Elgin, Sarah C.R.; Karpen, Gary H.

    2011-01-01

    Eukaryotic genomes are packaged in two basic forms, euchromatin and heterochromatin. We have examined the composition and organization of Drosophila melanogaster heterochromatin in different cell types using ChIP-array analysis of histone modifications and chromosomal proteins. As anticipated, the pericentric heterochromatin and chromosome 4 are on average enriched for the “silencing” marks H3K9me2, H3K9me3, HP1a, and SU(VAR)3-9, and are generally depleted for marks associated with active transcription. The locations of the euchromatin–heterochromatin borders identified by these marks are similar in animal tissues and most cell lines, although the amount of heterochromatin is variable in some cell lines. Combinatorial analysis of chromatin patterns reveals distinct profiles for euchromatin, pericentric heterochromatin, and the 4th chromosome. Both silent and active protein-coding genes in heterochromatin display complex patterns of chromosomal proteins and histone modifications; a majority of the active genes exhibit both “activation” marks (e.g., H3K4me3 and H3K36me3) and “silencing” marks (e.g., H3K9me2 and HP1a). The hallmark of active genes in heterochromatic domains appears to be a loss of H3K9 methylation at the transcription start site. We also observe complex epigenomic profiles of intergenic regions, repeated transposable element (TE) sequences, and genes in the heterochromatic extensions. An unexpectedly large fraction of sequences in the euchromatic chromosome arms exhibits a heterochromatic chromatin signature, which differs in size, position, and impact on gene expression among cell types. We conclude that patterns of heterochromatin/euchromatin packaging show greater complexity and plasticity than anticipated. This comprehensive analysis provides a foundation for future studies of gene activity and chromosomal functions that are influenced by or dependent upon heterochromatin. PMID:21177972

  1. Ribosomal protein insufficiency and the minute syndrome in Drosophila: a dose-response relationship.

    PubMed Central

    Saebøe-Larssen, S; Lyamouri, M; Merriam, J; Oksvold, M P; Lambertsson, A

    1998-01-01

    Minutes comprise > 50 phenotypically similar mutations scattered throughout the genome of Drosophila, many of which are identified as mutations in ribosomal protein (rp) genes. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. By mobilizing a P element inserted in the 5' UTR of M(3)95A, the gene encoding ribosomal protein S3 (RPS3), we have generated two homozygous viable heteroalleles that are partial revertants with respect to the Minute phenotype. Molecular characterization revealed both alleles to be imprecise excisions, leaving 40 and 110 bp, respectively, at the P-element insertion site. The weaker allele (40 bp insert) is associated with a approximately 15% decrease in RPS3 mRNA abundance and displays a moderate Minute phenotype. In the stronger allele (110 bp insert) RPS3 mRNA levels are reduced by approximately 60%, resulting in an extreme Minute phenotype that includes many morphological abnormalities as well as sterility in both males and females due to disruption of early gametogenesis. The results show that there is a correlation between reduced RPS3 mRNA levels and the severity of the Minute phenotype, in which faulty differentiation of somatic tissues and arrest of gametogenesis represent the extreme case. That heteroalleles in M(3)95A can mimic the phenotypic variations that exist between different Minute/rp-gene mutations strongly suggests that all phenotypes primarily are caused by reductions in maximum protein synthesis rates, but that the sensitivity for reduced levels of the individual rp-gene products is different. PMID:9539436

  2. Plasticity in patterns of histone modifications and chromosomal proteins in Drosophila heterochromatin.

    PubMed

    Riddle, Nicole C; Minoda, Aki; Kharchenko, Peter V; Alekseyenko, Artyom A; Schwartz, Yuri B; Tolstorukov, Michael Y; Gorchakov, Andrey A; Jaffe, Jacob D; Kennedy, Cameron; Linder-Basso, Daniela; Peach, Sally E; Shanower, Gregory; Zheng, Haiyan; Kuroda, Mitzi I; Pirrotta, Vincenzo; Park, Peter J; Elgin, Sarah C R; Karpen, Gary H

    2011-02-01

    Eukaryotic genomes are packaged in two basic forms, euchromatin and heterochromatin. We have examined the composition and organization of Drosophila melanogaster heterochromatin in different cell types using ChIP-array analysis of histone modifications and chromosomal proteins. As anticipated, the pericentric heterochromatin and chromosome 4 are on average enriched for the "silencing" marks H3K9me2, H3K9me3, HP1a, and SU(VAR)3-9, and are generally depleted for marks associated with active transcription. The locations of the euchromatin-heterochromatin borders identified by these marks are similar in animal tissues and most cell lines, although the amount of heterochromatin is variable in some cell lines. Combinatorial analysis of chromatin patterns reveals distinct profiles for euchromatin, pericentric heterochromatin, and the 4th chromosome. Both silent and active protein-coding genes in heterochromatin display complex patterns of chromosomal proteins and histone modifications; a majority of the active genes exhibit both "activation" marks (e.g., H3K4me3 and H3K36me3) and "silencing" marks (e.g., H3K9me2 and HP1a). The hallmark of active genes in heterochromatic domains appears to be a loss of H3K9 methylation at the transcription start site. We also observe complex epigenomic profiles of intergenic regions, repeated transposable element (TE) sequences, and genes in the heterochromatic extensions. An unexpectedly large fraction of sequences in the euchromatic chromosome arms exhibits a heterochromatic chromatin signature, which differs in size, position, and impact on gene expression among cell types. We conclude that patterns of heterochromatin/euchromatin packaging show greater complexity and plasticity than anticipated. This comprehensive analysis provides a foundation for future studies of gene activity and chromosomal functions that are influenced by or dependent upon heterochromatin.

  3. Context-dependent transcriptional interpretation of mitogen activated protein kinase signaling in the Drosophila embryo

    PubMed Central

    Kim, Yoosik; Iagovitina, Antonina; Ishihara, Keisuke; Fitzgerald, Kate M.; Deplancke, Bart; Papatsenko, Dmitri; Shvartsman, Stanislav Y.

    2013-01-01

    Terminal regions of the Drosophila embryo are patterned by the localized activation of Mitogen Activated Protein Kinase (MAPK), which induces zygotic genes through relief of their repression by transcriptional repressor Capicua. The levels of MAPK activation at the anterior and posterior termini are close to each other, but the expression patterns of MAPK-target genes, such as zerknüllt (zen) and tailless (tll), display strong anterior-posterior (AP) asymmetry. This region-specific response to MAPK activation provides a clear example of context-dependent interpretation of inductive signaling, a common developmental effect that remains poorly understood. In the past, the AP asymmetry of zen expression was attributed to a mechanism that depends on MAPK substrate competition. We present data suggesting that the asymmetric expression of tll is generated by a different mechanism, based on feedforward control and multiple enhancers of the tll gene. A simple mathematical model of this mechanism correctly predicts how the wild-type expression pattern of tll changes in mutants affecting the anterior, dorsoventral, and terminal patterning systems and some of their direct targets. PMID:23822503

  4. Signal Integration by the IκB Protein Pickle Shapes Drosophila Innate Host Defense.

    PubMed

    Morris, Otto; Liu, Xi; Domingues, Celia; Runchel, Christopher; Chai, Andrea; Basith, Shaherin; Tenev, Tencho; Chen, Haiyang; Choi, Sangdun; Pennetta, Giuseppa; Buchon, Nicolas; Meier, Pascal

    2016-09-14

    Pattern recognition receptors are activated following infection and trigger transcriptional programs important for host defense. Tight regulation of NF-κB activation is critical to avoid detrimental and misbalanced responses. We describe Pickle, a Drosophila nuclear IκB that integrates signaling inputs from both the Imd and Toll pathways by skewing the transcriptional output of the NF-κB dimer repertoire. Pickle interacts with the NF-κB protein Relish and the histone deacetylase dHDAC1, selectively repressing Relish homodimers while leaving other NF-κB dimer combinations unscathed. Pickle's ability to selectively inhibit Relish homodimer activity contributes to proper host immunity and organismal health. Although loss of pickle results in hyper-induction of Relish target genes and improved host resistance to pathogenic bacteria in the short term, chronic inactivation of pickle causes loss of immune tolerance and shortened lifespan. Pickle therefore allows balanced immune responses that protect from pathogenic microbes while permitting the establishment of beneficial commensal host-microbe relationships.

  5. Chromatin remodeling protein INO80 has a role in regulation of homeotic gene expression in Drosophila.

    PubMed

    Bhatia, Shipra; Pawar, Hema; Dasari, Vasanthi; Mishra, Rakesh K; Chandrashekaran, Shanti; Brahmachari, Vani

    2010-06-01

    The homologues of yeast INO80 are identified across phyla from Caenorhabditis elegans to human. In Drosophila it has been shown that dINO80 forms a complex with Pleiohomeotic but does not interact with Hox PRE (polycomb responsive element). As some proteins of the INO80 complex are implicated in homeotic gene regulation, we examined if dINO80 is involved in regulation of homeotic genes. We find that dINO80 null mutants generated by imprecise excision of P-element are late embryonic lethals and show homeotic transformation. We detect misexpression of homeotic genes like Sex-comb reduced, Antennapedia, Ultrabithorax and Abdominal-B in dIno80 mutant embryos by immunostaining which is further substantiated by quantitative PCR. Polycomb phenotype in dIno80-Pc is enhanced in double mutants. Concurrently, the localization of dINO80 to sequences upstream of misexpressed genes in vivo shows that dINO80 is involved in homeotic gene regulation and probably through its interactions with PcG-trxG complexes.

  6. Regulation of notch endosomal sorting and signaling by Drosophila Nedd4 family proteins.

    PubMed

    Wilkin, Marian B; Carbery, Ann-Marie; Fostier, Maggy; Aslam, Hanna; Mazaleyrat, Sabine L; Higgs, Jenny; Myat, Anna; Evans, Dana A P; Cornell, Michael; Baron, Martin

    2004-12-29

    The Notch receptor mediates a short-range signal that regulates many cell fate decisions. The misregulation of Notch has been linked to cancer and to developmental disorders. Upon binding to its ligands, Delta (Dl) or Serrate (Ser), the Notch ectodomain is shed by the action of an ADAM protease. The Notch intracellular domain is subsequently released proteolytically from the membrane by Presenilin and translocates to the nucleus to activate the transcription factor, Suppressor of Hairless. We show in Drosophila that Notch signaling is limited by the activity of two Nedd4 family HECT domain proteins, Suppressor of deltex [Su(dx)] and DNedd4. We rule out models by which Su(dx) downregulates Notch through modulating Deltex or by limiting the adherens junction accumulation of Notch. Instead, we show that Su(dx) regulates the postendocytic sorting of Notch within the early endosome to an Hrs- and ubiquitin-enriched subdomain en route to the late endosome. We propose a model in which endocytic sorting of Notch mediates a decision between its activation and downregulation. Such intersections between trafficking routes may provide key points at which other signals can modulate Notch activity in both normal development and in the pathological misactivation of Notch.

  7. Molecular Social Interactions: Drosophila melanogaster Seminal Fluid Proteins as a Case Study

    PubMed Central

    Sirot, Laura K.; LaFlamme, Brooke A.; Sitnik, Jessica L.; Rubinstein, C. Dustin; Avila, Frank W.; Chow, Clement Y.; Wolfner, Mariana F.

    2014-01-01

    Studies of social behavior generally focus on interactions between two or more individual animals. However, these interactions are not simply between whole animals, but also occur between molecules that were produced by the interacting individuals. Such “molecular social interactions” can both influence and be influenced by the organismal-level social interactions. We illustrate this by reviewing the roles played by seminal fluid proteins (Sfps) in molecular social interactions between males and females of the fruit fly Drosophila melanogaster. Sfps, which are produced by males and transferred to females during mating, are involved in inherently social interactions with female-derived molecules, and they influence social interactions between males and females and between a female’s past and potential future mates. Here, we explore four examples of molecular social interactions involving D. melanogaster Sfps: processes that influence mating, sperm storage, ovulation, and ejaculate transfer. We consider the molecular and organismal players involved in each interaction and the consequences of their interplay for the reproductive success of both sexes. We conclude with a discussion of the ways in which Sfps can both shape and be shaped by (in an evolutionary sense) the molecular social interactions in which they are involved. PMID:20109658

  8. Modulation of feeding behavior by odorant-binding proteins in Drosophila melanogaster.

    PubMed

    Swarup, Shilpa; Morozova, Tatiana V; Sridhar, Sruthipriya; Nokes, Michael; Anholt, Robert R H

    2014-02-01

    Nutrient intake and avoidance of toxins are essential for survival and controlled by attractive and aversive feeding responses. Drosophila melanogaster presents one of the best characterized systems for studies on chemosensation, which is mediated by multigene families of chemoreceptors, including olfactory receptors, gustatory receptors, and odorant-binding proteins (OBPs). Although the response profiles of gustatory receptors have been well studied, the contribution of OBPs to food intake is largely unknown. As most aversive ("bitter") tastants are hydrophobic, we hypothesized that OBPs may fulfill an essential function in transporting bitter tastants to gustatory receptors to modulate feeding behavior. Here, we used 16 RNAi lines that inhibit expression of individual target Obp genes and show that OBPs modulate sucrose intake in response to a panel of nine bitter compounds. Similar to their function in olfaction, OBPs appear to interact with bitter compounds in a combinatorial and sex-dependent manner. RNAi-mediated reduction in expression of individual Obp genes resulted either in enhanced or reduced intake of sucrose in the presence of bitter compounds, consistent with roles for OBPs in transporting tastants to bitter taste receptors, sequestering them to limit their access to these receptors, or interacting directly with gustatory neurons that respond to sucrose.

  9. Tre1, a G Protein-Coupled Receptor, Directs Transepithelial Migration of Drosophila Germ Cells

    PubMed Central

    Bainton, Roland J; Heberlein, Ulrike

    2003-01-01

    In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG) is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR), Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target. PMID:14691551

  10. Control of synaptic connectivity by a network of Drosophila IgSF cell surface proteins

    PubMed Central

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Jeon, Mili; Birnbaum, Michael E.; Bellen, Hugo J.; Garcia, K. Christopher; Zinn, Kai

    2015-01-01

    Summary We have defined a network of interacting Drosophila cell surface proteins in which a 21-member IgSF subfamily, the Dprs, binds to a 9-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the 6 dpr and DIP genes examined here is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. Our findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity. PMID:26687361

  11. Natural Variation in Odorant Recognition Among Odorant-Binding Proteins in Drosophila melanogaster

    PubMed Central

    Wang, Ping; Lyman, Richard F.; Mackay, Trudy F. C.; Anholt, Robert R. H.

    2010-01-01

    Chemical recognition is essential for survival and reproduction. Adaptive evolution has resulted in diverse chemoreceptor families, in which polymorphisms contribute to individual variation in chemosensation. To gain insights into the genetic determinants of individual variation in odorant recognition, we measured olfactory responses to two structurally similar odorants in a population of wild-derived inbred lines of Drosophila melanogaster. Odorant-binding proteins (OBPs) are the first components of the insect olfactory system to encounter odorants. Previously four single-nucleotide polymorphisms (SNPs) in the Obp99 group were associated with variation in olfactory responses to benzaldehyde. Here, we identify six different SNPs that are associated with variation in responses to a structurally similar odorant, acetophenone, in the same Obp genes. Five SNPs are in coding regions of Obp99b and Obp99d and one SNP is in the 3′-untranslated region of Obp99a (A610G). We found that the 610G allele is associated with higher response scores to acetophenone than the 610A allele, but with lower expression of Obp99a, suggesting that binding of acetophenone to Opb99a might limit rather than facilitate access to odorant receptors. Our results show that overlapping sets of OBPs contribute to odorant recognition for structurally similar odorants, but that different SNPs are associated with odorant-specific individual variation. Thus, dual olfactory recognition where OBPs regulate odorant access to receptors may enhance olfactory discrimination. PMID:20026676

  12. Control of Synaptic Connectivity by a Network of Drosophila IgSF Cell Surface Proteins.

    PubMed

    Carrillo, Robert A; Özkan, Engin; Menon, Kaushiki P; Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Jeon, Mili; Birnbaum, Michael E; Bellen, Hugo J; Garcia, K Christopher; Zinn, Kai

    2015-12-17

    We have defined a network of interacting Drosophila cell surface proteins in which a 21-member IgSF subfamily, the Dprs, binds to a nine-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the six dpr and DIP genes examined here is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. Our findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity.

  13. Reduction of protein translation and activation of autophagy protect against PINK1 pathogenesis in Drosophila melanogaster.

    PubMed

    Liu, Song; Lu, Bingwei

    2010-12-09

    Mutations in PINK1 and Parkin cause familial, early onset Parkinson's disease. In Drosophila melanogaster, PINK1 and Parkin mutants show similar phenotypes, such as swollen and dysfunctional mitochondria, muscle degeneration, energy depletion, and dopaminergic (DA) neuron loss. We previously showed that PINK1 and Parkin genetically interact with the mitochondrial fusion/fission pathway, and PINK1 and Parkin were recently proposed to form a mitochondrial quality control system that involves mitophagy. However, the in vivo relationships among PINK1/Parkin function, mitochondrial fission/fusion, and autophagy remain unclear; and other cellular events critical for PINK1 pathogenesis remain to be identified. Here we show that PINK1 genetically interacted with the protein translation pathway. Enhanced translation through S6K activation significantly exacerbated PINK1 mutant phenotypes, whereas reduction of translation showed suppression. Induction of autophagy by Atg1 overexpression also rescued PINK1 mutant phenotypes, even in the presence of activated S6K. Downregulation of translation and activation of autophagy were already manifested in PINK1 mutant, suggesting that they represent compensatory cellular responses to mitochondrial dysfunction caused by PINK1 inactivation, presumably serving to conserve energy. Interestingly, the enhanced PINK1 mutant phenotype in the presence of activated S6K could be fully rescued by Parkin, apparently in an autophagy-independent manner. Our results reveal complex cellular responses to PINK1 inactivation and suggest novel therapeutic strategies through manipulation of the compensatory responses.

  14. TSPO, a Mitochondrial Outer Membrane Protein, Controls Ethanol-Related Behaviors in Drosophila

    PubMed Central

    Lin, Ran; Rittenhouse, Danielle; Sweeney, Katelyn; Potluri, Prasanth; Wallace, Douglas C.

    2015-01-01

    The heavy consumption of ethanol can lead to alcohol use disorders (AUDs) which impact patients, their families, and societies. Yet the genetic and physiological factors that predispose humans to AUDs remain unclear. One hypothesis is that alterations in mitochondrial function modulate neuronal sensitivity to ethanol exposure. Using Drosophila genetics we report that inactivation of the mitochondrial outer membrane translocator protein 18kDa (TSPO), also known as the peripheral benzodiazepine receptor, affects ethanol sedation and tolerance in male flies. Knockdown of dTSPO in adult male neurons results in increased sensitivity to ethanol sedation, and this effect requires the dTSPO depletion-mediated increase in reactive oxygen species (ROS) production and inhibition of caspase activity in fly heads. Systemic loss of dTSPO in male flies blocks the development of tolerance to repeated ethanol exposures, an effect that is not seen when dTSPO is only inactivated in neurons. Female flies are naturally more sensitive to ethanol than males, and female fly heads have strikingly lower levels of dTSPO mRNA than males. Hence, mitochondrial TSPO function plays an important role in ethanol sensitivity and tolerance. Since a large array of benzodiazepine analogues have been developed that interact with the peripheral benzodiazepine receptor, the mitochondrial TSPO might provide an important new target for treating AUDs. PMID:26241038

  15. Dynamic regulation of basement membrane protein levels promotes egg chamber elongation in Drosophila

    PubMed Central

    Isabella, Adam J.; Horne-Badovinac, Sally

    2015-01-01

    Basement membranes (BMs) are sheet-like extracellular matrices that provide essential support to epithelial tissues. Recent evidence suggests that regulated changes in BM architecture can direct tissue morphogenesis, but the mechanisms by which cells remodel BMs are largely unknown. The Drosophila egg chamber is an organ-like structure that transforms from a spherical to an ellipsoidal shape as it matures. This elongation coincides with a stage-specific increase in Type IV Collagen (Col IV) levels in the BM surrounding the egg chamber; however, the mechanisms and morphogenetic relevance of this remodeling event have not been established. Here, we identify the Collagen-binding protein SPARC as a negative regulator of egg chamber elongation, and show that SPARC down-regulation is necessary for the increase in Col IV levels to occur. We find that SPARC interacts with Col IV prior to secretion and propose that, through this interaction, SPARC blocks the incorporation of newly synthesized Col IV into the BM. We additionally observe a decrease in Perlecan levels during elongation, and show that Perlecan is a negative regulator of this process. These data provide mechanistic insight into SPARC’s conserved role in matrix dynamics and demonstrate that regulated changes in BM composition influence organ morphogenesis. PMID:26348027

  16. Male accessory gland proteins affect differentially female sexual receptivity and remating in closely related Drosophila species.

    PubMed

    Denis, Béatrice; Claisse, Gaëlle; Le Rouzic, Arnaud; Wicker-Thomas, Claude; Lepennetier, Gildas; Joly, Dominique

    2017-05-01

    In sexual species, mating success depends on the male's capacity to find sexual partners and on female receptivity to mating. Mating is under evolutionary constraints to prevent interspecific mating and to maximize the reproductive success of both sexes. In Drosophila melanogaster, female receptivity to mating is mainly controlled by Sex peptide (SP, i.e. Acp70A) produced by the male accessory glands with other proteins (Acps). The transfer of SP during copulation dramatically reduces female receptivity to mating and prevents remating with other males. To date, female postmating responses are well-known in D. melanogaster but have been barely investigated in closely-related species or strains exhibiting different mating systems (monoandrous versus polyandrous). Here, we describe the diversity of mating systems in two strains of D. melanogaster and the three species of the yakuba complex. Remating delay and sexual receptivity were measured in cross-experiments following SP orthologs or Acp injections within females. Interestingly, we discovered strong differences between the two strains of D. melanogaster as well as among the three species of the yakuba complex. These results suggest that reproductive behavior is under the control of complex sexual interactions between the sexes and evolves rapidly, even among closely-related species. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. The Drosophila secreted protein Argos regulates signal transduction in the Ras/MAPK pathway.

    PubMed

    Sawamoto, K; Okabe, M; Tanimura, T; Mikoshiba, K; Nishida, Y; Okano, H

    1996-08-25

    The Drosophila argos gene encodes a secreted protein with an EGF motif which acts as an inhibitor of cellular differentiation in multiple developmental processes. To investigate the cellular pathways regulated by Argos, we screened for mutations which could modify the phenotype caused by overexpression of argos. We show that the effects of argos overexpression on the eye and wing vein development are suppressed by gain-of-function mutations of the MAPKK/D-MEK gene (Dsor1/D-mek) and the MAPK/ERK-A gene (rolled) and were enhanced by loss-of-function mutations of Star. Loss-of-function mutations in components of the Ras/MAPK signaling cascade act as dominant suppressors of the phenotype caused by the argos null mutations. A loss-of-function argos mutation enhanced the overproduction of R7 neurons caused by gain-of-function alleles of Son of sevenless and Dsor1. Conversely, overexpression of argos inhibited formation of the extra R7 cells that was caused by high-level MAPK/ERK-A activity. A phenotype of the sev; argos double mutants revealed that sev is epistatic to argos. These results provide evidence that Argos negatively regulates signal transduction events in the Ras/MAPK cascade.

  18. HP1 Recruitment in the Absence of Argonaute Proteins in Drosophila

    PubMed Central

    Moshkovich, Nellie; Lei, Elissa P.

    2010-01-01

    Highly repetitive and transposable element rich regions of the genome must be stabilized by the presence of heterochromatin. A direct role for RNA interference in the establishment of heterochromatin has been demonstrated in fission yeast. In metazoans, which possess multiple RNA–silencing pathways that are both functionally distinct and spatially restricted, whether RNA silencing contributes directly to heterochromatin formation is not clear. Previous studies in Drosophila melanogaster have suggested the involvement of both the AGO2-dependent endogenous small interfering RNA (endo-siRNA) as well as Piwi-interacting RNA (piRNA) silencing pathways. In order to determine if these Argonaute genes are required for heterochromatin formation, we utilized transcriptional reporters and chromatin immunoprecipitation of the critical factor Heterochromatin Protein 1 (HP1) to monitor the heterochromatic state of piRNA clusters, which generate both endo-siRNAs and the bulk of piRNAs. Surprisingly, we find that mutation of AGO2 or piwi increases silencing at piRNA clusters corresponding to an increase of HP1 association. Furthermore, loss of piRNA production from a single piRNA cluster results in genome-wide redistribution of HP1 and reduction of silencing at a distant heterochromatic site, suggesting indirect effects on HP1 recruitment. Taken together, these results indicate that heterochromatin forms independently of endo-siRNA and piRNA pathways. PMID:20300658

  19. Direct interaction of the Polycomb protein with Antennapedia regulatory sequences in polytene chromosomes of Drosophila melanogaster.

    PubMed Central

    Zink, B; Engström, Y; Gehring, W J; Paro, R

    1991-01-01

    The Polycomb (Pc) gene is responsible for the elaboration and maintenance of the expression pattern of the homeotic genes during development of Drosophila. In mutant Pc- embryos, homeotic transcripts are ectopically expressed, leading to abdominal transformations in all segments. From this it was suggested that PC+ acts as a repressor of homeotic gene transcription. We have mapped the cis-acting control sequences of the homeotic Antennapedia (Antp) gene regulated by Pc. Using Antp P1 and P2 promoter fragments linked to the E. coli lacZ reporter gene we show different expression patterns of beta-galactosidase (beta-gal) in transformed Pc+ and Pc- embryos. In addition we are able to visualize by immunocytochemical techniques on polytene chromosomes the direct binding of the Pc protein to the transposed cis-regulatory promoter fragments. However, short Antp P1 promoter constructs which are--due to position effects--ectopically activated in salivary glands, do not reveal a Pc binding signal. Images PMID:1671215

  20. The Drosophila fork head domain protein crocodile is required for the establishment of head structures.

    PubMed Central

    Häcker, U; Kaufmann, E; Hartmann, C; Jürgens, G; Knöchel, W; Jäckle, H

    1995-01-01

    The fork head (fkh) domain defines the DNA-binding region of a family of transcription factors which has been implicated in regulating cell fate decisions across species lines. We have cloned and molecularly characterized the crocodile (croc) gene which encodes a new family member from Drosophila. croc is expressed in the head anlagen of the blastoderm embryo under the control of the anterior, the dorsoventral and the terminal maternal organizer systems. The croc mutant phenotype indicates that the croc wild-type gene is required to function as an early patterning gene in the anterior-most blastoderm head segment anlage and for the establishment of a specific head skeletal structure that derives from the non-adjacent intercalary segment at a later stage of embryogenesis. As an early patterning gene, croc exerts unusual properties which do not allow it to be grouped among the established segmentation genes. A single-site mutation within the croc fkh domain, which causes a replacement of the first out of four conserved amino acid residues thought to be involved in the coordinate binding of Mg2+, abolishes the DNA binding of the protein in vitro. In view of the resulting lack-of-function mutant phenotype, it appears likely that metal binding by the affected region of the fkh domain is crucial for proper folding of the DNA-binding structure. Images PMID:7489720

  1. The two Drosophila cytochrome C proteins can function in both respiration and caspase activation.

    PubMed

    Arama, Eli; Bader, Maya; Srivastava, Mayank; Bergmann, Andreas; Steller, Hermann

    2006-01-11

    Cytochrome C has two apparently separable cellular functions: respiration and caspase activation during apoptosis. While a role of the mitochondria and cytochrome C in the assembly of the apoptosome and caspase activation has been established for mammalian cells, the existence of a comparable function for cytochrome C in invertebrates remains controversial. Drosophila possesses two cytochrome c genes, cyt-c-d and cyt-c-p. We show that only cyt-c-d is required for caspase activation in an apoptosis-like process during spermatid differentiation, whereas cyt-c-p is required for respiration in the soma. However, both cytochrome C proteins can function interchangeably in respiration and caspase activation, and the difference in their genetic requirements can be attributed to differential expression in the soma and testes. Furthermore, orthologues of the apoptosome components, Ark (Apaf-1) and Dronc (caspase-9), are also required for the proper removal of bulk cytoplasm during spermatogenesis. Finally, several mutants that block caspase activation during spermatogenesis were isolated in a genetic screen, including mutants with defects in spermatid mitochondrial organization. These observations establish a role for the mitochondria in caspase activation during spermatogenesis.

  2. Imp and Syp RNA-binding proteins govern decommissioning of Drosophila neural stem cells.

    PubMed

    Yang, Ching-Po; Samuels, Tamsin J; Huang, Yaling; Yang, Lu; Ish-Horowicz, David; Davis, Ilan; Lee, Tzumin

    2017-10-01

    The termination of the proliferation of Drosophila neural stem cells, also known as neuroblasts (NBs), requires a 'decommissioning' phase that is controlled in a lineage-specific manner. Most NBs, with the exception of those of the mushroom body (MB), are decommissioned by the ecdysone receptor and mediator complex, causing them to shrink during metamorphosis, followed by nuclear accumulation of Prospero and cell cycle exit. Here, we demonstrate that the levels of Imp and Syp RNA-binding proteins regulate NB decommissioning. Descending Imp and ascending Syp expression have been shown to regulate neuronal temporal fate. We show that Imp levels decline slower in the MB than in other central brain NBs. MB NBs continue to express Imp into pupation, and the presence of Imp prevents decommissioning partly by inhibiting the mediator complex. Late-larval induction of transgenic Imp prevents many non-MB NBs from decommissioning in early pupae. Moreover, the presence of abundant Syp in aged NBs permits Prospero accumulation that, in turn, promotes cell cycle exit. Together, our results reveal that progeny temporal fate and progenitor decommissioning are co-regulated in protracted neuronal lineages. © 2017. Published by The Company of Biologists Ltd.

  3. Genetic analysis of rolled, which encodes a Drosophila mitogen-activated protein kinase.

    PubMed Central

    Lim, Y M; Nishizawa, K; Nishi, Y; Tsuda, L; Inoue, Y H; Nishida, Y

    1999-01-01

    Genetic and molecular characterization of the dominant suppressors of D-raf(C110) on the second chromosome identified two gain-of-function alleles of rolled (rl), which encodes a mitogen-activated protein (MAP) kinase in Drosophila. One of the alleles, rl(Su23), was found to bear the same molecular lesion as rl(Sem), which has been reported to be dominant female sterile. However, rl(Su23) and the current stock of rl(Sem) showed only a weak dominant female sterility. Detailed analyses of the rl mutations demonstrated moderate dominant activities of these alleles in the Torso (Tor) signaling pathway, which explains the weak dominant female sterility observed in this study. The dominant rl mutations failed to suppress the terminal class maternal-effect mutations, suggesting that activation of Rl is essential, but not sufficient, for Tor signaling. Involvement of rl in cell proliferation was also demonstrated by clonal analysis. Branching and integration of signals in the MAP kinase cascade is discussed. PMID:10511556

  4. Deletion of Drosophila muscle LIM protein decreases flight muscle stiffness and power generation.

    PubMed

    Clark, Kathleen A; Lesage-Horton, Heather; Zhao, Cuiping; Beckerle, Mary C; Swank, Douglas M

    2011-08-01

    Muscle LIM protein (MLP) can be found at the Z-disk of sarcomeres where it is hypothesized to be involved in sensing muscle stretch. Loss of murine MLP results in dilated cardiomyopathy, and mutations in human MLP lead to cardiac hypertrophy, indicating a critical role for MLP in maintaining normal cardiac function. Loss of MLP in Drosophila (mlp84B) also leads to muscle dysfunction, providing a model system to examine MLP's mechanism of action. Mlp84B-null flies that survive to adulthood are not able to fly or beat their wings. Transgenic expression of the mlp84B gene in the Mlp84B-null background rescues flight ability and restores wing beating ability. Mechanical analysis of skinned flight muscle fibers showed a 30% decrease in oscillatory power production and a slight increase in the frequency at which maximum power is generated for fibers lacking Mlp84B compared with rescued fibers. Mlp84B-null muscle fibers displayed a 25% decrease in passive, active, and rigor stiffness compared with rescued fibers, but no significant decrease in isometric tension generation was observed. Muscle ultrastructure of Mlp84B-null muscle fibers is grossly normal; however, the null fibers have a slight decrease, 11%, in thick filament number per unit cross-sectional area. Our data indicate that MLP contributes to muscle stiffness and is necessary for maximum work and power generation.

  5. Context-dependent transcriptional interpretation of mitogen activated protein kinase signaling in the Drosophila embryo

    NASA Astrophysics Data System (ADS)

    Kim, Yoosik; Iagovitina, Antonina; Ishihara, Keisuke; Fitzgerald, Kate M.; Deplancke, Bart; Papatsenko, Dmitri; Shvartsman, Stanislav Y.

    2013-06-01

    Terminal regions of the Drosophila embryo are patterned by the localized activation of Mitogen Activated Protein Kinase (MAPK), which induces zygotic genes through relief of their repression by transcriptional repressor Capicua. The levels of MAPK activation at the anterior and posterior termini are close to each other, but the expression patterns of MAPK-target genes, such as zerknüllt (zen) and tailless (tll), display strong anterior-posterior (AP) asymmetry. This region-specific response to MAPK activation provides a clear example of context-dependent interpretation of inductive signaling, a common developmental effect that remains poorly understood. In the past, the AP asymmetry of zen expression was attributed to a mechanism that depends on MAPK substrate competition. We present data suggesting that the asymmetric expression of tll is generated by a different mechanism, based on feedforward control and multiple enhancers of the tll gene. A simple mathematical model of this mechanism correctly predicts how the wild-type expression pattern of tll changes in mutants affecting the anterior, dorsoventral, and terminal patterning systems and some of their direct targets.

  6. Development of the Cellular Immune System of Drosophila Requires the Membrane Attack Complex/Perforin-Like Protein Torso-Like.

    PubMed

    Forbes-Beadle, Lauren; Crossman, Tova; Johnson, Travis K; Burke, Richard; Warr, Coral G; Whisstock, James C

    2016-10-01

    Pore-forming members of the membrane attack complex/perforin-like (MACPF) protein superfamily perform well-characterized roles as mammalian immune effectors. For example, complement component 9 and perforin function to directly form pores in the membrane of Gram-negative pathogens or virally infected/transformed cells, respectively. In contrast, the only known MACPF protein in Drosophila melanogaster, Torso-like, plays crucial roles during development in embryo patterning and larval growth. Here, we report that in addition to these functions, Torso-like plays an important role in Drosophila immunity. However, in contrast to a hypothesized effector function in, for example, elimination of Gram-negative pathogens, we find that torso-like null mutants instead show increased susceptibility to certain Gram-positive pathogens such as Staphylococcus aureus and Enterococcus faecalis We further show that this deficit is due to a severely reduced number of circulating immune cells and, as a consequence, an impaired ability to phagocytose bacterial particles. Together these data suggest that Torso-like plays an important role in controlling the development of the Drosophila cellular immune system. Copyright © 2016 by the Genetics Society of America.

  7. Ataxin 2-binding protein 1 is a context-specific positive regulator of Notch signaling during neurogenesis in Drosophila melanogaster.

    PubMed

    Shukla, Jay Prakash; Deshpande, Girish; Shashidhara, L S

    2017-03-01

    The role of the Notch pathway during the lateral inhibition that underlies binary cell fate choice is extensively studied, but the context specificity that generates diverse outcomes is less well understood. In the peripheral nervous system of Drosophila melanogaster, differential Notch signaling between cells of the proneural cluster orchestrates sensory organ specification. Here we report functional analysis of Drosophila Ataxin 2-binding protein 1 (A2BP1) during this process. Its human ortholog is linked to type 2 spinocerebellar ataxia and other complex neuronal disorders. Downregulation of Drosophila A2BP1 in the proneural cluster increases adult sensory bristle number, whereas its overexpression results in loss of bristles. We show that A2BP1 regulates sensory organ specification by potentiating Notch signaling. Supporting its direct involvement, biochemical analysis shows that A2BP1 is part of the Suppressor of Hairless [Su(H)] complex in the presence and absence of Notch. However, in the absence of Notch signaling, the A2BP1 interacting fraction of Su(H) does not associate with the repressor proteins Groucho and CtBP. We propose a model explaining the requirement of A2BP1 as a positive regulator of context-specific Notch activity.

  8. A Drosophila homolog of cyclase-associated proteins collaborates with the Abl tyrosine kinase to control midline axon pathfinding.

    PubMed

    Wills, Zachary; Emerson, Mark; Rusch, Jannette; Bikoff, Jay; Baum, Buzz; Perrimon, Norbert; Van Vactor, David

    2002-11-14

    We demonstrate that Drosophila capulet (capt), a homolog of the adenylyl cyclase-associated protein that binds and regulates actin in yeast, associates with Abl in Drosophila cells, suggesting a functional relationship in vivo. We find a robust and specific genetic interaction between capt and Abl at the midline choice point where the growth cone repellent Slit functions to restrict axon crossing. Genetic interactions between capt and slit support a model where Capt and Abl collaborate as part of the repellent response. Further support for this model is provided by genetic interactions that both capt and Abl display with multiple members of the Roundabout receptor family. These studies identify Capulet as part of an emerging pathway linking guidance signals to regulation of cytoskeletal dynamics and suggest that the Abl pathway mediates signals downstream of multiple Roundabout receptors.

  9. Reversible symptoms and clearance of mutant prion protein in an inducible model of a genetic prion disease in Drosophila melanogaster.

    PubMed

    Murali, A; Maue, R A; Dolph, P J

    2014-07-01

    Prion diseases are progressive disorders that affect the central nervous system leading to memory loss, personality changes, ataxia and neurodegeneration. In humans, these disorders include Creutzfeldt-Jakob disease, kuru and Gerstmann-Straüssler-Scheinker (GSS) syndrome, the latter being a dominantly inherited prion disease associated with missense mutations in the gene that codes for the prion protein. The exact mechanism by which mutant prion proteins affect the central nervous system and cause neurological disease is not well understood. We have generated an inducible model of GSS disease in Drosophila melanogaster by temporally expressing a misfolded form of the murine prion protein in cholinergic neurons. Flies accumulating this mutant protein develop motor abnormalities which are associated with electrophysiological defects in cholinergic neurons. We find that, upon blocking the expression of the mutant protein, both behavioral and electrophysiological defects can be reversed. This represents the first case of reversibility reported in a model of genetic prion disease. Additionally, we observe that endogenous mechanisms exist within Drosophila that are capable of clearing the accumulated prion protein.

  10. The Protein O-glucosyltransferase Rumi Modifies Eyes Shut to Promote Rhabdomere Separation in Drosophila

    PubMed Central

    Harvey, Beth M.; Leonardi, Jessica; Chen, Yi-Jiun; Hong, Yang; Haltiwanger, Robert S.; Jafar-Nejad, Hamed

    2014-01-01

    The protein O-glucosyltransferase Rumi/POGLUT1 regulates Drosophila Notch signaling by adding O-glucose residues to the Notch extracellular domain. Rumi has other predicted targets including Crumbs (Crb) and Eyes shut (Eys), both of which are involved in photoreceptor development. However, whether Rumi is required for the function of Crb and Eys remains unknown. Here we report that in the absence of Rumi or its enzymatic activity, several rhabdomeres in each ommatidium fail to separate from one another in a Notch-independent manner. Mass spectral analysis indicates the presence of O-glucose on Crb and Eys. However, mutating all O-glucosylation sites in a crb knock-in allele does not cause rhabdomere attachment, ruling out Crb as a biologically-relevant Rumi target in this process. In contrast, eys and rumi exhibit a dosage-sensitive genetic interaction. In addition, although in wild-type ommatidia most of the Eys protein is found in the inter-rhabdomeral space (IRS), in rumi mutants a significant fraction of Eys remains in the photoreceptor cells. The intracellular accumulation of Eys and the IRS defect worsen in rumi mutants raised at a higher temperature, and are accompanied by a ∼50% decrease in the total level of Eys. Moreover, removing one copy of an endoplasmic reticulum chaperone enhances the rhabdomere attachment in rumi mutant animals. Altogether, our data suggest that O-glucosylation of Eys by Rumi ensures rhabdomere separation by promoting proper Eys folding and stability in a critical time window during the mid-pupal stage. Human EYS, which is mutated in patients with autosomal recessive retinitis pigmentosa, also harbors multiple Rumi target sites. Therefore, the role of O-glucose in regulating Eys may be conserved. PMID:25412384

  11. Silver nanoparticles induced heat shock protein 70, oxidative stress and apoptosis in Drosophila melanogaster

    SciTech Connect

    Ahamed, Maqusood; Posgai, Ryan; Gorey, Timothy J.; Nielsen, Mark; Hussain, Saber M.; Rowe, John J.

    2010-02-01

    Due to the intensive commercial application of silver nanoparticles (Ag NPs), risk assessment of this nanoparticle is of great importance. Our previous in vitro study demonstrated that Ag NPs caused DNA damage and apoptosis in mouse embryonic stem cells and fibroblasts. However, toxicity of Ag NPs in vivo is largely lacking. This study was undertaken to examine the toxic effects of well-characterized polysaccharide coated 10 nm Ag NPs on heat shock stress, oxidative stress, DNA damage and apoptosis in Drosophila melanogaster. Third instar larvae of D. melanogaster were fed a diet of standard cornmeal media mixed with Ag NPs at the concentrations of 50 and 100 mug/ml for 24 and 48 h. Ag NPs up-regulated the expression of heat shock protein 70 and induced oxidative stress in D. melanogaster. Malondialdehyde level, an end product of lipid peroxidation was significantly higher while antioxidant glutathione content was significantly lower in Ag NPs exposed organisms. Activities of antioxidant enzyme superoxide dismutase and catalase were also significantly higher in the organisms exposed to Ag NPs. Furthermore, Ag NPs up-regulated the cell cycle checkpoint p53 and cell signaling protein p38 that are involved in the DNA damage repair pathway. Moreover, activities of caspase-3 and caspase-9, markers of apoptosis were significantly higher in Ag NPs exposed organisms. The results indicate that Ag NPs in D. melanogaster induce heat shock stress, oxidative stress, DNA damage and apoptosis. This study suggests that the organism is stressed and thus warrants more careful assessment of Ag NPs using in vivo models to determine if chronic exposure presents developmental and reproductive toxicity.

  12. Gag Proteins of Drosophila Telomeric Retrotransposons: Collaborative Targeting to Chromosome Ends

    PubMed Central

    Fuller, Adelaide M.; Cook, Elizabeth G.; Kelley, Kerry J.; Pardue, Mary-Lou

    2010-01-01

    TAHRE, the least abundant of the three retrotransposons forming telomeres in Drosophila melanogaster, has high sequence similarity to the gag gene and untranslated regions of HeT-A, the most abundant telomere-specific retrotransposon. Despite TAHRE's apparent evolutionary relationship to HeT-A, we find TAHRE Gag cannot locate to telomere-associated “Het dots” unless collaborating with HeT-A Gag. TAHRE Gag is carried into nuclei by HeT-A or TART Gag, but both TART and TAHRE Gags need HeT-A Gag to localize to Het dots. When coexpressed with the appropriate fragment of HeT-A and/or TART Gags, TAHRE Gag multimerizes with either protein. HeT-A and TART Gags form homo- and heteromultimers using a region containing major homology region (MHR) and zinc knuckle (CCHC) motifs, separated by a pre_C2HC motif (motifs common to other retroelements). This region's sequence is strongly conserved among the three telomeric Gags, with precise spacing of conserved residues. Nontelomeric Gags neither interact with the telomeric Gags nor have this conserved spacing. TAHRE Gag is much less able to enter the nucleus by itself than HeT-A or TART Gags. The overall telomeric localization efficiency for each of the three telomeric Gag proteins correlates with the relative abundance of that element in telomere arrays, suggesting an explanation for the relative rarity of TAHRE elements in telomere arrays and supporting the hypothesis that Gag targeting to telomeres is important for the telomere-specific transposition of these elements. PMID:20026680

  13. Hearing in Drosophila Requires TilB, a Conserved Protein Associated With Ciliary Motility

    PubMed Central

    Kavlie, Ryan G.; Kernan, Maurice J.; Eberl, Daniel F.

    2010-01-01

    Cilia were present in the earliest eukaryotic ancestor and underlie many biological processes ranging from cell motility and propulsion of extracellular fluids to sensory physiology. We investigated the contribution of the touch insensitive larva B (tilB) gene to cilia function in Drosophila melanogaster. Mutants of tilB exhibit dysfunction in sperm flagella and ciliated dendrites of chordotonal organs that mediate hearing and larval touch sensitivity. Mutant sperm axonemes as well as sensory neuron dendrites of Johnston's organ, the fly's auditory organ, lack dynein arms. Through deficiency mapping and sequencing candidate genes, we identified tilB mutations in the annotated gene CG14620. A genomic CG14620 transgene rescued deafness and male sterility of tilB mutants. TilB is a 395-amino-acid protein with a conserved N-terminal leucine-rich repeat region at residues 16–164 and a coiled-coil domain at residues 171–191. A tilB-Gal4 transgene driving fluorescently tagged TilB proteins elicits cytoplasmic expression in embryonic chordotonal organs, in Johnston's organ, and in sperm flagella. TilB does not appear to affect tubulin polyglutamylation or polyglycylation. The phenotypes and expression of tilB indicate function in cilia construction or maintenance, but not in intraflagellar transport. This is also consistent with phylogenetic association of tilB homologs with presence of genes encoding axonemal dynein arm components. Further elucidation of tilB functional mechanisms will provide greater understanding of cilia function and will facilitate understanding ciliary diseases. PMID:20215474

  14. The Drosophila telomere-capping protein Verrocchio binds single-stranded DNA and protects telomeres from DNA damage response.

    PubMed

    Cicconi, Alessandro; Micheli, Emanuela; Vernì, Fiammetta; Jackson, Alison; Gradilla, Ana Citlali; Cipressa, Francesca; Raimondo, Domenico; Bosso, Giuseppe; Wakefield, James G; Ciapponi, Laura; Cenci, Giovanni; Gatti, Maurizio; Cacchione, Stefano; Raffa, Grazia Daniela

    2017-04-07

    Drosophila telomeres are sequence-independent structures maintained by transposition to chromosome ends of three specialized retroelements rather than by telomerase activity. Fly telomeres are protected by the terminin complex that includes the HOAP, HipHop, Moi and Ver proteins. These are fast evolving, non-conserved proteins that localize and function exclusively at telomeres, protecting them from fusion events. We have previously suggested that terminin is the functional analogue of shelterin, the multi-protein complex that protects human telomeres. Here, we use electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) to show that Ver preferentially binds single-stranded DNA (ssDNA) with no sequence specificity. We also show that Moi and Ver form a complex in vivo. Although these two proteins are mutually dependent for their localization at telomeres, Moi neither binds ssDNA nor facilitates Ver binding to ssDNA. Consistent with these results, we found that Ver-depleted telomeres form RPA and γH2AX foci, like the human telomeres lacking the ssDNA-binding POT1 protein. Collectively, our findings suggest that Drosophila telomeres possess a ssDNA overhang like the other eukaryotes, and that the terminin complex is architecturally and functionally similar to shelterin. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Amyloid precursor protein in Drosophila glia regulates sleep and genes involved in glutamate recycling.

    PubMed

    Farca Luna, Abud Jose; Perier, Magali; Seugnet, Laurent

    2017-03-17

    The Amyloid Precursor Protein (App) plays a crucial role in Alzheimer disease (AD) via the production and deposition of toxic β-amyloid peptides. App is heavily expressed in neurons where the vast majority of studies investigating its function have been carried out, while almost nothing is known about its function in glia, where it is also expressed, and can potentially participate in the regulation of neuronal physiology. In this report, we investigated whether Appl, the Drosophila homolog of App, could influence sleep-wake regulation when its function is manipulated in glial cells. Appl inhibition in astrocyte-like and cortex glia resulted in higher sleep amounts and longer sleep bout duration during the night, while overexpression had the opposite effect. These sleep phenotypes were not the result of developmental defects, and were correlated with changes in expression in Glutamine Synthetase (GS) in astrocyte-like glia, and in changes in the gap-junction component innexin2 in cortex glia. Downregulating both GS and innexin2, but not either one individually, resulted in higher sleep amounts, similarly to Appl inhibition. Consistent with these results the expression of GS and innexin2 are increased following sleep deprivation indicating that these two genes are dynamically linked to vigilance states. Interestingly, the reduction of GS expression and the sleep phenotype observed upon Appl inhibition could be rescued by increasing the expression of the glutamate transporter dEaat1. In contrast, reducing dEaat1 expression severely disrupted sleep. These results associate glutamate recycling, sleep and a glial function for the App family proteins.StatementThe Amyloid Precursor Protein (App) has been intensively studied for its implication in Alzheimer Disease (AD). The attributed functions of App are linked to the physiology and cellular biology of neurons where the protein is predominantly expressed. Consequences on glia in AD are generally thought to be secondary

  16. High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis.

    PubMed

    Sridharan, Vinod; Heimiller, Joseph; Robida, Mark D; Singh, Ravinder

    2016-01-01

    The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during spermatogenesis. The heph2 mutation in this gene results in a specific defect in spermatogenesis, causing aberrant spermatid individualization and male sterility. However, the array of molecular defects in the mutant remains uncharacterized. Using an unbiased high throughput sequencing approach, we have identified transcripts that are misregulated in this mutant. Aberrant transcripts show altered expression levels, exon skipping, and alternative 5' ends. We independently verified these findings by reverse-transcription and polymerase chain reaction (RT-PCR) analysis. Our analysis shows misregulation of transcripts that have been connected to spermatogenesis, including components of the actomyosin cytoskeletal apparatus. We show, for example, that the Myosin light chain 1 (Mlc1) transcript is aberrantly spliced. Furthermore, bioinformatics analysis reveals that Mlc1 contains a high affinity binding site(s) for dmPTB and that the site is conserved in many Drosophila species. We discuss that Mlc1 and other components of the actomyosin cytoskeletal apparatus offer important molecular links between the loss of dmPTB function and the observed developmental defect in spermatogenesis. This study provides the first comprehensive list of genes misregulated in vivo in the heph2 mutant in Drosophila and offers insight into the role of dmPTB during spermatogenesis.

  17. High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis

    PubMed Central

    Sridharan, Vinod; Heimiller, Joseph; Robida, Mark D.; Singh, Ravinder

    2016-01-01

    The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during spermatogenesis. The heph2 mutation in this gene results in a specific defect in spermatogenesis, causing aberrant spermatid individualization and male sterility. However, the array of molecular defects in the mutant remains uncharacterized. Using an unbiased high throughput sequencing approach, we have identified transcripts that are misregulated in this mutant. Aberrant transcripts show altered expression levels, exon skipping, and alternative 5’ ends. We independently verified these findings by reverse-transcription and polymerase chain reaction (RT-PCR) analysis. Our analysis shows misregulation of transcripts that have been connected to spermatogenesis, including components of the actomyosin cytoskeletal apparatus. We show, for example, that the Myosin light chain 1 (Mlc1) transcript is aberrantly spliced. Furthermore, bioinformatics analysis reveals that Mlc1 contains a high affinity binding site(s) for dmPTB and that the site is conserved in many Drosophila species. We discuss that Mlc1 and other components of the actomyosin cytoskeletal apparatus offer important molecular links between the loss of dmPTB function and the observed developmental defect in spermatogenesis. This study provides the first comprehensive list of genes misregulated in vivo in the heph2 mutant in Drosophila and offers insight into the role of dmPTB during spermatogenesis. PMID:26942929

  18. The Drosophila F-box protein dSkp2 regulates cell proliferation by targeting Dacapo for degradation.

    PubMed

    Dui, Wen; Wei, Bin; He, Feng; Lu, Wei; Li, Changqing; Liang, Xuehong; Ma, Jun; Jiao, Renjie

    2013-06-01

    Cell cycle progression is controlled by a complex regulatory network consisting of interacting positive and negative factors. In humans, the positive regulator Skp2, an F-box protein, has been a subject of intense investigation in part because of its oncogenic activity. By contrast, the molecular and developmental functions of its Drosophila homologue, dSkp2, are poorly understood. Here we investigate the role of dSkp2 by focusing on its functional relationship with Dacapo (Dap), the Drosophila homologue of the cyclin-dependent kinase inhibitors p21(cip1)/p27(kip1)/p57(kip2). We show that dSkp2 interacts physically with Dap and has a role in targeting Dap for ubiquitination and proteasome-mediated degradation. We present evidence that dSkp2 regulates cell cycle progression by antagonizing Dap in vivo. dSkp2 knockdown reduces cell density in the wing by prolonging the cell doubling time. In addition, the wing phenotype caused by dSkp2 knockdown resembles that caused by dap overexpression and can be partially suppressed by reducing the gene dose of dap. Our study thus documents a conserved functional relationship between dSkp2 and Dap in their control of cell cycle progression, suggesting the possibility of using Drosophila as a model system to study Skp2-mediated tumorigenesis.

  19. A highly conserved homologue of bovine neurocalcin in Drosophila melanogaster is a Ca(2+)-binding protein expressed in neuronal tissues.

    PubMed

    Teng, D H; Chen, C K; Hurley, J B

    1994-12-16

    Polymerase chain reaction was used to search for genes encoding recoverin-like proteins in Drosophila melanogaster. We identified a gene that codes for a cognate of bovine neurocalcin; hence, we have named it neurocalcin (nca). A cDNA of nca was isolated and sequenced. The deduced polypeptide product of the cDNA is 22 kDa in size, and its amino acid sequence is 88% identical to that of bovine neurocalcin. This deduced Drosophila neurocalcin (DrosNCa) protein has three putative EF-hands and has a sequence in its NH2 terminus required for fatty acylation. DrosNCa was expressed in Escherichia coli and subsequently purified by phenyl-Sepharose chromatography and Mono Q anion exchange fast protein liquid chromatography. This recombinant protein was capable of binding 45Ca2+ and exhibited Ca(2+)-dependent mobility shifts in both SDS-polyacrylamide gel electrophoresis and native gel electrophoresis. DrosNCa was tritiated when it was coexpressed in E. coli with N-myristoyl transferase in the presence of [3H]myristic acid. The nca transcript was approximately 1 kilobase long, and tissue in situ hybridization showed that this message was present in the brain of adult flies. Antibodies raised against recombinant DrosNCa cross-reacted with rat hippocalcin on an immunoblot but not with bovine recoverin. When immunohistochemical analysis was performed, staining was observed throughout the central nervous system of adult flies, particularly in the neuropil, where neurons synapse. The nca locus maps to or near 76F on the Drosophila third chromosome.

  20. The beta subunit of the Drosophila melanogaster ATP synthase: cDNA cloning, amino acid analysis and identification of the protein in adult flies.

    PubMed

    Peña, P; Garesse, R

    1993-09-15

    The cDNA encoding the Drosophila melanogaster beta subunit of H+ ATP synthase has been cloned and sequenced. The predicted mature protein is highly homologous to the equivalent beta subunits of other organisms and is preceded by a signal peptide of 31 amino acids, that although not conserved at primary sequence level has the characteristics of leader peptides present in other mitochondrial proteins. We have raised polyclonal antibodies that specifically recognize the beta H+ ATP synthase subunit present in Drosophila melanogaster protein extracts. This is the first time that a gene of the ATP synthase complex has been characterized in the invertebrate phyla.

  1. Local requirement of the Drosophila insulin binding protein imp-L2 in coordinating developmental progression with nutritional conditions.

    PubMed

    Sarraf-Zadeh, Ladan; Christen, Stefan; Sauer, Uwe; Cognigni, Paola; Miguel-Aliaga, Irene; Stocker, Hugo; Köhler, Katja; Hafen, Ernst

    2013-09-01

    In Drosophila, growth takes place during the larval stages until the formation of the pupa. Starvation delays pupariation to allow prolonged feeding, ensuring that the animal reaches an appropriate size to form a fertile adult. Pupariation is induced by a peak of the steroid hormone ecdysone produced by the prothoracic gland (PG) after larvae have reached a certain body mass. Local downregulation of the insulin/insulin-like growth factor signaling (IIS) activity in the PG interferes with ecdysone production, indicating that IIS activity in the PG couples the nutritional state to development. However, the underlying mechanism is not well understood. In this study we show that the secreted Imaginal morphogenesis protein-Late 2 (Imp-L2), a growth inhibitor in Drosophila, is involved in this process. Imp-L2 inhibits the activity of the Drosophila insulin-like peptides by direct binding and is expressed by specific cells in the brain, the ring gland, the gut and the fat body. We demonstrate that Imp-L2 is required to regulate and adapt developmental timing to nutritional conditions by regulating IIS activity in the PG. Increasing Imp-L2 expression at its endogenous sites using an Imp-L2-Gal4 driver delays pupariation, while Imp-L2 mutants exhibit a slight acceleration of development. These effects are strongly enhanced by starvation and are accompanied by massive alterations of ecdysone production resulting most likely from increased Imp-L2 production by neurons directly contacting the PG and not from elevated Imp-L2 levels in the hemolymph. Taken together our results suggest that Imp-L2-expressing neurons sense the nutritional state of Drosophila larvae and coordinate dietary information and ecdysone production to adjust developmental timing under starvation conditions.

  2. Midgut-enriched receptor protein tyrosine phosphatase PTP52F is required for Drosophila development during larva-pupa transition.

    PubMed

    Santhanam, Abirami; Liang, Suh-Yuen; Chen, Dong-Yuan; Chen, Guang-Chao; Meng, Tzu-Ching

    2013-01-01

    To date our understanding of Drosophila receptor protein tyrosine phosphatases (R-PTPs) in the regulation of signal transduction is limited. Of the seven R-PTPs identified in flies, six are involved in the axon guidance that occurs during embryogenesis. However, whether and how R-PTPs may control key steps of Drosophila development is not clear. In this study we investigated the potential role of Drosophila R-PTPs in developmental processes outside the neuronal system and beyond the embryogenesis stage. Through systematic data mining of available microarray databases, we found the mRNA level of PTP52F to be highly enriched in the midgut of flies at the larva-pupa transition. This finding was confirmed by gut tissue staining with a specific antibody. The unique spatiotemporal expression of PTP52F suggests that it is possibly involved in regulating metamorphosis during the transformation from larva to pupa. To test this hypothesis, we employed RNA interference to examine the defects of transgenic flies. We found that ablation of endogenous PTP52F led to high lethality characterized by the pharate adult phenotype, occurring due to post pupal eclosion failure. These results show that PTP52F plays an indispensable role during the larva-pupa transition. We also found that PTP52F could be reclassified as a member of the subtype R3 PTPs instead of as an unclassified R-PTP without a human ortholog, as suggested previously. Together, these findings suggest that Drosophila R-PTPs may control metamorphosis and other biological processes beyond our current knowledge.

  3. The F box protein partner of paired regulates stability of Drosophila centromeric histone H3, CenH3(CID).

    PubMed

    Moreno-Moreno, Olga; Medina-Giró, Sònia; Torras-Llort, Mònica; Azorín, Fernando

    2011-09-13

    Centromere identity and function is determined by the specific localization of CenH3 (reviewed in [1-7]). Several mechanisms regulate centromeric CenH3 localization, including proteasome-mediated degradation that, both in budding yeast and Drosophila, regulates CenH3 levels and prevents promiscuous misincorporation throughout chromatin [8, 9]. CenH3(CENP-A) proteolysis has also been reported in senescent human cells [10] or upon infection with herpes simplex virus 1 [11]. Little is known, however, about the actual mechanisms that regulate CenH3 proteolysis. Recent work in budding yeast identified Psh1 as an E3-ubiquitin ligase that mediates degradation of CenH3(Cse4p) [12, 13], but E3-ligases regulating CenH3 stability in metazoans are unknown. Here, we report that the F box protein partner of paired (Ppa), which is a variable subunit of the main E3-ligase SCF [14-17], mediates CenH3(CID) stability in Drosophila. Our results show that Ppa depletion results in increased CenH3(CID) levels. Ppa physically interacts with CenH3(CID) through the CATD(CID) that, in the fly, mediates Ppa-dependent CenH3(CID) stability. Altogether, these results strongly suggest that, in Drosophila, SCF(Ppa) regulates CenH3(CID) proteolysis. Interestingly, most known SCF complexes are inactive when, at mitosis, de novo CenH3(CID) deposition takes place at centromeres, suggesting that, in Drosophila, CenH3(CID) deposition and proteolysis are synchronized events.

  4. Drosophila Larp associates with poly(A)-binding protein and is required for male fertility and syncytial embryo development.

    PubMed

    Blagden, Sarah P; Gatt, Melanie K; Archambault, Vincent; Lada, Karolina; Ichihara, Keiko; Lilley, Kathryn S; Inoue, Yoshihiro H; Glover, David M

    2009-10-01

    As the influence of mRNA translation upon cell cycle regulation becomes clearer, we searched for genes that might specify such control in Drosophila. A maternal-effect lethal screen identified mutants in the Drosophila gene for Larp (La-related protein) which displayed maternal-effect lethality and male sterility. A role for La protein has already been implicated in mRNA translation whereas Larp has been proposed to regulate mRNA stability. Here we demonstrate that Larp exists in a physical complex with, and also interacts genetically with, the translation regulator poly(A)-binding protein (PABP). Most mutant alleles of pAbp are embryonic lethal. However hypomorphic pAbp alleles show similar meiotic defects to larp mutants. We find that larp mutant-derived syncytial embryos show a range of mitotic phenotypes, including failure of centrosomes to migrate around the nuclear envelope, detachment of centrosomes from spindle poles, the formation of multipolar spindle arrays and cytokinetic defects. We discuss why the syncytial mitotic cycles and male meiosis should have a particularly sensitive requirement for Larp proteins in regulating not only transcript stability but also potentially the translation of mRNAs.

  5. Drosophila p53-related protein kinase is required for PI3K/TOR pathway-dependent growth.

    PubMed

    Ibar, Consuelo; Cataldo, Vicente F; Vásquez-Doorman, Constanza; Olguín, Patricio; Glavic, Alvaro

    2013-03-01

    Cell growth and proliferation are pivotal for final organ and body size definition. p53-related protein kinase (Bud32/PRPK) has been identified as a protein involved in proliferation through its effects on transcription in yeast and p53 stabilization in human cell culture. However, the physiological function of Bud32/PRPK in metazoans is not well understood. In this work, we have analyzed the role of PRPK in Drosophila development. Drosophila PRPK is expressed in every tissue analyzed and is required to support proliferation and cell growth. The Prpk knockdown animals show phenotypes similar to those found in mutants for positive regulators of the PI3K/TOR pathway. This pathway has been shown to be fundamental for animal growth, transducing the hormonal and nutritional status into the protein translation machinery. Functional interactions have established that Prpk operates as a transducer of the PI3K/TOR pathway, being essential for TOR kinase activation and for the regulation of its targets (S6K and 4E-BP, autophagy and bulk endocytosis). This suggests that Prpk is crucial for stimulating the basal protein biosynthetic machinery in response to insulin signaling and to changes in nutrient availability.

  6. Phosphorylation at Serines 216 and 221 Is Important for Drosophila HeT-A Gag Protein Stability

    PubMed Central

    Brar, Sukhdev S.; Petrovich, Robert M.; Williams, Jason G.; Mason, James M.

    2013-01-01

    Telomeres from Drosophila appear to be very different from those of other organisms – in size and the mechanism of their maintenance. In the absence of the enzyme telomerase, Drosophila telomeres are maintained by retrotransposition of three elements, HeT-A, TART, and TAHRE, but details of their transposition mechanisms are not known. Here we characterized some biochemical characteristics of the HeT-A Gag protein encoded by the HeT-A element to understand this mechanism. The HeT-A Gag protein when overexpressed in S2 cells was localized to the nucleus but was resistant to high salt, detergents and nuclease extraction treatments. Analysis of the HeT-A Gag protein by tandem mass spectrophotometry revealed that serines 216 and 221 are phosphorylated. Substituting these serines with alanine or aspartic acid by site-directed mutagenesis did not result in any changes in HeT-A Gag translocation across the nucleus, suggesting that phosphorylation of these sites is not associated with HeT-A Gag translocation, but time course experiments showed that these phosphorylation sites are important for Gag-protein stability. PMID:24058682

  7. Drosophila CheB proteins involved in gustatory detection of pheromones are related to a human neurodegeneration factor.

    PubMed

    Pikielny, Claudio W

    2010-01-01

    The Drosophila CheBs proteins are expressed in a variety of sexually dimorphic subsets of taste hairs, some of which have been directly implicated in pheromone detection. Their remarkable collection of expression patterns suggests that CheBs have specialized roles in gustatory detection of pheromones. Indeed, mutations in the CheB42a gene specifically alter male response to female-specific cuticular hydrocarbons. Furthermore, CheBs belong to the large ML (MD-2-like) superfamily of lipid-binding proteins and share amino acids with an essential role in the function of human GM2-activator protein (GM2-AP), a protein whose absence results in neurodegeneration and death. As GM2-AP binds specifically to the GM2 ganglioside, we have proposed that CheB42a and other CheBs function by interacting directly with the lipid-like cuticular hydrocarbons of Drosophila melanogaster and modulating their detection by transmembrane receptors. Here I review the current knowledge of the CheB family and discuss possible models for their function. Copyright © 2010 Elsevier Inc. All rights reserved.

  8. Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction.

    PubMed

    McDermott, Suzanne M; Yang, Lu; Halstead, James M; Hamilton, Russell S; Meignin, Carine; Davis, Ilan

    2014-10-01

    Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. Here, we show that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. We use RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1, neurexin-1, futsch, highwire, discs large, and α-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. Our results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. We propose that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function.

  9. A human homolog of Drosophila lethal(3)malignant brain tumor (l(3)mbt) protein associates with condensed mitotic chromosomes.

    PubMed

    Koga, H; Matsui, S; Hirota, T; Takebayashi, S; Okumura, K; Saya, H

    1999-07-01

    The lethal(3)malignant brain tumor (D-l(3)mbt) gene is considered to be one of the tumor suppressor genes of Drosophila, and its recessive mutations are associated with malignant transformation of the neuroblasts in the larval brain. The structure of D-l(3)mbt protein is similar to Drosophila sex comb on midleg (Scm) protein which is a member of Polycomb group (PcG) proteins. We have isolated here the first human homolog of the D-l(3)mbt gene, designated h-l(3)mbt. Radiation hybrid mapping and fluorescence in situ hybridization (FISH) analysis localized the h-l(3)mbt gene to chromosome 20q12. The h-l(3)mbt transcript is expressed in most of the human adult normal tissues and cultured cell lines. However, some cancer cells markedly reduce the h-l(3)mbt protein expression. Immunocytochemical study revealed that the h-l(3)mbt protein shows a speckled and scattered distribution in interphase nuclei and completely associates with condensed chromosomes in mitotic cells. This subcellular localization has been shown to be different from that of Bmi1 protein which is a component of PcG complex. Furthermore, overexpression of h-l(3)mbt protein by using a Cre-mediated gene activation system leads to failures of proper chromosome segregation and cytokinesis, which result in formation of multinuclei in U251MG cells. These observations suggest that h-l(3)mbt protein has functions distinct from those of PcG proteins and may play a role in proper progression of cell division.

  10. Molecular mechanism underlying the regulatory specificity of a Drosophila homeodomain protein that specifies myoblast identity

    PubMed Central

    Busser, Brian W.; Shokri, Leila; Jaeger, Savina A.; Gisselbrecht, Stephen S.; Singhania, Aditi; Berger, Michael F.; Zhou, Bo; Bulyk, Martha L.; Michelson, Alan M.

    2012-01-01

    A subfamily of Drosophila homeodomain (HD) transcription factors (TFs) controls the identities of individual muscle founder cells (FCs). However, the molecular mechanisms by which these TFs generate unique FC genetic programs remain unknown. To investigate this problem, we first applied genome-wide mRNA expression profiling to identify genes that are activated or repressed by the muscle HD TFs Slouch (Slou) and Muscle segment homeobox (Msh). Next, we used protein-binding microarrays to define the sequences that are bound by Slou, Msh and other HD TFs that have mesodermal expression. These studies revealed that a large class of HDs, including Slou and Msh, predominantly recognize TAAT core sequences but that each HD also binds to unique sites that deviate from this canonical motif. To understand better the regulatory specificity of an individual FC identity HD, we evaluated the functions of atypical binding sites that are preferentially bound by Slou relative to other HDs within muscle enhancers that are either activated or repressed by this TF. These studies showed that Slou regulates the activities of particular myoblast enhancers through Slou-preferred sequences, whereas swapping these sequences for sites that are capable of binding to multiple HD family members does not support the normal regulatory functions of Slou. Moreover, atypical Slou-binding sites are overrepresented in putative enhancers associated with additional Slou-responsive FC genes. Collectively, these studies provide new insights into the roles of individual HD TFs in determining cellular identity, and suggest that the diversity of HD binding preferences can confer regulatory specificity. PMID:22296846

  11. The CPEB Protein Orb2 Has Multiple Functions during Spermatogenesis in Drosophila melanogaster

    PubMed Central

    Xu, Shuwa; Hafer, Nathaniel; Agunwamba, Blessing; Schedl, Paul

    2012-01-01

    Cytoplasmic Polyadenylation Element Binding (CPEB) proteins are translational regulators that can either activate or repress translation depending on the target mRNA and the specific biological context. There are two CPEB subfamilies and most animals have one or more genes from each. Drosophila has a single CPEB gene, orb and orb2, from each subfamily. orb expression is only detected at high levels in the germline and has critical functions in oogenesis but not spermatogenesis. By contrast, orb2 is broadly expressed in the soma; and previous studies have revealed important functions in asymmetric cell division, viability, motor function, learning, and memory. Here we show that orb2 is also expressed in the adult male germline and that it has essential functions in programming the progression of spermatogenesis from meiosis through differentiation. Like the translational regulators boule (bol) and off-schedule (ofs), orb2 is required for meiosis and orb2 mutant spermatocytes undergo a prolonged arrest during the meiotic G2-M transition. However, orb2 differs from boule and off-schedule in that this arrest occurs at a later step in meiotic progression after the synthesis of the meiotic regulator twine. orb2 is also required for the orderly differentiation of the spermatids after meiosis is complete. The differentiation defects in orb2 mutants include abnormal elongation of the spermatid flagellar axonemes, a failure in individualization and improper post-meiotic gene expression. Amongst the orb2 differentiation targets are orb and two other mRNAs, which are transcribed post-meiotically and localized to the tip of the flagellar axonemes. Additionally, analysis of a partial loss of function orb2 mutant suggests that the orb2 differentiation phenotypes are independent of the earlier arrest in meiosis. PMID:23209437

  12. Circadian expression of the presynaptic active zone protein Bruchpilot in the lamina of Drosophila melanogaster.

    PubMed

    Górska-Andrzejak, Jolanta; Makuch, Renata; Stefan, Joanna; Görlich, Alicja; Semik, Danuta; Pyza, Elzbieta

    2013-01-01

    In the fly's visual system, the morphology of cells and the number of synapses change during the day. In the present study we show that in the first optic neuropil (lamina) of Drosophila melanogaster, a presynaptic active zone protein Bruchpilot (BRP) exhibits a circadian rhythm in abundance. In day/night (or light/dark, LD) conditions the level of BRP increases two times, in the morning and in the evening. The same pattern of changes in the BRP level was detected in whole brain homogenates, thus indicating that the majority of synapses in the brain peaks twice during the day. However, these two peaks in BRP abundance, measured as the fluorescence intensity of immunolabeling, seem to be regulated differently. The peak in the morning is predominantly regulated by light and involves the transduction pathway in the retina photoreceptors. This peak is present neither in wild-type Canton-S flies in constant darkness (DD), nor in norpA(7) phototransduction mutant in LD. However, it also depends on the clock gene per, because it is abolished in the per(0) arrhythmic mutant. In turn, the peak of BRP in the evening is endogenously regulated by an input from the pacemaker located in the brain. This peak is present in Canton-S flies in DD, as well as in the norpA(7) mutant in LD, but is absent in per(01), tim,(01) and cry(01) mutants in LD. In addition both peaks seem to depend on clock gene-expressing photoreceptors and glial cells of the visual system.

  13. The Insulin-Like Proteins dILPs-2/5 Determine Diapause Inducibility in Drosophila

    PubMed Central

    Kyriacou, Charalambos P.; O’Connor, Michael B.; Costa, Rodolfo

    2016-01-01

    Diapause is an actively induced dormancy that has evolved in Metazoa to resist environmental stresses. In temperate regions, many diapausing insects overwinter at low temperatures by blocking embryonic, larval or adult development. Despite its Afro-tropical origin, Drosophila melanogaster migrated to temperate regions of Asia and Europe where females overwinter as adults by arresting gonadal development (reproductive diapause) at temperatures <13°C. Recent work in D. melanogaster has implicated the developmental hormones dILPs-2 and/or dILP3, and dILP5, homologues of vertebrate insulin/insulin-like growth factors (IGFs), in reproductive arrest. However, polymorphisms in timeless (tim) and couch potato (cpo) dramatically affect diapause inducibility and these dILP experiments could not exclude this common genetic variation contributing to the diapause phenotype. Here, we apply an extensive genetic dissection of the insulin signaling pathway which allows us to see both enhancements and reductions in egg development that are independent of tim and cpo variations. We show that a number of manipulations dramatically enhance diapause to ~100%. These include ablating, or reducing the excitability of the insulin-producing cells (IPCs) that express dILPs-2,3,5 employing the dilp2,3,5-/- triple mutant, desensitizing insulin signaling using a chico mutation, or inhibiting dILP2 and 5 in the hemolymph by over-expressing Imaginal Morphogenesis Protein-Late 2 (Imp-L2). In addition, triple mutant dilp2,3,5-/- females maintain high levels of diapause even when temperatures are raised in adulthood to 19°C. However at 22°C, these females all show egg development revealing that the effects are conditional on temperature and not a general female sterility. In contrast, over-expression of dilps-2/5 or enhancing IPC excitability, led to levels of ovarian arrest that approached zero, underscoring dILPs-2 and 5 as key antagonists of diapause. PMID:27689881

  14. The Drosophila suppressor of sable protein binds to RNA and associates with a subset of polytene chromosome bands.

    PubMed

    Murray, M V; Turnage, M A; Williamson, K J; Steinhauer, W R; Searles, L L

    1997-04-01

    Mutations of the Drosophila melanogaster suppressor of sable [su(s)] gene, which encodes a 150-kDa nuclear protein [Su(s)], increase the accumulation of specific transcripts in a manner that is not well understood but that appears to involve pre-mRNA processing. Here, we report biochemical analysis of purified, recombinant Su(s) [rSu(s)] expressed in baculovirus and in Escherichia coli as maltose binding protein (MBP) fusions and immunocytochemical analysis of endogenous Su(s). This work has shown that purified, baculovirus-expressed rSu(s) binds to RNA in vitro with a high affinity and limited specificity. Systematic evolution of ligands by exponential enrichment was used to identify preferred RNA targets of rSu(s), and a large proportion of RNAs isolated contain a full or partial match to the consensus sequence UCAGUAGUCU, which was confirmed to be a high-affinity rSu(s) binding site. An MBP-Su(s) fusion protein containing the N-terminal third of Su(s) binds RNAs containing this sequence with a higher specificity than full-length, baculovirus-expressed rSu(s). The consensus sequence resembles both a cryptic 5' splice site and a sequence that is found near the 5' end of some Drosophila transcripts. Immunolocalization studies showed that endogenous Su(s) is distributed in a reticulated pattern in Drosophila embryo and salivary gland nuclei. In salivary gland cells, Su(s) is found both in the nucleoplasm and in association with a subset of polytene chromosome bands. Considering these and previous results, we propose two models to explain how su(s) mutations affect nuclear pre-mRNA processing.

  15. PH Domain-Arf G Protein Interactions Localize the Arf-GEF Steppke for Cleavage Furrow Regulation in Drosophila.

    PubMed

    Lee, Donghoon M; Rodrigues, Francisco F; Yu, Cao Guo; Swan, Michael; Harris, Tony J C

    2015-01-01

    The recruitment of GDP/GTP exchange factors (GEFs) to specific subcellular sites dictates where they activate small G proteins for the regulation of various cellular processes. Cytohesins are a conserved family of plasma membrane GEFs for Arf small G proteins that regulate endocytosis. Analyses of mammalian cytohesins have identified a number of recruitment mechanisms for these multi-domain proteins, but the conservation and developmental roles for these mechanisms are unclear. Here, we report how the pleckstrin homology (PH) domain of the Drosophila cytohesin Steppke affects its localization and activity at cleavage furrows of the early embryo. We found that the PH domain is necessary for Steppke furrow localization, and for it to regulate furrow structure. However, the PH domain was not sufficient for the localization. Next, we examined the role of conserved PH domain amino acid residues that are required for mammalian cytohesins to bind PIP3 or GTP-bound Arf G proteins. We confirmed that the Steppke PH domain preferentially binds PIP3 in vitro through a conserved mechanism. However, disruption of residues for PIP3 binding had no apparent effect on GFP-Steppke localization and effects. Rather, residues for binding to GTP-bound Arf G proteins made major contributions to this Steppke localization and activity. By analyzing GFP-tagged Arf and Arf-like small G proteins, we found that Arf1-GFP, Arf6-GFP and Arl4-GFP, but not Arf4-GFP, localized to furrows. However, analyses of embryos depleted of Arf1, Arf6 or Arl4 revealed either earlier defects than occur in embryos depleted of Steppke, or no detectable furrow defects, possibly because of redundancies, and thus it was difficult to assess how individual Arf small G proteins affect Steppke. Nonetheless, our data show that the Steppke PH domain and its conserved residues for binding to GTP-bound Arf G proteins have substantial effects on Steppke localization and activity in early Drosophila embryos.

  16. Bcl-2 proteins and autophagy regulate mitochondrial dynamics during programmed cell death in the Drosophila ovary

    PubMed Central

    Tanner, Elizabeth A.; Blute, Todd A.; Brachmann, Carrie Baker; McCall, Kimberly

    2011-01-01

    The Bcl-2 family has been shown to regulate mitochondrial dynamics during cell death in mammals and C. elegans, but evidence for this in Drosophila has been elusive. Here, we investigate the regulation of mitochondrial dynamics during germline cell death in the Drosophila melanogaster ovary. We find that mitochondria undergo a series of events during the progression of cell death, with remodeling, cluster formation and uptake of clusters by somatic follicle cells. These mitochondrial dynamics are dependent on caspases, the Bcl-2 family, the mitochondrial fission and fusion machinery, and the autophagy machinery. Furthermore, Bcl-2 family mutants show a striking defect in cell death in the ovary. These data indicate that a mitochondrial pathway is a major mechanism for activation of cell death in Drosophila oogenesis. PMID:21177345

  17. Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis

    PubMed Central

    2013-01-01

    Background In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. Results Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof1/+; mnkp6/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. Conclusion mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry

  18. Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis.

    PubMed

    Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Ramaiah, M Janaki; Chowdhury, Debabani Roy; Bhadra, Utpal; Pal-Bhadra, Manika

    2013-01-24

    In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof¹/+; mnkp⁶/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry out the interaction of MOF

  19. The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus

    PubMed Central

    Kracklauer, Martin P.; Wiora, Heather M.; Deery, William J.; Chen, Xin; Bolival, Benjamin; Romanowicz, Dwight; Simonette, Rebecca A.; Fuller, Margaret T.; Fischer, Janice A.; Beckingham, Kathleen M.

    2010-01-01

    Maintaining the proximity of centrosomes to nuclei is important in several cellular contexts, and LINC complexes formed by SUN and KASH proteins are crucial in this process. Here, we characterize the presumed Drosophila ortholog of the mammalian SUN protein, sperm-associated antigen 4 (Spag4, previously named Giacomo), and demonstrate that Spag4 is required for centriole and nuclear attachment during spermatogenesis. Production of spag4 mRNA is limited to the testis, and Spag4 protein shows a dynamic pattern of association with the germline nuclei, including a concentration of protein at the site of attachment of the single spermatid centriole. In the absence of Spag4, nuclei and centrioles or basal bodies (BBs) dissociate from each other after meiosis. This role of Spag4 in centriolar attachment does not involve either of the two KASH proteins of the Drosophila genome (Klarsicht and MSP-300), but does require the coiled-coil protein Yuri Gagarin. Yuri shows an identical pattern of localization at the nuclear surface to Spag4 during spermatogenesis, and epistasis studies show that the activities of Yuri and dynein-dynactin are downstream of spag4 in this centriole attachment pathway. The later defects in spermatogenesis seen for yuri and spag4 mutants are similar, suggesting they could be secondary to initial disruption of events at the nuclear surface. PMID:20647369

  20. The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus.

    PubMed

    Kracklauer, Martin P; Wiora, Heather M; Deery, William J; Chen, Xin; Bolival, Benjamin; Romanowicz, Dwight; Simonette, Rebecca A; Fuller, Margaret T; Fischer, Janice A; Beckingham, Kathleen M

    2010-08-15

    Maintaining the proximity of centrosomes to nuclei is important in several cellular contexts, and LINC complexes formed by SUN and KASH proteins are crucial in this process. Here, we characterize the presumed Drosophila ortholog of the mammalian SUN protein, sperm-associated antigen 4 (Spag4, previously named Giacomo), and demonstrate that Spag4 is required for centriole and nuclear attachment during spermatogenesis. Production of spag4 mRNA is limited to the testis, and Spag4 protein shows a dynamic pattern of association with the germline nuclei, including a concentration of protein at the site of attachment of the single spermatid centriole. In the absence of Spag4, nuclei and centrioles or basal bodies (BBs) dissociate from each other after meiosis. This role of Spag4 in centriolar attachment does not involve either of the two KASH proteins of the Drosophila genome (Klarsicht and MSP-300), but does require the coiled-coil protein Yuri Gagarin. Yuri shows an identical pattern of localization at the nuclear surface to Spag4 during spermatogenesis, and epistasis studies show that the activities of Yuri and dynein-dynactin are downstream of spag4 in this centriole attachment pathway. The later defects in spermatogenesis seen for yuri and spag4 mutants are similar, suggesting they could be secondary to initial disruption of events at the nuclear surface.

  1. Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

    SciTech Connect

    Lee, Andrew Loyd

    1996-05-01

    Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the δ-Al-ε activation domain from c-jun and the Drosophila RNA-binding protein Sex-lethal. The second system is the Drosophila Sex-lethal (Sxl) protein, an RNA-binding protein which is the ``master switch`` in sex determination. Sxl contains two adjacent RNA-binding domains (RBDs) of the RNP consensus-type. The NMR spectrum of the second RBD (Sxl-RBD2) was assigned using multidimensional heteronuclear NMR, and an intermediate-resolution family of structures was calculated from primarily NOE distance restraints. The overall fold was determined to be similar to other RBDs: a βαβ-βαβ pattern of secondary structure, with the two helices packed against a 4-stranded anti-parallel β-sheet. In addition 15N T1, T2, and 15N/1H NOE relaxation measurements were carried out to characterize the backbone dynamics of Sxl-RBD2 in solution. RNA corresponding to the polypyrimidine tract of transformer pre-mRNA was generated and titrated into 3 different Sxl-RBD protein constructs. Combining Sxl-RBD1+2 (bht RBDs) with this RNA formed a specific, high affinity protein/RNA complex that is amenable to further NMR characterization. The backbone 1H, 13C, and 15N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and 15N relaxation experiments were carried out to characterize the backbone dynamics of this complex. The changes in chemical shift in Sxl-RBD1+2 upon binding RNA are observed using Sxl-RBD2 as a substitute for unbound Sxl-RBD1+2. This allowed the binding interface to be qualitatively mapped for the second domain.

  2. Mutational Analysis of Rab3 Function for Controlling Active Zone Protein Composition at the Drosophila Neuromuscular Junction.

    PubMed

    Chen, Shirui; Gendelman, Hannah K; Roche, John P; Alsharif, Peter; Graf, Ethan R

    2015-01-01

    At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function.

  3. Targeted Downregulation of s36 Protein Unearths its Cardinal Role in Chorion Biogenesis and Architecture during Drosophila melanogaster Oogenesis

    PubMed Central

    Velentzas, Athanassios D.; Velentzas, Panagiotis D.; Sagioglou, Niki E.; Konstantakou, Eumorphia G.; Anagnostopoulos, Athanasios K.; Tsioka, Maria M.; Mpakou, Vassiliki E.; Kollia, Zoe; Consoulas, Christos; Margaritis, Lukas H.; Papassideri, Issidora S.; Tsangaris, George Th.; Sarantopoulou, Evangelia; Cefalas, Alkiviadis-Constantinos; Stravopodis, Dimitrios J.

    2016-01-01

    Drosophila chorion represents a model biological system for the in vivo study of gene activity, epithelial development, extracellular-matrix assembly and morphogenetic-patterning control. It is produced during the late stages of oogenesis by epithelial follicle cells and develops into a highly organized multi-layered structure that exhibits regional specialization and radial complexity. Among the six major proteins involved in chorion’s formation, the s36 and s38 ones are synthesized first and regulated in a cell type-specific and developmental stage-dependent manner. In our study, an RNAi-mediated silencing of s36 chorionic-gene expression specifically in the follicle-cell compartment of Drosophila ovary unearths the essential, and far from redundant, role of s36 protein in patterning establishment of chorion’s regional specialization and radial complexity. Without perturbing the developmental courses of follicle- and nurse-cell clusters, the absence of s36 not only promotes chorion’s fragility but also induces severe structural irregularities on chorion’s surface and entirely impairs fly’s fertility. Moreover, we herein unveil a novel function of s36 chorionic protein in the regulation of number and morphogenetic integrity of dorsal appendages in follicles sporadically undergoing aged fly-dependent stress. PMID:27752139

  4. The Drosophila Retinoblastoma Binding Protein 6 Family Member Has Two Isoforms and Is Potentially Involved in Embryonic Patterning

    PubMed Central

    Hull, Rodney; Oosthuysen, Brent; Cajee, Umar-Faruq; Mokgohloa, Lehlogonolo; Nweke, Ekene; Antunes, Ricardo Jorge; Coetzer, Theresa H. T.; Ntwasa, Monde

    2015-01-01

    The human retinoblastoma binding protein 6 (RBBP6) is implicated in esophageal, lung, hepatocellular and colon cancers. Furthermore, RBBP6 was identified as a strong marker for colon cancer prognosis and as a predisposing factor in familial myeloproliferative neoplasms. Functionally, the mammalian protein interacts with p53 and enhances the activity of Mdm2, the prototypical negative regulator of p53. However, since RBBP6 (known as PACT in mice) exists in multiple isoforms and pact−/− mice exhibit a more severe phenotype than mdm2−/− mutants, it must possess some Mdm2-independent functions. The function of the invertebrate homologue is poorly understood. This is complicated by the absence of the Mdm2 gene in both Drosophila and Caenorhabditis elegans. We have experimentally identified the promoter region of Snama, the Drosophila homologue, analyzed potential transcription factor binding sites and confirmed the existence of an additional isoform. Using band shift and co-immunoprecipitation assays combined with mass spectrometry, we found evidence that this gene may be regulated by, amongst others, DREF, which regulates hundreds of genes related to cell proliferation. The potential transcription factors for Snama fall into distinct functional groups, including anteroposterior embryonic patterning and nucleic acid metabolism. Significantly, previous work in mice shows that pact−/− induces an anteroposterior phenotype in embryos when rescued by simultaneous deletion of p53. Taken together, these observations indicate the significance of RBBP6 proteins in carcinogenesis and in developmental defects. PMID:25955646

  5. Kank Is an EB1 Interacting Protein that Localises to Muscle-Tendon Attachment Sites in Drosophila

    PubMed Central

    Clohisey, Sara M. R.; Dzhindzhev, Nikola S.; Ohkura, Hiroyuki

    2014-01-01

    Little is known about how microtubules are regulated in different cell types during development. EB1 plays a central role in the regulation of microtubule plus ends. It directly binds to microtubule plus ends and recruits proteins which regulate microtubule dynamics and behaviour. We report the identification of Kank, the sole Drosophila orthologue of human Kank proteins, as an EB1 interactor that predominantly localises to embryonic attachment sites between muscle and tendon cells. Human Kank1 was identified as a tumour suppressor and has documented roles in actin regulation and cell polarity in cultured mammalian cells. We found that Drosophila Kank binds EB1 directly and this interaction is essential for Kank localisation to microtubule plus ends in cultured cells. Kank protein is expressed throughout fly development and increases during embryogenesis. In late embryos, it accumulates to sites of attachment between muscle and epidermal cells. A kank deletion mutant was generated. We found that the mutant is viable and fertile without noticeable defects. Further analysis showed that Kank is dispensable for muscle function in larvae. This is in sharp contrast to C. elegans in which the Kank orthologue VAB-19 is required for development by stabilising attachment structures between muscle and epidermal cells. PMID:25203404

  6. Exon junction complex proteins bind nascent transcripts independently of pre-mRNA splicing in Drosophila melanogaster

    PubMed Central

    Choudhury, Subhendu Roy; Singh, Anand K; McLeod, Tina; Blanchette, Marco; Jang, Boyun; Badenhorst, Paul; Kanhere, Aditi; Brogna, Saverio

    2016-01-01

    Although it is currently understood that the exon junction complex (EJC) is recruited on spliced mRNA by a specific interaction between its central protein, eIF4AIII, and splicing factor CWC22, we found that eIF4AIII and the other EJC core proteins Y14 and MAGO bind the nascent transcripts of not only intron-containing but also intronless genes on Drosophila polytene chromosomes. Additionally, Y14 ChIP-seq demonstrates that association with transcribed genes is also splicing-independent in Drosophila S2 cells. The association of the EJC proteins with nascent transcripts does not require CWC22 and that of Y14 and MAGO is independent of eIF4AIII. We also show that eIF4AIII associates with both polysomal and monosomal RNA in S2 cell extracts, whereas Y14 and MAGO fractionate separately. Cumulatively, our data indicate a global role of eIF4AIII in gene expression, which would be independent of Y14 and MAGO, splicing, and of the EJC, as currently understood. DOI: http://dx.doi.org/10.7554/eLife.19881.001 PMID:27879206

  7. The Cricket Paralysis Virus Suppressor Inhibits microRNA Silencing Mediated by the Drosophila Argonaute-2 Protein

    PubMed Central

    Besnard-Guérin, Corinne; Jacquier, Caroline; Pidoux, Josette; Deddouche, Safia; Antoniewsk, Christophe

    2015-01-01

    Small RNAs are potent regulators of gene expression. They also act in defense pathways against invading nucleic acids such as transposable elements or viruses. To counteract these defenses, viruses have evolved viral suppressors of RNA silencing (VSRs). Plant viruses encoded VSRs interfere with siRNAs or miRNAs by targeting common mediators of these two pathways. In contrast, VSRs identified in insect viruses to date only interfere with the siRNA pathway whose effector Argonaute protein is Argonaute-2 (Ago-2). Although a majority of Drosophila miRNAs exerts their silencing activity through their loading into the Argonaute-1 protein, recent studies highlighted that a fraction of miRNAs can be loaded into Ago-2, thus acting as siRNAs. In light of these recent findings, we re-examined the role of insect VSRs on Ago-2-mediated miRNA silencing in Drosophila melanogaster. Using specific reporter systems in cultured Schneider-2 cells and transgenic flies, we showed here that the Cricket Paralysis virus VSR CrPV1-A but not the Flock House virus B2 VSR abolishes silencing by miRNAs loaded into the Ago-2 protein. Thus, our results provide the first evidence that insect VSR have the potential to directly interfere with the miRNA silencing pathway. PMID:25793377

  8. Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases

    PubMed Central

    Niehues, Sven; Bussmann, Julia; Steffes, Georg; Erdmann, Ines; Köhrer, Caroline; Sun, Litao; Wagner, Marina; Schäfer, Kerstin; Wang, Guangxia; Koerdt, Sophia N.; Stum, Morgane; RajBhandary, Uttam L.; Thomas, Ulrich; Aberle, Hermann; Burgess, Robert W.; Yang, Xiang-Lei; Dieterich, Daniela; Storkebaum, Erik

    2015-01-01

    Dominant mutations in five tRNA synthetases cause Charcot–Marie–Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. Mutant GARS proteins display normal subcellular localization but markedly reduce global protein synthesis in motor and sensory neurons, or when ubiquitously expressed in adults, as revealed by FUNCAT and BONCAT. Translational slowdown is not attributable to altered tRNAGly aminoacylation, and cannot be rescued by Drosophila Gars overexpression, indicating a gain-of-toxic-function mechanism. Expression of CMT-mutant tyrosyl-tRNA synthetase also impairs translation, suggesting a common pathogenic mechanism. Finally, genetic reduction of translation is sufficient to induce CMT-like phenotypes, indicating a causal contribution of translational slowdown to CMT. PMID:26138142

  9. The Drosophila Meiotic Recombination Gene Mei-9 Encodes a Homologue of the Yeast Excision Repair Protein Rad1

    PubMed Central

    Sekelsky, J. J.; McKim, K. S.; Chin, G. M.; Hawley, R. S.

    1995-01-01

    Meiotic recombination and DNA repair are mediated by overlapping sets of genes. In the yeast Saccharomyces cerevisiae, many genes required to repair DNA double-strand breaks are also required for meiotic recombination. In contrast, mutations in genes required for nucleotide excision repair (NER) have no detectable effects on meiotic recombination in S. cerevisiae. The Drosophila melanogaster mei-9 gene is unique among known recombination genes in that it is required for both meiotic recombination and NER. We have analyzed the mei-9 gene at the molecular level and found that it encodes a homologue of the S. cerevisiae excision repair protein Rad1, the probable homologue of mammalian XPF/ERCC4. Hence, the predominant process of meiotic recombination in Drosophila proceeds through a pathway that is at least partially distinct from that of S. cerevisiae, in that it requires an NER protein. The biochemical properties of the Rad1 protein allow us to explain the observation that mei-9 mutants suppress reciprocal exchange without suppressing the frequency of gene conversion. PMID:8647398

  10. Impaired protein translation in Drosophila models for Charcot-Marie-Tooth neuropathy caused by mutant tRNA synthetases.

    PubMed

    Niehues, Sven; Bussmann, Julia; Steffes, Georg; Erdmann, Ines; Köhrer, Caroline; Sun, Litao; Wagner, Marina; Schäfer, Kerstin; Wang, Guangxia; Koerdt, Sophia N; Stum, Morgane; Jaiswal, Sumit; RajBhandary, Uttam L; Thomas, Ulrich; Aberle, Hermann; Burgess, Robert W; Yang, Xiang-Lei; Dieterich, Daniela; Storkebaum, Erik

    2015-07-03

    Dominant mutations in five tRNA synthetases cause Charcot-Marie-Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. Mutant GARS proteins display normal subcellular localization but markedly reduce global protein synthesis in motor and sensory neurons, or when ubiquitously expressed in adults, as revealed by FUNCAT and BONCAT. Translational slowdown is not attributable to altered tRNA(Gly) aminoacylation, and cannot be rescued by Drosophila Gars overexpression, indicating a gain-of-toxic-function mechanism. Expression of CMT-mutant tyrosyl-tRNA synthetase also impairs translation, suggesting a common pathogenic mechanism. Finally, genetic reduction of translation is sufficient to induce CMT-like phenotypes, indicating a causal contribution of translational slowdown to CMT.

  11. A Drosophila haemocyte-specific protein, hemolectin, similar to human von Willebrand factor.

    PubMed Central

    Goto, A; Kumagai, T; Kumagai, C; Hirose, J; Narita, H; Mori, H; Kadowaki, T; Beck, K; Kitagawa, Y

    2001-01-01

    We identified a novel Drosophila protein of approximately 400 kDa, hemolectin (d-Hml), secreted from haemocyte-derived Kc167 cells. Its 11.7 kbp cDNA contains an open reading frame of 3843 amino acid residues, with conserved domains in von Willebrand factor (VWF), coagulation factor V/VIII and complement factors. The d-hml gene is located on the third chromosome (position 70C1-5) and consists of 26 exons. The major part of d-Hml consists of well-known motifs with the organization: CP1-EG1-CP2-EG2-CP3-VD1-VD2-VD'-VD3-VC1-VD"-VD"'-FC1-FC2-VC2-LA1-VD4-VD5-VC3-VB1-VB2-VC4-VC5-CK1 (CP, complement-control protein domain; EG, epidermal-growth-factor-like domain; VB, VC, VD, VWF type B-, C- and D-like domains; VD', VD", VD"', truncated C-terminal VDs; FC, coagulation factor V/VIII type C domain; LA, low-density-lipoprotein-receptor class A domain; CK, cysteine knot domain). The organization of VD1-VD2-VD'-VD3, essential for VWF to be processed by furin, to bind to coagulation factor VIII and to form interchain disulphide linkages, is conserved. The 400 kDa form of d-Hml was sensitive to acidic cleavage near the boundary between VD2 and VD', where the cleavage site of pro-VWF is located. Agarose-gel electrophoresis of metabolically radiolabelled d-Hml suggested that it is secreted from Kc167 cells mainly as dimers. Resembling VWF, 7.9% (305 residues) of cysteine residues on the d-Hml sequence had well-conserved positions in each motif. Coinciding with the development of phagocytic haemocytes, d-hml transcript was detected in late embryos and larvae. Its low-level expression in adult flies was induced by injury at any position on the body. PMID:11563973

  12. Variation in sperm displacement and its association with accessory gland protein loci in Drosophila melanogaster.

    PubMed

    Clark, A G; Aguadé, M; Prout, T; Harshman, L G; Langley, C H

    1995-01-01

    Genes that influence mating and/or fertilization success may be targets for strong natural selection. If females remate frequently relative to the duration of sperm storage and rate of sperm use, sperm displacement may be an important component of male reproductive success. Although it has long been known that mutant laboratory stocks of Drosophila differ in sperm displacement, the magnitude of the naturally occurring genetic variation in this character has not been systematically quantified. Here we report the results of a screen for variation in sperm displacement among 152 lines of Drosophilia melanogaster that were made homozygous for second and/or third chromosomes recovered from natural populations. Sperm displacement was assayed by scoring the progeny of cn;bw females that had been mated sequentially to cn;bw and tested males in either order. Highly significant differences were seen in both the ability to displace sperm that is resident in the female's reproductive tract and in the ability to resist displacement by subsequent sperm. Most lines exhibited nearly complete displacement, having nearly all progeny sired by the second male, but several lines had as few as half the progeny fathered by the second male. Lines that were identified in the screen for naturally occurring variation in sperm displacement were also characterized for single-strand conformation polymorphisms (SSCP) at seven accessory gland protein (Acp) genes, Glucose dehydrogenase (Gld), and Esterase-6 (Est-6). Acp genes encode proteins that are in some cases known to be transmitted to the female in the seminal fluid and are likely candidates for genes that might mediate the phenomenon of sperm displacement. Significant associations were found between particular Acp alleles at four different loci (Acp26Aa/Ab, Acp29B, Acp36DE and Acp53E) and the ability of males to resist displacement by subsequent sperm. There was no correlation between the ability to displace resident sperm and the ability

  13. EAST Organizes Drosophila Insulator Proteins in the Interchromosomal Nuclear Compartment and Modulates CP190 Binding to Chromatin

    PubMed Central

    Golovnin, Anton; Melnikova, Larisa; Shapovalov, Igor; Kostyuchenko, Margarita; Georgiev, Pavel

    2015-01-01

    Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. Here, we have shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization. PMID:26489095

  14. EAST Organizes Drosophila Insulator Proteins in the Interchromosomal Nuclear Compartment and Modulates CP190 Binding to Chromatin.

    PubMed

    Golovnin, Anton; Melnikova, Larisa; Shapovalov, Igor; Kostyuchenko, Margarita; Georgiev, Pavel

    2015-01-01

    Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. Here, we have shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization.

  15. The Kl-3 Loop of the Y Chromosome of Drosophila Melanogaster Binds a Tektin-like Protein

    PubMed Central

    Pisano, C.; Bonaccorsi, S.; Gatti, M.

    1993-01-01

    Primary spermatocyte nuclei of Drosophila melanogaster exhibit three giant lampbrush-like loops formed by the kl-5, kl-3 and ks-1 Y-chromosome fertility factors. These structures contain and abundantly transcribe highly repetitive, simple sequence DNAs and accumulate large amounts of non-Y-encoded proteins. By immunizing mice with the 53-kD fraction (enriched in β(2)-tubulin) excised from a sodium dodecyl sulfate-polyacrylamide gel loaded with Drosophila testis proteins we raised a polyclonal antibody, designated as T53-1, which decorates the kl-3 loop and the sperm flagellum. Two dimensional immunoblot analysis showed that the T53-1 antibody reacts with a single protein of about 53 kD, different from the tubulins and present both in X/Y and X/O males. Moreover, the antigen recognized by the T53-1 antibody proved to be testis-specific because it was detected in testes and seminal vesicles but not in other male tissues or in females. The characteristics of the protein recognized by the T53-1 antibody suggested that it might be a member of a class of axonemal proteins, the tektins, known to form Sarkosyl-urea insoluble filaments in the wall of flagellar microtubules. Purification of the Sarkosyl-urea insoluble fraction of D. melanogaster sperm revealed that it contains four polypeptides having molecular masses ranging from 51 to 57 kD. One of these polypeptides reacts strongly with the T53-1 antibody but none of them reacts with antitubulin antibodies. These results indicate that the kl-3 loop binds a non-Y encoded, testis-specific, tektin-like protein which is a constituent of the sperm flagellum. This finding supports the hypothesis that the Y loops fulfill a protein-binding function required for the proper assembly of the axoneme components. PMID:8454204

  16. Low Levels of p53 Protein and Chromatin Silencing of p53 Target Genes Repress Apoptosis in Drosophila Endocycling Cells

    PubMed Central

    Zhang, Bingqing; Mehrotra, Sonam; Ng, Wei Lun; Calvi, Brian R.

    2014-01-01

    Apoptotic cell death is an important response to genotoxic stress that prevents oncogenesis. It is known that tissues can differ in their apoptotic response, but molecular mechanisms are little understood. Here, we show that Drosophila polyploid endocycling cells (G/S cycle) repress the apoptotic response to DNA damage through at least two mechanisms. First, the expression of all the Drosophila p53 protein isoforms is strongly repressed at a post-transcriptional step. Second, p53-regulated pro-apoptotic genes are epigenetically silenced in endocycling cells, preventing activation of a paused RNA Pol II by p53-dependent or p53-independent pathways. Over-expression of the p53A isoform did not activate this paused RNA Pol II complex in endocycling cells, but over-expression of the p53B isoform with a longer transactivation domain did, suggesting that dampened p53B protein levels are crucial for apoptotic repression. We also find that the p53A protein isoform is ubiquitinated and degraded by the proteasome in endocycling cells. In mitotic cycling cells, p53A was the only isoform expressed to detectable levels, and its mRNA and protein levels increased after irradiation, but there was no evidence for an increase in protein stability. However, our data suggest that p53A protein stability is regulated in unirradiated cells, which likely ensures that apoptosis does not occur in the absence of stress. Without irradiation, both p53A protein and a paused RNA pol II were pre-bound to the promoters of pro-apoptotic genes, preparing mitotic cycling cells for a rapid apoptotic response to genotoxic stress. Together, our results define molecular mechanisms by which different cells in development modulate their apoptotic response, with broader significance for the survival of normal and cancer polyploid cells in mammals. PMID:25211335

  17. Basic Leucine Zipper Protein Cnc-C Is a Substrate and Transcriptional Regulator of the Drosophila 26S Proteasome▿ †

    PubMed Central

    Grimberg, Kristian Björk; Beskow, Anne; Lundin, Daniel; Davis, Monica M.; Young, Patrick

    2011-01-01

    While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n’ collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis. PMID:21149573

  18. Basic leucine zipper protein Cnc-C is a substrate and transcriptional regulator of the Drosophila 26S proteasome.

    PubMed

    Grimberg, Kristian Björk; Beskow, Anne; Lundin, Daniel; Davis, Monica M; Young, Patrick

    2011-02-01

    While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n' collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis.

  19. Lie Group Analysis of the Photo-Induced Fluorescence of Drosophila Oogenesis with the Asymmetrically Localized Gurken Protein

    PubMed Central

    Wang, Jen-Cheng; Wang, Pei-Yu; Chen, Hung-Ing; Wu, Kai-Ling; Pai, Li-Mei; Nee, Tzer-En

    2013-01-01

    Lie group analysis of the photo-induced fluorescence of Drosophila oogenesis with the asymmetrically localized Gurken protein has been performed systematically to assess the roles of ligand-receptor complexes in follicle cells. The (2×2) matrix representations resulting from the polarized tissue spectra were employed to characterize the asymmetrical Gurken distributions. It was found that the fluorescence of the wild-type egg shows the Lie point symmetry X23 at early stages of oogenesis. However, due to the morphogen regulation by intracellular proteins and extracellular proteins, the fluorescence of the embryogenesis with asymmetrically localized Gurken expansions exhibits specific symmetry features: Lie point symmetry Z1 and Lie point symmetry X1. The novel approach developed herein was successfully used to validate that the invariant-theoretical characterizations are consonant with the observed asymmetric fluctuations during early embryological development. PMID:23840317

  20. A Gap Junction Protein, Inx2, Modulates Calcium Flux to Specify Border Cell Fate during Drosophila oogenesis.

    PubMed

    Sahu, Aresh; Ghosh, Ritabrata; Deshpande, Girish; Prasad, Mohit

    2017-01-01

    Intercellular communication mediated by gap junction (GJ) proteins is indispensable during embryogenesis, tissue regeneration and wound healing. Here we report functional analysis of a gap junction protein, Innexin 2 (Inx2), in cell type specification during Drosophila oogenesis. Our data reveal a novel involvement of Inx2 in the specification of Border Cells (BCs), a migratory cell type, whose identity is determined by the cell autonomous STAT activity. We show that Inx2 influences BC fate specification by modulating STAT activity via Domeless receptor endocytosis. Furthermore, detailed experimental analysis has uncovered that Inx2 also regulates a calcium flux that transmits across the follicle cells. We propose that Inx2 mediated calcium flux in the follicle cells stimulates endocytosis by altering Dynamin (Shibire) distribution which is in turn critical for careful calibration of STAT activation and, thus for BC specification. Together our data provide unprecedented molecular insights into how gap junction proteins can regulate cell-type specification.

  1. The inhibitor of DNA replication encoded by the Drosophila gene plutonium is a small, ankyrin repeat protein.

    PubMed Central

    Axton, J M; Shamanski, F L; Young, L M; Henderson, D S; Boyd, J B; Orr-Weaver, T L

    1994-01-01

    The plutonium (plu) gene product controls DNA replication early in Drosophila development. plu mutant females lay unfertilized eggs that have undergone extensive DNA synthesis. In fertilized embryos from plu mutant mothers, S-phase is uncoupled from mitosis. The gene is expressed only in ovaries and embryos, null alleles are strict maternal effect mutations, and the phenotype of inappropriate DNA replication is the consequence of loss-of-gene function. plu therefore negatively regulates S-phase at a time in early development when commitment to S-phase does not depend on cyclic transcription. plu encodes a protein with two ankyrin-like repeats, a domain for protein-protein interaction. plu is immediately adjacent to, but distinct from, the PCNA gene. Images PMID:8313891

  2. Lie Group Analysis of the Photo-Induced Fluorescence of Drosophila Oogenesis with the Asymmetrically Localized Gurken Protein.

    PubMed

    Wang, Jen-Cheng; Wang, Pei-Yu; Chen, Hung-Ing; Wu, Kai-Ling; Pai, Li-Mei; Nee, Tzer-En

    2013-01-01

    Lie group analysis of the photo-induced fluorescence of Drosophila oogenesis with the asymmetrically localized Gurken protein has been performed systematically to assess the roles of ligand-receptor complexes in follicle cells. The (2×2) matrix representations resulting from the polarized tissue spectra were employed to characterize the asymmetrical Gurken distributions. It was found that the fluorescence of the wild-type egg shows the Lie point symmetry X 23 at early stages of oogenesis. However, due to the morphogen regulation by intracellular proteins and extracellular proteins, the fluorescence of the embryogenesis with asymmetrically localized Gurken expansions exhibits specific symmetry features: Lie point symmetry Z 1 and Lie point symmetry X 1. The novel approach developed herein was successfully used to validate that the invariant-theoretical characterizations are consonant with the observed asymmetric fluctuations during early embryological development.

  3. A Gap Junction Protein, Inx2, Modulates Calcium Flux to Specify Border Cell Fate during Drosophila oogenesis

    PubMed Central

    Ghosh, Ritabrata; Deshpande, Girish

    2017-01-01

    Intercellular communication mediated by gap junction (GJ) proteins is indispensable during embryogenesis, tissue regeneration and wound healing. Here we report functional analysis of a gap junction protein, Innexin 2 (Inx2), in cell type specification during Drosophila oogenesis. Our data reveal a novel involvement of Inx2 in the specification of Border Cells (BCs), a migratory cell type, whose identity is determined by the cell autonomous STAT activity. We show that Inx2 influences BC fate specification by modulating STAT activity via Domeless receptor endocytosis. Furthermore, detailed experimental analysis has uncovered that Inx2 also regulates a calcium flux that transmits across the follicle cells. We propose that Inx2 mediated calcium flux in the follicle cells stimulates endocytosis by altering Dynamin (Shibire) distribution which is in turn critical for careful calibration of STAT activation and, thus for BC specification. Together our data provide unprecedented molecular insights into how gap junction proteins can regulate cell-type specification. PMID:28114410

  4. Maintenance of Drosophila germline stem cell sexual identity in oogenesis and tumorigenesis.

    PubMed

    Shapiro-Kulnane, Laura; Smolko, Anne Elizabeth; Salz, Helen Karen

    2015-03-15

    Adult stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. In Drosophila females, germline stem cells (GSCs) require Sex lethal (Sxl) to exit the stem cell state and to enter the differentiation pathway. Without Sxl GSCs do not differentiate and instead form tumors. Previous studies have shown that these tumors are not caused by a failure in the self-renewal/differentiation switch. Here, we show that Sxl is also necessary for the cell-autonomous maintenance of germ cell female identity and demonstrate that tumors are caused by the acquisition of male characteristics. Germ cells without Sxl protein exhibit a global derepression of testis genes, including Phf7, a male germline sexual identity gene. Phf7 is a key effector of the tumor-forming pathway, as it is both necessary and sufficient for tumor formation. In the absence of Sxl protein, inappropriate Phf7 expression drives tumor formation through a cell-autonomous mechanism that includes sex-inappropriate activation of Jak/Stat signaling. Remarkably, tumor formation requires a novel response to external signals emanating from the GSC niche, highlighting the importance of interactions between mutant cells and the surrounding normal cells that make up the tumor microenvironment. Derepression of testis genes, and inappropriate Phf7 expression, is also observed in germ cell tumors arising from the loss of bag of marbles (bam), demonstrating that maintenance of female sexual identity requires the concerted actions of Sxl and bam. Our work reveals that GSCs must maintain their sexual identity as they are reprogrammed into a differentiated cell, or risk tumorigenesis.

  5. The Drosophila suppressor of sable gene encodes a polypeptide with regions similar to those of RNA-binding proteins.

    PubMed Central

    Voelker, R A; Gibson, W; Graves, J P; Sterling, J F; Eisenberg, M T

    1991-01-01

    The nucleotide sequence of the Drosophila melanogaster suppressor of sable [su(s)] gene has been determined. Comparison of genomic and cDNA sequences indicates that an approximately 7,860-nucleotide primary transcript is processed into an approximately 5-kb message, expressed during all stages of the life cycle, that contains an open reading frame capable of encoding a 1,322-amino-acid protein of approximately 150 kDa. The putative protein contains an RNA recognition motif-like region and a highly charged arginine-, lysine-, serine-, aspartic or glutamic acid-rich region that is similar to a region contained in several RNA-processing proteins. In vitro translation of in vitro-transcribed RNA from a complete cDNA yields a product whose size agrees with the size predicted by the open reading frame. Antisera against su(s) fusion proteins recognize the in vitro-translated protein and detect a protein of identical size in the nuclear fractions from tissue culture cells and embryos. The protein is also present in smaller amounts in cytoplasmic fractions of embryos. That the su(s) protein has regions similar in structure to RNA-processing protein is consistent with its known role in affecting the transcript levels of those alleles that it suppresses. Images PMID:1703632

  6. Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila

    PubMed Central

    Morelli, Elena; Ginefra, Pierpaolo; Mastrodonato, Valeria; Beznoussenko, Galina V; Rusten, Tor Erik; Bilder, David; Stenmark, Harald; Mironov, Alexandre A; Vaccari, Thomas

    2015-01-01

    How autophagic degradation is linked to endosomal trafficking routes is little known. Here we screened a collection of uncharacterized Drosophila mutants affecting membrane transport to identify new genes that also have a role in autophagy. We isolated a loss of function mutant in Snap29 (Synaptosomal-associated protein 29 kDa), the gene encoding the Drosophila homolog of the human protein SNAP29 and have characterized its function in vivo. Snap29 contains 2 soluble NSF attachment protein receptor (SNARE) domains and a asparagine-proline-phenylalanine (NPF motif) at its N terminus and rescue experiments indicate that both SNARE domains are required for function, whereas the NPF motif is in part dispensable. We find that Snap29 interacts with SNARE proteins, localizes to multiple trafficking organelles, and is required for protein trafficking and for proper Golgi apparatus morphology. Developing tissue lacking Snap29 displays distinctive epithelial architecture defects and accumulates large amounts of autophagosomes, highlighting a major role of Snap29 in autophagy and secretion. Mutants for autophagy genes do not display epithelial architecture or secretion defects, suggesting that the these alterations of the Snap29 mutant are unlikely to be caused by the impairment of autophagy. In contrast, we find evidence of elevated levels of hop-Stat92E (hopscotch-signal transducer and activator of transcription protein at 92E) ligand, receptor, and associated signaling, which might underlie the epithelial defects. In summary, our findings support a role of Snap29 at key steps of membrane trafficking, and predict that signaling defects may contribute to the pathogenesis of cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma (CEDNIK), a human congenital syndrome due to loss of Snap29. PMID:25551675

  7. Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila.

    PubMed

    Morelli, Elena; Ginefra, Pierpaolo; Mastrodonato, Valeria; Beznoussenko, Galina V; Rusten, Tor Erik; Bilder, David; Stenmark, Harald; Mironov, Alexandre A; Vaccari, Thomas

    2014-01-01

    How autophagic degradation is linked to endosomal trafficking routes is little known. Here we screened a collection of uncharacterized Drosophila mutants affecting membrane transport to identify new genes that also have a role in autophagy. We isolated a loss of function mutant in Snap29 (Synaptosomal-associated protein 29 kDa), the gene encoding the Drosophila homolog of the human protein SNAP29 and have characterized its function in vivo. Snap29 contains 2 soluble NSF attachment protein receptor (SNARE) domains and a asparagine-proline-phenylalanine (NPF motif) at its N terminus and rescue experiments indicate that both SNARE domains are required for function, whereas the NPF motif is in part dispensable. We find that Snap29 interacts with SNARE proteins, localizes to multiple trafficking organelles, and is required for protein trafficking and for proper Golgi apparatus morphology. Developing tissue lacking Snap29 displays distinctive epithelial architecture defects and accumulates large amounts of autophagosomes, highlighting a major role of Snap29 in autophagy and secretion. Mutants for autophagy genes do not display epithelial architecture or secretion defects, suggesting that the these alterations of the Snap29 mutant are unlikely to be caused by the impairment of autophagy. In contrast, we find evidence of elevated levels of hop-Stat92E (hopscotch-signal transducer and activator of transcription protein at 92E) ligand, receptor, and associated signaling, which might underlie the epithelial defects. In summary, our findings support a role of Snap29 at key steps of membrane trafficking, and predict that signaling defects may contribute to the pathogenesis of cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma (CEDNIK), a human congenital syndrome due to loss of Snap29.

  8. Meiosis in male Drosophila

    PubMed Central

    McKee, Bruce D.; Yan, Rihui; Tsai, Jui-He

    2012-01-01

    Meiosis entails sorting and separating both homologous and sister chromatids. The mechanisms for connecting sister chromatids and homologs during meiosis are highly conserved and include specialized forms of the cohesin complex and a tightly regulated homolog synapsis/recombination pathway designed to yield regular crossovers between homologous chromatids. Drosophila male meiosis is of special interest because it dispenses with large segments of the standard meiotic script, particularly recombination, synapsis and the associated structures. Instead, Drosophila relies on a unique protein complex composed of at least two novel proteins, SNM and MNM, to provide stable connections between homologs during meiosis I. Sister chromatid cohesion in Drosophila is mediated by cohesins, ring-shaped complexes that entrap sister chromatids. However, unlike other eukaryotes Drosophila does not rely on the highly conserved Rec8 cohesin in meiosis, but instead utilizes two novel cohesion proteins, ORD and SOLO, which interact with the SMC1/3 cohesin components in providing meiotic cohesion. PMID:23087836

  9. Overexpression of tumor suppressor protein OSCP1/NOR1 induces ER stress and apoptosis during development of Drosophila melanogaster

    PubMed Central

    Huu, Nguyen Tho; Yoshida, Hideki; Yamaguchi, Masamitsu

    2015-01-01

    OSCP1/NOR1 (organic solute carrier partner 1/oxidored-nitro domain-containing protein 1) is known as a transporter of various organic solutes into cells and also is reported to act as a tumor suppressor protein. Although overexpression of OSCP1 has been shown to play multiple roles in mammalian cell lines, its biological significance in living organisms is not fully understood. To explore the effects of OSCP1/NOR1 on development, we performed genetic studies in flies featuring overexpression of its Drosophila orthologue, dOSCP1. Overexpression of dOSCP1 in eye imaginal discs induced a rough eye phenotype in adult flies, likely resulting from a delay in S phase progression and induction of caspase-dependent apoptosis followed by compensatory proliferation. However, it did not appear to be involved in differentiation of R7 photoreceptor cells. We also found that overexpression of dOSCP1 caused endoplasmic reticulum stress in salivary gland cells. These results indicate that overexpression of dOSCP1 exerts effects on various biological processes during Drosophila development. PMID:26175940

  10. The apoptotic engulfment protein Ced-6 participates in clathrin-mediated yolk uptake in Drosophila egg chambers

    PubMed Central

    Jha, Anupma; Watkins, Simon C.; Traub, Linton M.

    2012-01-01

    Clathrin-mediated endocytosis and phagocytosis are both selective surface internalization processes but have little known mechanistic similarity or interdependence. Here we show that the phosphotyrosine-binding (PTB) domain protein Ced-6, a well-established phagocytosis component that operates as a transducer of so-called “eat-me” signals during engulfment of apoptotic cells and microorganisms, is expressed in the female Drosophila germline and that Ced-6 expression correlates with ovarian follicle development. Ced-6 exhibits all the known biochemical properties of a clathrin-associated sorting protein, yet ced-6–null flies are semifertile despite massive accumulation of soluble yolk precursors in the hemolymph. This is because redundant sorting signals within the cytosolic domain of the Drosophila vitellogenin receptor Yolkless, a low density lipoprotein receptor superfamily member, occur; a functional atypical dileucine signal binds to the endocytic AP-2 clathrin adaptor directly. Nonetheless, the Ced-6 PTB domain specifically recognizes the noncanonical Yolkless FXNPXA sorting sequence and in HeLa cells promotes the rapid, clathrin-dependent uptake of a Yolkless chimera lacking the distal dileucine signal. Ced-6 thus operates in vivo as a clathrin adaptor. Because the human Ced-6 orthologue GULP similarly binds to clathrin machinery, localizes to cell surface clathrin-coated structures, and is enriched in placental clathrin-coated vesicles, new possibilities for Ced-6/Gulp operation during phagocytosis must be considered. PMID:22398720

  11. The Cutoff protein regulates piRNA cluster expression and piRNA production in the Drosophila germline

    PubMed Central

    Pane, Attilio; Jiang, Peng; Zhao, Dorothy Yanling; Singh, Mona; Schüpbach, Trudi

    2011-01-01

    In a broad range of organisms, Piwi-interacting RNAs (piRNAs) have emerged as core components of a surveillance system that protects the genome by silencing transposable and repetitive elements. A vast proportion of piRNAs is produced from discrete genomic loci, termed piRNA clusters, which are generally embedded in heterochromatic regions. The molecular mechanisms and the factors that govern their expression are largely unknown. Here, we show that Cutoff (Cuff), a Drosophila protein related to the yeast transcription termination factor Rai1, is essential for piRNA production in germline tissues. Cuff accumulates at centromeric/pericentromeric positions in germ-cell nuclei and strongly colocalizes with the major heterochromatic domains. Remarkably, we show that Cuff is enriched at the dual-strand piRNA cluster 1/42AB and is likely to be involved in regulation of transcript levels of similar loci dispersed in the genome. Consistent with this observation, Cuff physically interacts with the Heterochromatin Protein 1 (HP1) variant Rhino (Rhi). Our results unveil a link between Cuff activity, heterochromatin assembly and piRNA cluster expression, which is critical for stem-cell and germ-cell development in Drosophila. PMID:21952049

  12. Drosophila Condensin II subunit Chromosome-associated protein D3 regulates cell fate determination through non-cell-autonomous signaling

    PubMed Central

    Klebanow, Lindsey R.; Peshel, Emanuela C.; Schuster, Andrew T.; De, Kuntal; Sarvepalli, Kavitha; Lemieux, Madeleine E.; Lenoir, Jessica J.; Moore, Adrian W.; McDonald, Jocelyn A.

    2016-01-01

    The pattern of the Drosophila melanogaster adult wing is heavily influenced by the expression of proteins that dictate cell fate decisions between intervein and vein during development. dSRF (Blistered) expression in specific regions of the larval wing disc promotes intervein cell fate, whereas EGFR activity promotes vein cell fate. Here, we report that the chromatin-organizing protein CAP-D3 acts to dampen dSRF levels at the anterior/posterior boundary in the larval wing disc, promoting differentiation of cells into the anterior crossvein. CAP-D3 represses KNOT expression in cells immediately adjacent to the anterior/posterior boundary, thus blocking KNOT-mediated repression of EGFR activity and preventing cell death. Maintenance of EGFR activity in these cells depresses dSRF levels in the neighboring anterior crossvein progenitor cells, allowing them to differentiate into vein cells. These findings uncover a novel transcriptional regulatory network influencing Drosophila wing vein development, and are the first to identify a Condensin II subunit as an important regulator of EGFR activity and cell fate determination in vivo. PMID:27317808

  13. Drosophila Condensin II subunit Chromosome-associated protein D3 regulates cell fate determination through non-cell-autonomous signaling.

    PubMed

    Klebanow, Lindsey R; Peshel, Emanuela C; Schuster, Andrew T; De, Kuntal; Sarvepalli, Kavitha; Lemieux, Madeleine E; Lenoir, Jessica J; Moore, Adrian W; McDonald, Jocelyn A; Longworth, Michelle S

    2016-08-01

    The pattern of the Drosophila melanogaster adult wing is heavily influenced by the expression of proteins that dictate cell fate decisions between intervein and vein during development. dSRF (Blistered) expression in specific regions of the larval wing disc promotes intervein cell fate, whereas EGFR activity promotes vein cell fate. Here, we report that the chromatin-organizing protein CAP-D3 acts to dampen dSRF levels at the anterior/posterior boundary in the larval wing disc, promoting differentiation of cells into the anterior crossvein. CAP-D3 represses KNOT expression in cells immediately adjacent to the anterior/posterior boundary, thus blocking KNOT-mediated repression of EGFR activity and preventing cell death. Maintenance of EGFR activity in these cells depresses dSRF levels in the neighboring anterior crossvein progenitor cells, allowing them to differentiate into vein cells. These findings uncover a novel transcriptional regulatory network influencing Drosophila wing vein development, and are the first to identify a Condensin II subunit as an important regulator of EGFR activity and cell fate determination in vivo. © 2016. Published by The Company of Biologists Ltd.

  14. Biochemical identification of new proteins involved in splicing repression at the Drosophila P-element exonic splicing silencer

    PubMed Central

    Horan, Lucas; Yasuhara, Jiro C.; Kohlstaedt, Lori A.; Rio, Donald C.

    2015-01-01

    Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5′ exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5′ exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites. PMID:26545814

  15. Chip, a widely expressed chromosomal protein required for segmentation and activity of a remote wing margin enhancer in Drosophila

    PubMed Central

    Morcillo, Patrick; Rosen, Christina; Baylies, Mary K.; Dorsett, Dale

    1997-01-01

    The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by the Drosophila suppressor of Hairy-wing protein (Suhw) that binds to gypsy retrovirus insertions between enhancers and promoters. Suhw bound to a gypsy insertion in the cut gene also appears to act interchromosomally to antagonize enhancer–promoter interactions on the homologous chromosome when activity of the Chip gene is reduced. This implicates Chip in enhancer–promoter communication. We cloned Chip and find that it encodes a homolog of the recently discovered mouse Nli/Ldb1/Clim-2 and Xenopus Xldb1 proteins that bind nuclear LIM domain proteins. Chip protein interacts with the LIM domains in the Apterous homeodomain protein, and Chip interacts genetically with apterous, showing that these interactions are important for Apterous function in vivo. Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins. Chip is present in all nuclei examined and at numerous sites along the salivary gland polytene chromosomes. Embryos without Chip activity lack segments and show abnormal gap and pair–rule gene expression, although no LIM domain proteins are known to regulate segmentation. We conclude that Chip is a ubiquitous chromosomal factor required for normal expression of diverse genes at many stages of development. We suggest that Chip cooperates with different LIM domain proteins and other factors to structurally support remote enhancer–promoter interactions. PMID:9334334

  16. Molecular Characterization and Evolution of a Gene Family Encoding Both Female- and Male-Specific Reproductive Proteins in Drosophila

    PubMed Central

    Sirot, Laura K.; Findlay, Geoffrey D.; Sitnik, Jessica L.; Frasheri, Dorina; Avila, Frank W.; Wolfner, Mariana F.

    2014-01-01

    Gene duplication is an important mechanism for the evolution of new reproductive proteins. However, in most cases, each resulting paralog continues to function within the same sex. To investigate the possibility that seminal fluid proteins arise through duplicates of female reproductive genes that become “co-opted” by males, we screened female reproductive genes in Drosophila melanogaster for cases of duplication in which one of the resulting paralogs produces a protein in males that is transferred to females during mating. We identified a set of three tandemly duplicated genes that encode secreted serine-type endopeptidase homologs, two of which are expressed primarily in the female reproductive tract (RT), whereas the third is expressed specifically in the male RT and encodes a seminal fluid protein. Evolutionary and gene expression analyses across Drosophila species suggest that this family arose from a single-copy gene that was female-specific; after duplication, one paralog evolved male-specific expression. Functional tests of knockdowns of each gene in D. melanogaster show that one female-expressed gene is essential for full fecundity, and both female-expressed genes contribute singly or in combination to a female’s propensity to remate. In contrast, knockdown of the male-expressed paralog had no significant effect on female fecundity or remating. These data are consistent with a model in which members of this gene family exert effects on females by acting on a common, female-expressed target. After duplication and male co-option of one paralog, the evolution of the interacting proteins could have resulted in differential strengths or effects of each paralog. PMID:24682282

  17. Amyloid-β Peptide Exacerbates the Memory Deficit Caused by Amyloid Precursor Protein Loss-of-Function in Drosophila

    PubMed Central

    Bourdet, Isabelle; Lampin-Saint-Amaux, Aurélie; Preat, Thomas; Goguel, Valérie

    2015-01-01

    The amyloid precursor protein (APP) plays a central role in Alzheimer’s disease (AD). APP can undergo two exclusive proteolytic pathways: cleavage by the α-secretase initiates the non-amyloidogenic pathway while cleavage by the β-secretase initiates the amyloidogenic pathway that leads, after a second cleavage by the γ-secretase, to amyloid-β (Aβ) peptides that can form toxic extracellular deposits, a hallmark of AD. The initial events leading to AD are still unknown. Importantly, aside from Aβ toxicity whose molecular mechanisms remain elusive, several studies have shown that APP plays a positive role in memory, raising the possibility that APP loss-of-function may participate to AD. We previously showed that APPL, the Drosophila APP ortholog, is required for associative memory in young flies. In the present report, we provide the first analysis of the amyloidogenic pathway’s influence on memory in the adult. We show that transient overexpression of the β-secretase in the mushroom bodies, the center for olfactory memory, did not alter memory. In sharp contrast, β-secretase overexpression affected memory when associated with APPL partial loss-of-function. Interestingly, similar results were observed with Drosophila Aβ peptide. Because Aβ overexpression impaired memory only when combined to APPL partial loss-of-function, the data suggest that Aβ affects memory through the APPL pathway. Thus, memory is altered by two connected mechanisms—APPL loss-of-function and amyloid peptide toxicity—revealing in Drosophila a functional interaction between APPL and amyloid peptide. PMID:26274614

  18. Structural and functional analyses of PAS domain interactions of the clock proteins Drosophila PERIOD and mouse PERIOD2.

    PubMed

    Hennig, Sven; Strauss, Holger M; Vanselow, Katja; Yildiz, Ozkan; Schulze, Sabrina; Arens, Julia; Kramer, Achim; Wolf, Eva

    2009-04-28

    PERIOD proteins are central components of the Drosophila and mammalian circadian clocks. The crystal structure of a Drosophila PERIOD (dPER) fragment comprising two PER-ARNT-SIM (PAS) domains (PAS-A and PAS-B) and two additional C-terminal alpha-helices (alphaE and alphaF) has revealed a homodimer mediated by intermolecular interactions of PAS-A with tryptophane 482 in PAS-B and helix alphaF. Here we present the crystal structure of a monomeric PAS domain fragment of dPER lacking the alphaF helix. Moreover, we have solved the crystal structure of a PAS domain fragment of the mouse PERIOD homologue mPER2. The mPER2 structure shows a different dimer interface than dPER, which is stabilized by interactions of the PAS-B beta-sheet surface including tryptophane 419 (equivalent to Trp482dPER). We have validated and quantitatively analysed the homodimer interactions of dPER and mPER2 by site-directed mutagenesis using analytical gel filtration, analytical ultracentrifugation, and co-immunoprecipitation experiments. Furthermore we show, by yeast-two-hybrid experiments, that the PAS-B beta-sheet surface of dPER mediates interactions with TIMELESS (dTIM). Our study reveals quantitative and qualitative differences between the homodimeric PAS domain interactions of dPER and its mammalian homologue mPER2. In addition, we identify the PAS-B beta-sheet surface as a versatile interaction site mediating mPER2 homodimerization in the mammalian system and dPER-dTIM heterodimer formation in the Drosophila system.

  19. High-resolution two-dimensional gel analysis of proteins in wing imaginal discs: A data base of Drosophila

    SciTech Connect

    Santaren, J.F.; Garcia-Bellido, A. )

    1990-08-01

    An improved method of high-resolution two-dimensional gel electrophoresis has been used to study the patterns of protein synthesis in wing imaginal discs of late instar larvae of Drosophila melanogaster. A small number of discs were radiolabeled with a mixture of {sup 14}C-labeled amino acids or with ({sup 35}S)methionine and the pattern of labeled proteins was analyzed. One thousand and twenty-five polypeptides (787 acidic (IEF) and 238 basic (NEPHGE)) from wing discs of several wild-type strains have so far been separated and cataloged. All these polypeptides have been numbered and presented in a reference map for further studies. When comparing patterns of label we have found small quantitative differences in rate of synthesis between individuals of the same strain, not due to sexual differences, and very few quantitative and qualitative differences between groups of individuals of different strains.

  20. Seven Genes of the Enhancer of Split Complex of Drosophila Melanogaster Encode Helix-Loop-Helix Proteins

    PubMed Central

    Knust, E.; Schrons, H.; Grawe, F.; Campos-Ortega, J. A.

    1992-01-01

    Enhancer of split [E(spl)] is one of the neurogenic loci of Drosophila and, as such, is required for normal segregation of neural and epidermal cell progenitors. Genetic observations indicate that the E(spl) locus is in fact a gene complex comprising a cluster of related genes and that other genes of the region are also required for normal early neurogenesis. Three of the genes of the complex were known to encode helix-loop-helix (HLH) proteins and to be transcribed in nearly identical patterns. Here, we show that four other genes in the vicinity also encode HLH proteins and, during neuroblast segregation, three of them are expressed in the same pattern. We show by germ-line transformation that these three genes are also necessary to allow epidermal development of the neuroectodermal cells. PMID:1427040

  1. Tubby domain superfamily protein is required for the formation of the 7S SNARE complex in Drosophila.

    PubMed

    Yoon, Eun Jang; Jeong, Yong Taek; Lee, Ji Eun; Moon, Seok Jun; Kim, Chul Hoon

    2017-01-22

    Tubby domain superfamily protein (TUSP) is a distant member of the Tubby-like protein (TULP) family. Although other TULPs play important roles in sensation, metabolism, and development, the molecular functions of TUSP are completely unknown. Here, we explore the function of TUSP in the Drosophila nervous system where it is expressed in all neurons. Tusp mutant flies exhibit a temperature-sensitive paralysis. This paralysis can be rescued by tissue-specific expression of Tusp in the giant fibers and peripherally synapsing interneurons of the giant fiber system, a well-characterized neuronal circuit that mediates rapid escape behavior in flies. Consistent with this paralytic phenotype, we observed a profound reduction in the assembly of the ternary 7S SNARE complex that is required for neurotransmitter release despite seeing no changes in the expression of each individual SNARE complex component. Together, these data suggest TUSP is a novel regulator of SNARE assembly and, therefore, of neurotransmitter release.

  2. Cloning and mapping of a human gene (TBX2) sharing a highly conserved protein motif with a Drosophila omb gene

    SciTech Connect

    Campbell, C.; Goodrich, K.; Casey, G.; Beatty, B.

    1995-07-20

    We have identified and cloned a human gene (TBX2) that exhibits strong sequence homology within a putative DNA binding domain to the drosophila optomotor-blind (omb) gene and lesser homology to the DNA binding domain of the murine brachyury or T gene. Unlike omb, which is expressed in neural tissue, or T, which is not expressed in adult animals, TBX2 is expressed primarily in adult in kidney, lung, and placenta as multiple transcripts of between {approximately} 2 and 4 kb. At least part of this transcript heterogenity appears to be due to alternative polyadenylation. This is the first reported human member of a new family of highly evolutionarily conserved DNA binding proteins, the Tbx or T-box proteins. The human gene has been mapped by somatic cell hybrid mapping and chromosomal in situ hybridization to chromosome 17q23, a region frequently altered in ovarian carcinomas. 19 refs., 6 figs.

  3. The Drosophila SOX-domain protein Dichaete is required for the development of the central nervous system midline.

    PubMed

    Soriano, N S; Russell, S

    1998-10-01

    SOX-domain proteins are a class of developmentally important transcriptional regulators related to the mammalian testis determining factor SRY. In common with other SOX-domain genes, the Drosophila Dichaete gene has a dynamic expression profile in the developing central nervous system, including cells of the ventral midline. We find defects in the differentiation of midline glia and concomitant axonal defects in Dichaete mutants that are rescued by driving Dichaete expression in the midline. Since Dichaete is required for the correct specification or differentiation of midline glia, we have used the ventral midline as a model system to study SOX gene function in vivo and demonstrate a genetic interaction between Dichaete and the POU domain gene ventral veinless. In mammals, a protein related to Dichaete, SOX2, also interacts with POU transcription factors. The midline phenotypes of Dichaete mutations are rescued by expression of mouse SOX2. Our data suggest that SOX gene structure, function and interactions have been conserved during evolution.

  4. Microsomal triacylglycerol transfer protein (MTP) is required to expand tracheal lumen in Drosophila in a cell-autonomous manner.

    PubMed

    Baer, Magdalena M; Palm, Wilhelm; Eaton, Suzanne; Leptin, Maria; Affolter, Markus

    2012-12-15

    The Drosophila tracheal system is a useful model for dissecting the molecular mechanisms controlling the assembly and expansion of tubular organs. We have identified microsomal triacylglycerol transfer protein (MTP) as a new player involved in the lumen expansion in unicellular tubes. MTP is an endoplasmic reticulum resident protein that can transfer triglycerides and phospholipids between membranes in vitro. MTP lipid transfer activity is crucial for the assembly and secretion of apoB family lipoproteins, which are carriers of lipids between different tissues. Here we describe an unexpected role of MTP in tracheal development, which we postulate to be independent of its known function in lipoprotein secretion. We propose that, in tracheal cells, MTP is involved in regulation of de novo apical membrane delivery to the existing lumen and thus promotes proper expansion of the larval tracheal system.

  5. The insulator protein Suppressor of Hairy wing is required for proper ring canal development during oogenesis in Drosophila.

    PubMed

    Hsu, Shih-Jui; Plata, Maria P; Ernest, Ben; Asgarifar, Saghi; Labrador, Mariano

    2015-07-01

    Chromatin insulators orchestrate gene transcription during embryo development and cell differentiation by stabilizing interactions between distant genomic sites. Mutations in genes encoding insulator proteins are generally lethal, making in vivo functional analyses of insulator proteins difficult. In Drosophila, however, mutations in the gene encoding the Suppressor of Hairy wing insulator protein [Su(Hw)] are viable and female sterile, providing an opportunity to study insulator function during oocyte development. Whereas previous reports suggest that the function of Su(Hw) in oogenesis is independent of its insulator activity, many aspects of the role of Su(Hw) in Drosophila oogenesis remain unexplored. Here we show that mutations in su(Hw) result in smaller ring canal lumens and smaller outer ring diameters, which likely obstruct molecular and vesicle passage from nurse cells to the oocyte. Fluorescence microscopy reveals that lack of Su(Hw) leads to excess accumulation of Kelch (Kel) and Filament-actin (F-actin) proteins in the ring canal structures of developing egg chambers. Furthermore, we found that misexpression of the Src oncogene at 64B (Src64B) may cause ring canal development defects as microarray analysis and real-time RT-PCR revealed there is a three fold decrease in Src64B expression in su(Hw) mutant ovaries. Restoration of Src64B expression in su(Hw) mutant female germ cells rescued the ring phenotype but did not restore fertility. We conclude that loss of su(Hw) affects expression of many oogenesis related genes and down-regulates Src64B, resulting in ring canal defects potentially contributing to obstruction of molecular flow and an eventual failure of egg chamber organization.

  6. Drosophila KASH-domain protein Klarsicht regulates microtubule stability and integrin receptor localization during collective cell migration.

    PubMed

    Myat, M M; Rashmi, R N; Manna, D; Xu, N; Patel, U; Galiano, M; Zielinski, K; Lam, A; Welte, M A

    2015-11-01

    During collective migration of the Drosophila embryonic salivary gland, cells rearrange to form a tube of a distinct shape and size. Here, we report a novel role for the Drosophila Klarsicht-Anc-Syne Homology (KASH) domain protein Klarsicht (Klar) in the regulation of microtubule (MT) stability and integrin receptor localization during salivary gland migration. In wild-type salivary glands, MTs became progressively stabilized as gland migration progressed. In embryos specifically lacking the KASH domain containing isoforms of Klar, salivary gland cells failed to rearrange and migrate, and these defects were accompanied by decreased MT stability and altered integrin receptor localization. In muscles and photoreceptors, KASH isoforms of Klar work together with Klaroid (Koi), a SUN domain protein, to position nuclei; however, loss of Koi had no effect on salivary gland migration, suggesting that Klar controls gland migration through novel interactors. The disrupted cell rearrangement and integrin localization observed in klar mutants could be mimicked by overexpressing Spastin (Spas), a MT severing protein, in otherwise wild-type salivary glands. In turn, promoting MT stability by reducing spas gene dosage in klar mutant embryos rescued the integrin localization, cell rearrangement and gland migration defects. Klar genetically interacts with the Rho1 small GTPase in salivary gland migration and is required for the subcellular localization of Rho1. We also show that Klar binds tubulin directly in vitro. Our studies provide the first evidence that a KASH-domain protein regulates the MT cytoskeleton and integrin localization during collective cell migration. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Drosophila melanogaster NPC2 Proteins Bind Bacterial Cell Wall Components and May Function in Immune Signal Pathways

    PubMed Central

    Shi, Xiu-Zhen; Zhong, Xue; Yu, Xiao-Qiang

    2012-01-01

    ML (MD-2 (myeloid differentiation factor 2)-related Lipid-recognition) is a conserved domain identified in MD-2, MD-1, NPC2 (Niemann-Pick disease type C2), and mite major allergen protein from animals, plants, and fungi. Vertebrate members of the ML family proteins, such as NPC2 and MD-2, play important roles in lipid metabolism and immune signaling pathway. MD-2 is an essential co-receptor in the lipopolysaccharide (LPS)/Toll-like receptor 4 (TLR4) signaling pathway. Insects contain multiple ML genes, arbitrarily named md-2- or npc2-like genes. However, whether insect ML genes have functions similar to vertebrate md-2 is unknown. In Drosophila melanogaster, there are eight npc2 genes (npc2a-h), and they can be further divided into three subgroups based on the numbers of cysteine residues (6, 7 and 8 Cys) in the mature proteins. The purpose of this study is to investigate whether any Drosophila npc2 genes may have functions in immune signaling pathways. We chose npc2a, npc2e and npc2h genes representing the three subgroups for this study. We showed that recombinant NPC2a, NPC2e and NPC2h not only bound to LPS and lipid A, but also bound to peptidoglycan (PG) and lipoteichoic acid (LTA), a property that has not been reported previously for vertebrate NPC2 or MD-2. More importantly, we showed that over-expression of NPC2a and NPC2e activated diptericin promoter reporter in S2 cells stimulated by PG, suggesting that NPC2e and NPC2a may play a role in the immune deficiency (Imd) pathway. This is the first in vitro study about NPC2 proteins in innate immunity of D. melanogaster. PMID:22580186

  8. The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila

    PubMed Central

    Duncan, Jason E.; Lytle, Nikki K.; Zuniga, Alfredo; Goldstein, Lawrence S. B.

    2013-01-01

    Axonal transport, a form of long-distance, bi-directional intracellular transport that occurs between the cell body and synaptic terminal, is critical in maintaining the function and viability of neurons. We have identified a requirement for the stathmin (stai) gene in the maintenance of axonal microtubules and regulation of axonal transport in Drosophila. The stai gene encodes a cytosolic phosphoprotein that regulates microtubule dynamics by partitioning tubulin dimers between pools of soluble tubulin and polymerized microtubules, and by directly binding to microtubules and promoting depolymerization. Analysis of stai function in Drosophila, which has a single stai gene, circumvents potential complications with studies performed in vertebrate systems in which mutant phenotypes may be compensated by genetic redundancy of other members of the stai gene family. This has allowed us to identify an essential function for stai in the maintenance of the integrity of axonal microtubules. In addition to the severe disruption in the abundance and architecture of microtubules in the axons of stai mutant Drosophila, we also observe additional neurological phenotypes associated with loss of stai function including a posterior paralysis and tail-flip phenotype in third instar larvae, aberrant accumulation of transported membranous organelles in stai deficient axons, a progressive bang-sensitive response to mechanical stimulation reminiscent of the class of Drosophila mutants used to model human epileptic seizures, and a reduced adult lifespan. Reductions in the levels of Kinesin-1, the primary anterograde motor in axonal transport, enhance these phenotypes. Collectively, our results indicate that stai has an important role in neuronal function, likely through the maintenance of microtubule integrity in the axons of nerves of the peripheral nervous system necessary to support and sustain long-distance axonal transport. PMID:23840848

  9. RDGBα, a PtdIns-PtdOH transfer protein, regulates G-protein-coupled PtdIns(4,5)P2 signalling during Drosophila phototransduction

    PubMed Central

    Yadav, Shweta; Garner, Kathryn; Georgiev, Plamen; Li, Michelle; Gomez-Espinosa, Evelyn; Panda, Aniruddha; Mathre, Swarna; Okkenhaug, Hanneke; Cockcroft, Shamshad; Raghu, Padinjat

    2015-01-01

    ABSTRACT Many membrane receptors activate phospholipase C (PLC) during signalling, triggering changes in the levels of several plasma membrane lipids including phosphatidylinositol (PtdIns), phosphatidic acid (PtdOH) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. It is widely believed that exchange of lipids between the plasma membrane and endoplasmic reticulum (ER) is required to restore lipid homeostasis during PLC signalling, yet the mechanism remains unresolved. RDGBα (hereafter RDGB) is a multi-domain protein with a PtdIns transfer protein (PITP) domain (RDGB-PITPd). We find that, in vitro, the RDGB-PITPd binds and transfers both PtdOH and PtdIns. In Drosophila photoreceptors, which experience high rates of PLC activity, RDGB function is essential for phototransduction. We show that binding of PtdIns to RDGB-PITPd is essential for normal phototransduction; however, this property is insufficient to explain the in vivo function because another Drosophila PITP (encoded by vib) that also binds PtdIns cannot rescue the phenotypes of RDGB deletion. In RDGB mutants, PtdIns(4,5)P2 resynthesis at the plasma membrane following PLC activation is delayed and PtdOH levels elevate. Thus RDGB couples the turnover of both PtdIns and PtdOH, key lipid intermediates during G-protein-coupled PtdIns(4,5)P2 turnover. PMID:26203165

  10. Localization of the Drosophila checkpoint control protein Bub3 to the kinetochore requires Bub1 but not Zw10 or Rod.

    PubMed

    Basu, J; Logarinho, E; Herrmann, S; Bousbaa, H; Li, Z; Chan, G K; Yen, T J; Sunkel, C E; Goldberg, M L

    1998-12-01

    We report here the isolation and molecular characterization of the Drosophila homolog of the mitotic checkpoint control protein Bub3. The Drosophila Bub3 protein is associated with the centromere/kinetochore of chromosomes in larval neuroblasts whose spindle assembly checkpoints have been activated by incubation with the microtubule-depolymerizing agent colchicine. Drosophila Bub3 is also found at the kinetochore regions in mitotic larval neuroblasts and in meiotic primary and secondary spermatocytes, with the strong signal seen during prophase and prometaphase becoming increasingly weaker after the chromosomes have aligned at the metaphase plate. We further show that the localization of Bub3 to the kinetochore is disrupted by mutations in the gene encoding the Drosophila homolog of the spindle assembly checkpoint protein Bub1. Combined with recent findings showing that the kinetochore localization of Bub1 conversely depends upon Bub3, these results support the hypothesis that the spindle assembly checkpoint proteins exist as a multiprotein complex recruited as a unit to the kinetochore. In contrast, we demonstrate that the kinetochore constituents Zw10 and Rod are not needed for the binding of Bub3 to the kinetochore. This suggests that the kinetochore is assembled in at least two relatively independent pathways.

  11. β-Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP-activated protein kinase-dependent increase in autophagy.

    PubMed

    Yang, Si; Long, Li-Hong; Li, Di; Zhang, Jian-Kang; Jin, Shan; Wang, Fang; Chen, Jian-Guo

    2015-12-01

    Previous studies have demonstrated that AMP-activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc-51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β-guanidinopropionic acid (β-GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β-GPA and aging remains elusive. In this study, we hypothesized that feeding β-GPA to adult Drosophila produces the lifespan extension via activation of AMPK-dependent autophagy. It was found that dietary administration of β-GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule-associated protein 1A/1B-light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β-GPA treatment, indicating that autophagic activity plays a role in the effect of β-GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β-GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β-GPA treatment significantly elevated the expression of phospho-T172-AMPK levels, while inhibition of AMPK by either AMPK-RNAi or compound C significantly attenuated the expression of autophagy-related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β-GPA can induce an extension of the lifespan of Drosophila via AMPK-Atg1-autophagy signaling pathway. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.