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Sample records for drosophila deoxyribonucleoside kinase

  1. Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

    SciTech Connect

    Knecht, Wolfgang; Mikkelsen, Nils Egil; Clausen, Anders Ranegaard; Willer, Mette; Gojkovic, Zoran

    2009-05-01

    Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2 A resolution structure of Dm-dNK in complex with gemcitabine shows that the residues Tyr70 and Arg105 play a crucial role in the firm positioning of gemcitabine by extra interactions made by the fluoride atoms. This explains why gemcitabine is a good substrate for Dm-dNK.

  2. In vivo selection of active deoxyribonucleoside kinase by a mutagenic nucleoside analog.

    PubMed

    Kamiya, Hiroyuki; Ito, Mana; Nishi, Kosuke; Harashima, Hideyoshi

    2016-06-20

    A novel in vivo selection method for active deoxyribonucleoside kinase proteins is described here. A pool of randomly mutated genes for deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) was prepared and inserted into an expression vector. Enzymatically active mutants were selected by repeated cycles, including (i) introduction into Escherichia coli, (ii) treatment of the E. coli pool with a mutagenic deoxyribonucleoside (2-hydroxy-dA), and (iii) selection of antibiotic-resistant colonies resulting from mutations by phosphorylated 2-hydroxy-dA and the subsequent isolation of the plasmid DNAs. The ratio of the resistant colonies increased by two orders of magnitude from the first cycle to the fifth cycle, and then reached a plateau. Fifteen Dm-dNK mutants selected after the seventh and eighth evolution cycles were actually active in vivo. Moreover, one of the mutant proteins was as active as the wild-type protein in vitro. These results indicate that this novel in vivo evolution method was useful and that similar strategies would be applicable to other deoxyribonucleoside kinases. In addition, the distribution of mutated amino acids suggests important residues/regions in the Dm-dNK protein. PMID:27131895

  3. Receptor Tyrosine Kinases in Drosophila Development

    PubMed Central

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

    Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

  4. Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B.

    PubMed Central

    Lizcano, Jose M; Alrubaie, Saif; Kieloch, Agnieszka; Deak, Maria; Leevers, Sally J; Alessi, Dario R

    2003-01-01

    An important mechanism by which insulin regulates cell growth and protein synthesis is through activation of the p70 ribosomal S6 protein kinase (S6K). In mammalian cells, insulin-induced PI3K (phosphoinositide 3-kinase) activation, generates the lipid second messenger PtdIns(3,4,5) P (3), which is thought to play a key role in triggering the activation of S6K. Although the major components of the insulin-signalling pathway are conserved in Drosophila, recent studies suggested that S6K activation does not require PI3K in this system. To investigate further the role of dPI3K (Drosophila PI3K) in dS6K (Drosophila S6K) activation, we examined the effect of two structurally distinct PI3K inhibitors on insulin-induced dS6K activation in Kc167 and S2 Drosophila cell lines. We found that both inhibitors prevented insulin-stimulated phosphorylation and activation of dS6K. To investigate further the role of the dPI3K pathway in regulating dS6K activation, we also used dsRNAi (double-stranded RNA-mediated interference) to decrease expression of dPI3K and the PtdIns(3,4,5) P (3) phosphatase dPTEN ( Drosophila phosphatase and tensin homologue deleted on chromosome 10) in Kc167 and S2 cells. Knock-down of dPI3K prevented dS6K activation, whereas knock-down of dPTEN, which would be expected to increase PtdIns(3,4,5) P (3) levels, stimulated dS6K activity. Moreover, when the expression of the dPI3K target, dPKB (Drosophila protein kinase B), was decreased to undetectable levels, we found that insulin could no longer trigger dS6K activation. This observation provides the first direct demonstration that dPKB is required for insulin-stimulated dS6K activation. We also present evidence that the amino-acid-induced activation of dS6K in the absence of insulin, thought to be mediated by dTOR (Drosophila target of rapamycin), which is unaffected by the inhibition of dPI3K by wortmannin. The results of the present study support the view that, in Drosophila cells, dPI3K and dPKB, as well d

  5. Characterization of a Drosophila Ortholog of the Cdc7 Kinase

    PubMed Central

    Stephenson, Robert; Hosler, Marcus R.; Gavande, Navnath S.; Ghosh, Arun K.; Weake, Vikki M.

    2015-01-01

    Cdc7 is a serine-threonine kinase that phosphorylates components of the pre-replication complex during DNA replication initiation. Cdc7 is highly conserved, and Cdc7 orthologs have been characterized in organisms ranging from yeast to humans. Cdc7 is activated specifically during late G1/S phase by binding to its regulatory subunit, Dbf4. Drosophila melanogaster contains a Dbf4 ortholog, Chiffon, which is essential for chorion amplification in Drosophila egg chambers. However, no Drosophila ortholog of Cdc7 has yet been characterized. Here, we report the functional and biochemical characterization of a Drosophila ortholog of Cdc7. Co-expression of Drosophila Cdc7 and Chiffon is able to complement a growth defect in yeast containing a temperature-sensitive Cdc7 mutant. Cdc7 and Chiffon physically interact and can be co-purified from insect cells. Cdc7 phosphorylates the known Cdc7 substrates Mcm2 and histone H3 in vitro, and Cdc7 kinase activity is stimulated by Chiffon and inhibited by the Cdc7-specific inhibitor XL413. Drosophila egg chamber follicle cells deficient for Cdc7 have a defect in two types of DNA replication, endoreplication and chorion gene amplification. However, follicle cells deficient for Chiffon have a defect in chorion gene amplification but still undergo endocycling. Our results show that Cdc7 interacts with Chiffon to form a functional Dbf4-dependent kinase complex and that Cdc7 is necessary for DNA replication in Drosophila egg chamber follicle cells. Additionally, we show that Chiffon is a member of an expanding subset of DNA replication initiation factors that are not strictly required for endoreplication in Drosophila. PMID:25451925

  6. Kinase active Misshapen regulates Notch signaling in Drosophila melanogaster.

    PubMed

    Mishra, Abhinava K; Sachan, Nalani; Mutsuddi, Mousumi; Mukherjee, Ashim

    2015-11-15

    Notch signaling pathway represents a principal cellular communication system that plays a pivotal role during development of metazoans. Drosophila misshapen (msn) encodes a protein kinase, which is related to the budding yeast Ste20p (sterile 20 protein) kinase. In a genetic screen, using candidate gene approach to identify novel kinases involved in Notch signaling, we identified msn as a novel regulator of Notch signaling. Data presented here suggest that overexpression of kinase active form of Msn exhibits phenotypes similar to Notch loss-of-function condition and msn genetically interacts with components of Notch signaling pathway. Kinase active form of Msn associates with Notch receptor and regulate its signaling activity. We further show that kinase active Misshapen leads to accumulation of membrane-tethered form of Notch. Moreover, activated Msn also depletes Armadillo and DE-Cadherin from adherens junctions. Thus, this study provides a yet unknown mode of regulation of Notch signaling by Misshapen. PMID:26431585

  7. DFak56 is a novel Drosophila melanogaster focal adhesion kinase.

    PubMed

    Palmer, R H; Fessler, L I; Edeen, P T; Madigan, S J; McKeown, M; Hunter, T

    1999-12-10

    The mammalian focal adhesion kinase (FAK) family of nonreceptor protein-tyrosine kinases have been implicated in controlling a multitude of cellular responses to the engagement of cell surface integrins and G protein-coupled receptors. We describe here a Drosophila melanogaster FAK homologue, DFak56, which maps to band 56D on the right arm of the second chromosome. Full-length DFak56 cDNA encodes a phosphoprotein of 140 kDa, which shares strong sequence similarity not only with mammalian p125(FAK) but also with the more recently described mammalian Pyk2 (also known as CAKbeta, RAFTK, FAK2, and CADTK) FAK family member. DFak56 has intrinsic tyrosine kinase activity and is phosphorylated on tyrosine in vivo. As is the case for FAK, tyrosine phosphorylation of DFak56 is increased upon plating Drosophila embryo cells on extracellular matrix proteins. In situ hybridization and immunofluorescence staining analysis showed that DFak56 is ubiquitously expressed with particularly high levels within the developing central nervous system. We utilized the UAS-GAL4 expression system to express DFak56 and analyze its function in vivo. Overexpression of DFak56 in the wing imaginal disc results in wing blistering in adults, a phenotype also observed with both position-specific integrin loss of function and position-specific integrin overexpression. Our results imply a role for DFak56 in adhesion-dependent signaling pathways in vivo during D. melanogaster development. PMID:10585440

  8. Ribosomal S6 Kinase Cooperates with Casein Kinase 2 to Modulate the Drosophila Circadian Molecular Oscillator

    PubMed Central

    Akten, Bikem; Tangredi, Michelle M.; Jauch, Eike; Roberts, Mary A.; Ng, Fanny; Raabe, Thomas; Jackson, F. Rob

    2009-01-01

    There is a universal requirement for post-translational regulatory mechanisms in circadian clock systems. Previous work in Drosophila has identified several kinases, phosphatases and an E3 ligase that are critical for determining the nuclear translocation and/or stability of clock proteins. The present study evaluated the function of p90 ribosomal S6 kinase (RSK) in the Drosophila circadian system. In mammals, RSK1 is a light- and clock-regulated kinase known to be activated by the MAPK pathway, but there is no direct evidence that it functions as a component of the circadian system. Here, we show that Drosophila S6KII RNA displays rhythms in abundance, indicative of circadian control. Importantly, an S6KII null mutant exhibits a short-period circadian phenotype that can be rescued by expression of the wild-type gene in clock neurons, indicating a role for S6KII in the molecular oscillator. Peak PER clock protein expression is elevated in the mutant, indicative of enhanced stability, whereas per mRNA level is decreased, consistent with enhanced feedback repression. Gene reporter assays show that decreased S6KII is associated with increased PER repression. Surprisingly, we demonstrate a physical interaction between S6KII and the Casein Kinase 2 regulatory subunit (CK2β), suggesting a functional relationship between the two kinases. In support of such a relationship, there are genetic interactions between S6KII and CK2 mutations, in vivo, which indicate that CK2 activity is required for S6KII action. We propose that the two kinases cooperate within clock neurons to fine-tune circadian period, improving the precision of the clock mechanism. PMID:19144847

  9. p38 MAP kinase regulates circadian rhythms in Drosophila.

    PubMed

    Vrailas-Mortimer, Alysia D; Ryan, Sarah M; Avey, Matthew J; Mortimer, Nathan T; Dowse, Harold; Sanyal, Subhabrata

    2014-12-01

    The large repertoire of circadian rhythms in diverse organisms depends on oscillating central clock genes, input pathways for entrainment, and output pathways for controlling rhythmic behaviors. Stress-activated p38 MAP Kinases (p38K), although sparsely investigated in this context, show circadian rhythmicity in mammalian brains and are considered part of the circadian output machinery in Neurospora. We find that Drosophila p38Kb is expressed in clock neurons, and mutants in p38Kb either are arrhythmic or have a longer free-running periodicity, especially as they age. Paradoxically, similar phenotypes are observed through either transgenic inhibition or activation of p38Kb in clock neurons, suggesting a requirement for optimal p38Kb function for normal free-running circadian rhythms. We also find that p38Kb genetically interacts with multiple downstream targets to regulate circadian locomotor rhythms. More specifically, p38Kb interacts with the period gene to regulate period length and the strength of rhythmicity. In addition, we show that p38Kb suppresses the arrhythmic behavior associated with inhibition of a second p38Kb target, the transcription factor Mef2. Finally, we find that manipulating p38K signaling in free-running conditions alters the expression of another downstream target, MNK/Lk6, which has been shown to cycle with the clock and to play a role in regulating circadian rhythms. These data suggest that p38Kb may affect circadian locomotor rhythms through the regulation of multiple downstream pathways. PMID:25403440

  10. Drosophila Wee1 kinase rescues fission yeast from mitotic catastrophe and phosphorylates Drosophila Cdc2 in vitro.

    PubMed Central

    Campbell, S D; Sprenger, F; Edgar, B A; O'Farrell, P H

    1995-01-01

    Cdc2 kinase activity is required for triggering entry into mitosis in all known eukaryotes. Elaborate mechanisms have evolved for regulating Cdc2 activity so that mitosis occurs in a timely manner, when preparations for its execution are complete. In Schizosaccharomyces pombe, Wee1 and a related Mik1 kinase are Cdc2-inhibitory kinases that are required for preventing premature activation of the mitotic program. To identify Cdc2-inhibitory kinases in Drosophila, we screened for cDNA clones that rescue S. pombe wee1- mik1- mutants from lethal mitotic catastrophe. One of the genes identified in this screen, Drosophila wee1 (Dwee1), encodes a new Wee1 homologue. Dwee1 kinase is closely related to human and Xenopus Wee1 homologues, and can inhibit Cdc2 activity by phosphorylating a critical tyrosine residue. Dwee1 mRNA is maternally provided to embryos, and is zygotically expressed during the postblastoderm divisions of embryogenesis. Expression remains high in the proliferating cells of the central nervous system well after cells in the rest of the embryo have ceased dividing. The loss of zygotically expressed Dwee1 does not lead to mitotic catastrophe during postblastoderm cycles 14 to 16. This result may indicate that maternally provided Dwee1 is sufficient for regulating Cdc2 during embryogenesis, or it may reflect the presence of a redundant Cdc2 inhibitory kinase, as in fission yeast. Images PMID:8573790

  11. Transgene Expression of Drosophila melanogaster Nucleoside Kinase Reverses Mitochondrial Thymidine Kinase 2 Deficiency*♦

    PubMed Central

    Krishnan, Shuba; Zhou, Xiaoshan; Paredes, João A.; Kuiper, Raoul V.; Curbo, Sophie; Karlsson, Anna

    2013-01-01

    A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK+/− transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK+/−TK2−/− mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK+/−TK2−/− mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency. PMID:23288848

  12. Fray, a Drosophila serine/threonine kinase homologous to mammalian PASK, is required for axonal ensheathment

    NASA Technical Reports Server (NTRS)

    Leiserson, W. M.; Harkins, E. W.; Keshishian, H.

    2000-01-01

    Fray is a serine/threonine kinase expressed by the peripheral glia of Drosophila, whose function is required for normal axonal ensheathment. Null fray mutants die early in larval development and have nerves with severe swelling and axonal defasciculation. The phenotype is associated with a failure of the ensheathing glia to correctly wrap peripheral axons. When the fray cDNA is expressed in the ensheathing glia of fray mutants, normal nerve morphology is restored. Fray belongs to a novel family of Ser/Thr kinases, the PF kinases, whose closest relatives are the PAK kinases. Rescue of the Drosophila mutant phenotype with PASK, the rat homolog of Fray, demonstrates a functional homology among these proteins and suggests that the Fray signaling pathway is widely conserved.

  13. Pantethine rescues a Drosophila model for pantothenate kinase-associated neurodegeneration.

    PubMed

    Rana, Anil; Seinen, Erwin; Siudeja, Katarzyna; Muntendam, Remco; Srinivasan, Balaji; van der Want, Johannes J; Hayflick, Susan; Reijngoud, Dirk-Jan; Kayser, Oliver; Sibon, Ody C M

    2010-04-13

    Pantothenate kinase-associated neurodegeneration (PKAN), a progressive neurodegenerative disorder, is associated with impairment of pantothenate kinase function. Pantothenate kinase is the first enzyme required for de novo synthesis of CoA, an essential metabolic cofactor. The pathophysiology of PKAN is not understood, and there is no cure to halt or reverse the symptoms of this devastating disease. Recently, we and others presented a PKAN Drosophila model, and we demonstrated that impaired function of pantothenate kinase induces a neurodegenerative phenotype and a reduced lifespan. We have explored this Drosophila model further and have demonstrated that impairment of pantothenate kinase is associated with decreased levels of CoA, mitochondrial dysfunction, and increased protein oxidation. Furthermore, we searched for compounds that can rescue pertinent phenotypes of the Drosophila PKAN model and identified pantethine. Pantethine feeding restores CoA levels, improves mitochondrial function, rescues brain degeneration, enhances locomotor abilities, and increases lifespan. We show evidence for the presence of a de novo CoA biosynthesis pathway in which pantethine is used as a precursor compound. Importantly, this pathway is effective in the presence of disrupted pantothenate kinase function. Our data suggest that pantethine may serve as a starting point to develop a possible treatment for PKAN. PMID:20351285

  14. DNA sequence, structure, and tyrosine kinase activity of the Drosophila melanogaster abelson proto-oncogene homolog

    SciTech Connect

    Henkemeyer, M.J.; Bennett, R.L.; Gertler, F.B.; Hoffmann, F.M.

    1988-02-01

    The authors report their molecular characterization of the Drosophila melanogaster Abelson gene (abl), a gene in which recessive loss-of-function mutations result in lethality at the pupal stage of development. This essential gene consists of 10 exons extending over 26 kilobase pairs of genomic DNA. The DNA sequence encodes a protein of 1,520 amino acids with strong sequence similarity to the human c-abl proto-oncogene beginning in the type 1b 5' exon and extending through the region essential for tyrosine kinase activity. When the tyrosine kinase homologous region was expressed in Escherichia coli, phosphorylation of proteins on tyrosine residues was observed with an antiphosphotyrosine antibody. These results show that the abl gene is highly conserved through evolution and encodes a functional tyrosine protein kinase required for Drosophila development.

  15. Drosophila ATM and ATR checkpoint kinases control partially redundant pathways for telomere maintenance

    PubMed Central

    Bi, Xiaolin; Srikanta, Deepa; Fanti, Laura; Pimpinelli, Sergio; Badugu, RamaKrishna; Kellum, Rebecca; Rong, Yikang S.

    2005-01-01

    In higher eukaryotes, the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) checkpoint kinases play distinct, but partially overlapping, roles in DNA damage response. Yet their interrelated function has not been defined for telomere maintenance. We discover in Drosophila that the two proteins control partially redundant pathways for telomere protection: the loss of ATM leads to the fusion of some telomeres, whereas the loss of both ATM and ATR renders all telomeres susceptible to fusion. The ATM-controlled pathway includes the Mre11 and Nijmegen breakage syndrome complex but not the Chk2 kinase, whereas the ATR-regulated pathway includes its partner ATR-interacting protein but not the Chk1 kinase. This finding suggests that ATM and ATR regulate different molecular events at the telomeres compared with the sites of DNA damage. This compensatory relationship between ATM and ATR is remarkably similar to that observed in yeast despite the fact that the biochemistry of telomere elongation is completely different in the two model systems. We provide evidence suggesting that both the loading of telomere capping proteins and normal telomeric silencing requires ATM and ATR in Drosophila and propose that ATM and ATR protect telomere integrity by safeguarding chromatin architecture that favors the loading of telomere-elongating, capping, and silencing proteins. PMID:16203987

  16. Drosophila translational elongation factor-1gamma is modified in response to DOA kinase activity and is essential for cellular viability.

    PubMed

    Fan, Yujie; Schlierf, Michael; Gaspar, Ana Cuervo; Dreux, Catherine; Kpebe, Arlette; Chaney, Linda; Mathieu, Aurelie; Hitte, Christophe; Grémy, Olivier; Sarot, Emeline; Horn, Mark; Zhao, Yunlong; Kinzy, Terri Goss; Rabinow, Leonard

    2010-01-01

    Drosophila translational elongation factor-1gamma (EF1gamma) interacts in the yeast two-hybrid system with DOA, the LAMMER protein kinase of Drosophila. Analysis of mutant EF1gamma alleles reveals that the locus encodes a structurally conserved protein essential for both organismal and cellular survival. Although no genetic interactions were detected in combinations with mutations in EF1alpha, an EF1gamma allele enhanced mutant phenotypes of Doa alleles. A predicted LAMMER kinase phosphorylation site conserved near the C terminus of all EF1gamma orthologs is a phosphorylation site in vitro for both Drosophila DOA and tobacco PK12 LAMMER kinases. EF1gamma protein derived from Doa mutant flies migrates with altered mobility on SDS gels, consistent with it being an in vivo substrate of DOA kinase. However, the aberrant mobility appears to be due to a secondary protein modification, since the mobility of EF1gamma protein obtained from wild-type Drosophila is unaltered following treatment with several nonspecific phosphatases. Expression of a construct expressing a serine-to-alanine substitution in the LAMMER kinase phosphorylation site into the fly germline rescued null EF1gamma alleles but at reduced efficiency compared to a wild-type construct. Our data suggest that EF1gamma functions in vital cellular processes in addition to translational elongation and is a LAMMER kinase substrate in vivo. PMID:19841092

  17. Aurora Kinases Phosphorylate Lgl to Induce Mitotic Spindle Orientation in Drosophila Epithelia

    PubMed Central

    Bell, Graham P.; Fletcher, Georgina C.; Brain, Ruth; Thompson, Barry J.

    2015-01-01

    Summary The Lethal giant larvae (Lgl) protein was discovered in Drosophila as a tumor suppressor in both neural stem cells (neuroblasts) and epithelia. In neuroblasts, Lgl relocalizes to the cytoplasm at mitosis, an event attributed to phosphorylation by mitotically activated aPKC kinase and thought to promote asymmetric cell division. Here we show that Lgl also relocalizes to the cytoplasm at mitosis in epithelial cells, which divide symmetrically. The Aurora A and B kinases directly phosphorylate Lgl to promote its mitotic relocalization, whereas aPKC kinase activity is required only for polarization of Lgl. A form of Lgl that is a substrate for aPKC, but not Aurora kinases, can restore cell polarity in lgl mutants but reveals defects in mitotic spindle orientation in epithelia. We propose that removal of Lgl from the plasma membrane at mitosis allows Pins/LGN to bind Dlg and thus orient the spindle in the plane of the epithelium. Our findings suggest a revised model for Lgl regulation and function in both symmetric and asymmetric cell divisions. PMID:25484300

  18. Cyclin E-dependent protein kinase activity regulates niche retention of Drosophila ovarian follicle stem cells

    PubMed Central

    Wang, Zhu A.; Kalderon, Daniel

    2009-01-01

    Whether stem cells have unique cell cycle machineries and how they integrate with niche interactions remains largely unknown. We identified a hypomorphic cyclin E allele WX that strongly impairs the maintenance of follicle stem cells (FSCs) in the Drosophila ovary but does not reduce follicle cell proliferation or germline stem cell maintenance. CycEWX protein can still bind to the cyclin-dependent kinase catalytic subunit Cdk2, but forms complexes with reduced protein kinase activity measured in vitro. By creating additional CycE variants with different degrees of kinase dysfunction and expressing these and CycEWX at different levels, we found that higher CycE-Cdk2 kinase activity is required for FSC maintenance than to support follicle cell proliferation. Surprisingly, cycEWX FSCs were lost from their niches rather than arresting proliferation. Furthermore, FSC function was substantially restored by expressing either excess DE-cadherin or excess E2F1/DP, the transcription factor normally activated by CycE-Cdk2 phosphorylation of retinoblastoma proteins. These results suggest that FSC maintenance through niche adhesion is regulated by inputs that normally control S phase entry, possibly as a quality control mechanism to ensure adequate stem cell proliferation. We speculate that a positive connection between central regulators of the cell cycle and niche retention may be a common feature of highly proliferative stem cells. PMID:19966222

  19. Binding partners of the kinase domains in Drosophila obscurin and their effect on the structure of the flight muscle

    PubMed Central

    Katzemich, Anja; West, Ryan J. H.; Fukuzawa, Atsushi; Sweeney, Sean T.; Gautel, Mathias; Sparrow, John; Bullard, Belinda

    2015-01-01

    ABSTRACT Drosophila obscurin (Unc-89) is a titin-like protein in the M-line of the muscle sarcomere. Obscurin has two kinase domains near the C-terminus, both of which are predicted to be inactive. We have identified proteins binding to the kinase domains. Kinase domain 1 bound Bällchen (Ball, an active kinase), and both kinase domains 1 and 2 bound MASK (a 400-kDa protein with ankyrin repeats). Ball was present in the Z-disc and M-line of the indirect flight muscle (IFM) and was diffusely distributed in the sarcomere. MASK was present in both the M-line and the Z-disc. Reducing expression of Ball or MASK by siRNA resulted in abnormalities in the IFM, including missing M-lines and multiple Z-discs. Obscurin was still present, suggesting that the kinase domains act as a scaffold binding Ball and MASK. Unlike obscurin in vertebrate skeletal muscle, Drosophila obscurin is necessary for the correct assembly of the IFM sarcomere. We show that Ball and MASK act downstream of obscurin, and both are needed for development of a well defined M-line and Z-disc. The proteins have not previously been identified in Drosophila muscle. PMID:26251439

  20. 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…

  1. Drosophila tribbles antagonizes insulin signaling-mediated growth and metabolism via interactions with Akt kinase.

    PubMed

    Das, Rahul; Sebo, Zachary; Pence, Laramie; Dobens, Leonard L

    2014-01-01

    Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation. PMID:25329475

  2. Drosophila Tribbles Antagonizes Insulin Signaling-Mediated Growth and Metabolism via Interactions with Akt Kinase

    PubMed Central

    Das, Rahul; Sebo, Zachary; Pence, Laramie; Dobens, Leonard L.

    2014-01-01

    Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation. PMID:25329475

  3. 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.

  4. 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

  5. Adenylate kinase isozyme 2 is essential for growth and development of Drosophila melanogaster.

    PubMed

    Fujisawa, Koichi; Murakami, Ryutaro; Horiguchi, Taigo; Noma, Takafumi

    2009-05-01

    Adenylate kinases are phylogenetically widespread, highly conserved, and involved in energy metabolism and energy transfer. Of these, adenylate kinase (AK) isozyme 2 is uniquely localized in the mitochondrial intermembrane space and its physiological role remains largely unknown. In this study, we selected Drosophila melanogaster to analyze its role in vivo. AK isozyme cDNAs were cloned and their gene expressions were characterized in D. melanogaster. The deduced amino acid sequences contain highly conserved motifs for P-loop, NMP binding, and LID domains of AKs. In addition, the effects of AK2 gene knockout on phenotype of AK2 mutants were examined using P-element technology. Although homozygous AK2 mutated embryos developed without any visible defects, their growth ceased and they died before reaching the third instar larval stage. Maternally provided AK2 mRNA was detected in fertilized eggs, and weak AK2 activity was observed in first and second instar larvae of the homozygous AK2 mutants, suggesting that maternally provided AK2 is sufficient for embryonic development. Disappearance of AK2 activity during larval stages resulted in growth arrest and eventual death. These results demonstrate that AK2 plays a critical role in adenine nucleotide metabolism in the mitochondrial intermembrane space and is essential for growth in D. melanogaster. PMID:19416704

  6. A Broadly Conserved G-Protein-Coupled Receptor Kinase Phosphorylation Mechanism Controls Drosophila Smoothened Activity

    PubMed Central

    Maier, Dominic; Cheng, Shuofei; Faubert, Denis; Hipfner, David R.

    2014-01-01

    Hedgehog (Hh) signaling is essential for normal growth, patterning, and homeostasis of many tissues in diverse organisms, and is misregulated in a variety of diseases including cancer. Cytoplasmic Hedgehog signaling is activated by multisite phosphorylation of the seven-pass transmembrane protein Smoothened (Smo) in its cytoplasmic C-terminus. Aside from a short membrane-proximal stretch, the sequence of the C-terminus is highly divergent in different phyla, and the evidence suggests that the precise mechanism of Smo activation and transduction of the signal to downstream effectors also differs. To clarify the conserved role of G-protein-coupled receptor kinases (GRKs) in Smo regulation, we mapped four clusters of phosphorylation sites in the membrane-proximal C-terminus of Drosophila Smo that are phosphorylated by Gprk2, one of the two fly GRKs. Phosphorylation at these sites enhances Smo dimerization and increases but is not essential for Smo activity. Three of these clusters overlap with regulatory phosphorylation sites in mouse Smo and are highly conserved throughout the bilaterian lineages, suggesting that they serve a common function. Consistent with this, we find that a C-terminally truncated form of Drosophila Smo consisting of just the highly conserved core, including Gprk2 regulatory sites, can recruit the downstream effector Costal-2 and activate target gene expression, in a Gprk2-dependent manner. These results indicate that GRK phosphorylation in the membrane proximal C-terminus is an evolutionarily ancient mechanism of Smo regulation, and point to a higher degree of similarity in the regulation and signaling mechanisms of bilaterian Smo proteins than has previously been recognized. PMID:25009998

  7. The Novel Smad Protein Expansion Regulates Receptor Tyrosine Kinase Pathway to Control Drosophila Tracheal Tube Size

    PubMed Central

    Iordanou, Ekaterini; Chandran, Rachana R.; Yang, Yonghua; Essak, Mina; Blackstone, Nicholas; Jiang, Lan

    2014-01-01

    Tubes with distinct shapes and sizes are critical for the proper function of many tubular organs. Here we describe a unique phenotype caused by the loss of a novel, evolutionarily-conserved, Drosophila Smad-like protein, Expansion. In expansion mutants, unicellular and intracellular tracheal branches develop bubble-like cysts with enlarged apical membranes. Cysts in unicellular tubes are enlargements of the apical lumen, whereas cysts in intracellular tubes are cytoplasmic vacuole-like compartments. The cyst phenotype in expansion mutants is similar to, but weaker than, that observed in double mutants of Drosophila type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. Ptp4E and Ptp10D negatively regulate the receptor tyrosine kinase (RTK) pathways, especially epithelial growth factor receptor (EGFR) and fibroblast growth factor receptor/breathless (FGFR, Btl) signaling to maintain the proper size of unicellular and intracellular tubes. We show Exp genetically interacts with RTK signaling, the downstream targets of RPTPs. Cyst size and number in expansion mutants is enhanced by increased RTK signaling and suppressed by reduced RTK signaling. Genetic interaction studies strongly suggest that Exp negatively regulates RTK (EGFR, Btl) signaling to ensure proper tube sizes. Smad proteins generally function as intermediate components of the transforming growth factor-β (TGF-β, DPP) signaling pathway. However, no obvious genetic interaction between expansion and TGF-β (DPP) signaling was observed. Therefore, Expansion does not function as a typical Smad protein. The expansion phenotype demonstrates a novel role for Smad-like proteins in epithelial tube formation. PMID:24973580

  8. 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.

  9. Drosophila Spidey/Kar Regulates Oenocyte Growth via PI3-Kinase Signaling

    PubMed Central

    Cinnamon, Einat; Sawala, Annick; Tittiger, Claus; Paroush, Ze'ev

    2016-01-01

    Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. Here, we focus on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. We now show that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, we show that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, the findings in this study show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes. PMID:27500738

  10. Drosophila Spidey/Kar Regulates Oenocyte Growth via PI3-Kinase Signaling.

    PubMed

    Cinnamon, Einat; Makki, Rami; Sawala, Annick; Wickenberg, Leah P; Blomquist, Gary J; Tittiger, Claus; Paroush, Ze'ev; Gould, Alex P

    2016-08-01

    Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. Here, we focus on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. We now show that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, we show that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, the findings in this study show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes. PMID:27500738

  11. REQUIREMENT OF THE TEC FAMILY TYROSINE KINASE BTK29A FOR COURTSHIP MEMORY IN Drosophila MALES.

    PubMed

    Sunouchi, Kazuya; Koganezawa, Masayuki; Yamamoto, Daisuke

    2016-03-01

    A male Drosophila that is not successful in courtship will reduce his courtship efforts in the next encounter with a female. This courtship suppression persists for more than 1 h in wild-type males. The Btk29A(ficP) mutant males null for the Btk29A type 2 isoform, a fly homolog of the nonreceptor tyrosine kinase Btk, show no courtship suppression, while Btk29A hypomorphic males exhibit a rapid decline in courtship suppression, leading to its complete loss within 30 min. The males of a revertant stock or Btk29A(ficP) males that are also mutant for parkas, a gene encoding the presumptive negative regulator of Btk29A, exhibit normal courtship suppression. Since another behavioral assay has shown that Btk29A(ficP) mutants are sensitization-defective, we hypothesize that the mutant flies are unable to maintain the neural excitation state acquired by experience, resulting in the rapid loss of courtship suppression. PMID:26782301

  12. 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

  13. Spindle-F Is the Central Mediator of Ik2 Kinase-Dependent Dendrite Pruning in Drosophila Sensory Neurons

    PubMed Central

    Lai, Yu-Ting; Chiang, Kai-Wen; Hsieh, Hsin-Lun; Wu, Yi-Ping; Ke, Jian-Ming; Lee, Myong-Chol; Liao, Shih-Sian; Shih, Hsueh-Tzu; Tang, Chiou-Yang; Yang, Shi-Bing; Cheng, Hsu-Chen; Wu, June-Tai; Jan, Yuh-Nung; Lee, Hsiu-Hsiang

    2015-01-01

    During development, certain Drosophila sensory neurons undergo dendrite pruning that selectively eliminates their dendrites but leaves the axons intact. How these neurons regulate pruning activity in the dendrites remains unknown. Here, we identify a coiled-coil protein Spindle-F (Spn-F) that is required for dendrite pruning in Drosophila sensory neurons. Spn-F acts downstream of IKK-related kinase Ik2 in the same pathway for dendrite pruning. Spn-F exhibits a punctate pattern in larval neurons, whereas these Spn-F puncta become redistributed in pupal neurons, a step that is essential for dendrite pruning. The redistribution of Spn-F from puncta in pupal neurons requires the phosphorylation of Spn-F by Ik2 kinase to decrease Spn-F self-association, and depends on the function of microtubule motor dynein complex. Spn-F is a key component to link Ik2 kinase to dynein motor complex, and the formation of Ik2/Spn-F/dynein complex is critical for Spn-F redistribution and for dendrite pruning. Our findings reveal a novel regulatory mechanism for dendrite pruning achieved by temporal activation of Ik2 kinase and dynein-mediated redistribution of Ik2/Spn-F complex in neurons. PMID:26540204

  14. Mutations in Drosophila Greatwall/Scant Reveal Its Roles in Mitosis and Meiosis and Interdependence with Polo Kinase

    PubMed Central

    White-Cooper, Helen; Carpenter, Adelaide T. C; Glover, David M

    2007-01-01

    Polo is a conserved kinase that coordinates many events of mitosis and meiosis, but how it is regulated remains unclear. Drosophila females having only one wild-type allele of the polo kinase gene and the dominant Scant mutation produce embryos in which one of the centrosomes detaches from the nuclear envelope in late prophase. We show that Scant creates a hyperactive form of Greatwall (Gwl) with altered specificity in vitro, another protein kinase recently implicated in mitotic entry in Drosophila and Xenopus. Excess Gwl activity in embryos causes developmental failure that can be rescued by increasing maternal Polo dosage, indicating that coordination between the two mitotic kinases is crucial for mitotic progression. Revertant alleles of Scant that restore fertility to polo–Scant heterozygous females are recessive alleles or deficiencies of gwl; they show chromatin condensation defects and anaphase bridges in larval neuroblasts. One recessive mutant allele specifically disrupts a Gwl isoform strongly expressed during vitellogenesis. Females hemizygous for this allele are sterile, and their oocytes fail to arrest in metaphase I of meiosis; both homologues and sister chromatids separate on elongated meiotic spindles with little or no segregation. This allelic series of gwl mutants highlights the multiple roles of Gwl in both mitotic and meiotic progression. Our results indicate that Gwl activity antagonizes Polo and thus identify an important regulatory interaction of the cell cycle. PMID:17997611

  15. The Extracellular-Regulated Kinase Effector Lk6 is Required for Glutamate Receptor Localization at the Drosophila Neuromuscular Junction.

    PubMed

    Hussein, Nizar A; Delaney, Taylor L; Tounsel, Brittany L; Liebl, Faith L W

    2016-01-01

    The proper localization and synthesis of postsynaptic glutamate receptors are essential for synaptic plasticity. Synaptic translation initiation is thought to occur via the target of rapamycin (TOR) and mitogen-activated protein kinase signal-integrating kinase (Mnk) signaling pathways, which is downstream of extracellular-regulated kinase (ERK). We used the model glutamatergic synapse, the Drosophila neuromuscular junction, to better understand the roles of the Mnk and TOR signaling pathways in synapse development. These synapses contain non-NMDA receptors that are most similar to AMPA receptors. Our data show that Lk6, the Drosophila homolog of Mnk1 and Mnk2, is required in either presynaptic neurons or postsynaptic muscle for the proper localization of the GluRIIA glutamate receptor subunit. Lk6 may signal through eukaryotic initiation factor (eIF) 4E to regulate the synaptic levels of GluRIIA as either interfering with eIF4E binding to eIF4G or expression of a nonphosphorylatable isoform of eIF4E resulted in a significant reduction in GluRIIA at the synapse. We also find that Lk6 and TOR may independently regulate synaptic levels of GluRIIA. PMID:27199570

  16. The Extracellular-Regulated Kinase Effector Lk6 is Required for Glutamate Receptor Localization at the Drosophila Neuromuscular Junction

    PubMed Central

    Hussein, Nizar A.; Delaney, Taylor L.; Tounsel, Brittany L.; Liebl, Faith L.W.

    2016-01-01

    The proper localization and synthesis of postsynaptic glutamate receptors are essential for synaptic plasticity. Synaptic translation initiation is thought to occur via the target of rapamycin (TOR) and mitogen-activated protein kinase signal-integrating kinase (Mnk) signaling pathways, which is downstream of extracellular-regulated kinase (ERK). We used the model glutamatergic synapse, the Drosophila neuromuscular junction, to better understand the roles of the Mnk and TOR signaling pathways in synapse development. These synapses contain non-NMDA receptors that are most similar to AMPA receptors. Our data show that Lk6, the Drosophila homolog of Mnk1 and Mnk2, is required in either presynaptic neurons or postsynaptic muscle for the proper localization of the GluRIIA glutamate receptor subunit. Lk6 may signal through eukaryotic initiation factor (eIF) 4E to regulate the synaptic levels of GluRIIA as either interfering with eIF4E binding to eIF4G or expression of a nonphosphorylatable isoform of eIF4E resulted in a significant reduction in GluRIIA at the synapse. We also find that Lk6 and TOR may independently regulate synaptic levels of GluRIIA. PMID:27199570

  17. G-protein coupled Receptor Kinase 2 is required for rhythmic olfactory responses in Drosophila

    PubMed Central

    Tanoue, Shintaro; Krishnan, Parthasarathy; Chatterjee, Abhishek; Hardin, Paul E.

    2008-01-01

    Summary Background The Drosophila circadian clock controls rhythms in the amplitude of odor-induced electrophysiological responses that peak during the middle of night. These rhythms are dependent on clocks in olfactory sensory neurons (OSNs), which suggests that odorant receptors(ORs) or OR-dependent processes are under clock control. Since responses to odors are initiated by heteromeric OR complexes that form odor-gated and cyclic-nucleotide-activated cation channels, we tested whether regulators of ORs were under circadian clock control. Results The levels of G-protein coupled receptor kinase 2 (Gprk2) mRNA and protein cycle in a circadian clock-dependent manner with a peak around mid-night in antennae. Gprk2 overexpression in OSNs from wild-type or cyc01 flies elicits constant high amplitude electroantennogram (EAG) responses to ethyl acetate, whereas Gprk mutants produce constant low amplitude EAG responses. Odorant receptors (ORs) accumulate to high levels in the dendrites of OSNs around mid-night, and this dendritic localization of ORs is enhanced by Gprk2 at times when ORs are primarily localized in the cell body. Conclusion These results support a model in which circadian clock-dependent rhythms in Gprk2 abundance control the rhythmic accumulation of ORs in OSN dendrites, which in turn control rhythms in olfactory responses. The enhancement of OR function by GPRK2 contrasts with the traditional role of Gprks in desensitizing activated receptors, and suggests that GPRK2 functions through a fundamentally different mechanism to modulate OR activity. PMID:18499458

  18. Dual Role of Jun N-Terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development.

    PubMed

    Park, Sung Yeon; Stultz, Brian G; Hursh, Deborah A

    2015-12-01

    The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps. PMID:26500262

  19. LRRK2 Kinase Regulates Synaptic Morphology through Distinct Substrates at the Presynaptic and Postsynaptic Compartments of the Drosophila Neuromuscular Junction

    PubMed Central

    Lee, Seongsoo; Liu, Hsin-Ping; Lin, Wei-Yong; Guo, Huifu; Lu, Bingwei

    2011-01-01

    Mutations in leucine-rich repeat kinase 2 (LRRK2) are linked to familial as well as sporadic forms of Parkinson’s disease (PD), a neurodegenerative disease characterized by dysfunction and degeneration of dopaminergic and other types of neurons. The molecular and cellular mechanisms underlying LRRK2 action remain poorly defined. Here, we show that LRRK2 controls synaptic morphogenesis at the Drosophila neuromuscular junction. Loss of Drosophila LRRK2 results in synaptic overgrowth, whereas overexpression of DrosophilaLRRKorhumanLRRK2has opposite effects. Alteration ofLRRK2activity also affects neurotransmission. LRRK2exerts its effects on synaptic morphology by interacting with distinct downstream effectors at the presynaptic and postsynaptic compartments. At the postsynapse, LRRK2 interacts with the previously characterized substrate 4E-BP, an inhibitor of protein synthesis. At the presynapse, LRRK2 phosphorylates and negatively regulates the microtubule (MT)-binding protein Futsch. These results implicate synaptic dysfunction caused by deregulated protein synthesis and aberrant MT dynamics in LRRK2 pathogenesis and offer a new paradigm for understanding and ultimately treating PD. PMID:21159966

  20. Mutation of Drosophila Focal Adhesion Kinase Induces Bang-Sensitive Behavior and Disrupts Glial Function, Axonal Conduction and Synaptic Transmission

    PubMed Central

    Ueda, Atsushi; Grabbe, Caroline; Lee, Jihye; Lee, Jisue; Palmer, Ruth H.; Wu, Chun-Fang

    2009-01-01

    The role of the conserved Focal Adhesion Kinase (FAK) family of protein tyrosine kinases (PTKs) in the development and physiological functions of the CNS has long been an area of interest among neuroscientists. In this report, we observe that Drosophila mutants lacking Fak56 exhibit a decreased life span, accompanied by a bang-sensitive phenotype, which is characterised by sensitivity to mechanical and high-frequency electrical stimulation. Fak56 mutant animals display lower thresholds and higher rates of seizures in response to electroconvulsive stimuli, and direct measurements of action potential conduction in larval segmental nerves demonstrate a slowed propagation speed and failure during high-frequency nerve stimulation. In addition, neuromuscular junctions in Fak56 mutant animals display transmission blockade during high-frequency activity as a result of action potential failure. Endogenous Fak56 protein is abundant in glial cells ensheathing the axon bundles, and structural alterations of segmental nerve bundles can be observed in mutants. Manipulation of Fak56 function specifically in glial cells also disrupts action potential conduction and neurotransmission, suggesting a glial component in the Fak56 bang-sensitive phenotype. Furthermore, we show that increased intracellular calcium levels result in the dephosphorylation of endogenous Fak56 protein in Drosophila cell lines, in parallel with our observations of highly variable synaptic potentials at a higher Ca2+ level in Fak56 mutant larvae. Together these findings suggest that modulation of Fak56 function is important for action potential propagation and Ca2+-regulated neuromuscular transmission in vivo. PMID:18540882

  1. Expression of DFak56, a Drosophila homolog of vertebrate focal adhesion kinase, supports a role in cell migration in vivo

    PubMed Central

    Fox, George L.; Rebay, Ilaria; Hynes, Richard O.

    1999-01-01

    Focal adhesion kinase (FAK) is a highly conserved, cytoplasmic tyrosine kinase that has been implicated in promoting cell migration and transmission of antiapoptotic signals in vertebrate cells. In cultured cells, integrin engagement with the extracellular matrix promotes the recruitment of FAK to focal contacts and increases in its phosphotyrosine content and kinase activity, suggesting FAK is an intracellular mediator of integrin signaling. We have identified a Drosophila FAK homolog, DFak56, that is 33% identical to vertebrate FAK, with the highest degree of homology in domains critical for FAK function, including the kinase and focal adhesion targeting domains, and several protein–protein interaction motifs. Furthermore, when expressed in NIH 3T3 cells, DFak56 both localizes to focal contacts and displays the characteristic elevation of phosphotyrosine content in response to plating the cells on fibronectin. During embryogenesis, DFak56 is broadly expressed, and it becomes elevated in the gut and central nervous system at later stages. Consistent with a role in cell migration, we also observe that DFak56 is abundant in the border cells of developing egg chambers before the onset of, and during, their migration. PMID:10611323

  2. FAK Acts as a Suppressor of RTK-MAP Kinase Signalling in Drosophila melanogaster Epithelia and Human Cancer Cells

    PubMed Central

    Macagno, Juan Pablo; Diaz Vera, Jesica; Yu, Yachuan; MacPherson, Iain; Sandilands, Emma; Palmer, Ruth; Norman, Jim C.; Frame, Margaret; Vidal, Marcos

    2014-01-01

    Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours. PMID:24676055

  3. A homologue of AMP-activated protein kinase in Drosophila melanogaster is sensitive to AMP and is activated by ATP depletion.

    PubMed Central

    Pan, David A; Hardie, D Grahame

    2002-01-01

    We have identified single genes encoding homologues of the alpha, beta and gamma subunits of mammalian AMP-activated protein kinase (AMPK) in the genome of Drosophila melanogaster. Kinase activity could be detected in extracts of a Drosophila cell line using the SAMS peptide, which is a relatively specific substrate for the AMPK/SNF1 kinases in mammals and yeast. Expression of double stranded (ds) RNAs targeted at any of the putative alpha, beta or gamma subunits ablated this activity, and abolished expression of the alpha subunit. The Drosophila kinase (DmAMPK) was activated by AMP in cell-free assays (albeit to a smaller extent than mammalian AMPK), and by stresses that deplete ATP (oligomycin and hypoxia), as well as by carbohydrate deprivation, in intact cells. Using a phosphospecific antibody, we showed that activation was associated with phosphorylation of a threonine residue (Thr-184) within the 'activation loop' of the alpha subunit. We also identified a homologue of acetyl-CoA carboxylase (DmACC) in Drosophila and, using a phosphospecific antibody, showed that the site corresponding to the regulatory AMPK site on the mammalian enzyme became phosphorylated in response to oligomycin or hypoxia. By immunofluorescence microscopy of oligomycin-treated Dmel2 cells using the phosphospecific antibody, the phosphorylated DmAMPK alpha subunit was mainly detected in the nucleus. Our results show that the AMPK system is highly conserved between insects and mammals. Drosophila cells now represent an attractive system to study this pathway, because of the small, well-defined genome and the ability to ablate expression of specific gene products using interfering dsRNAs. PMID:12093363

  4. The Octopamine Receptor OAMB Mediates Ovulation via Ca2+/Calmodulin-Dependent Protein Kinase II in the Drosophila Oviduct Epithelium

    PubMed Central

    Lee, Hyun-Gwan; Rohila, Suman; Han, Kyung-An

    2009-01-01

    Ovulation is an essential physiological process in sexual reproduction; however, the underlying cellular mechanisms are poorly understood. We have previously shown that OAMB, a Drosophila G-protein-coupled receptor for octopamine (the insect counterpart of mammalian norepinephrine), is required for ovulation induced upon mating. OAMB is expressed in the nervous and reproductive systems and has two isoforms (OAMB-AS and OAMB-K3) with distinct capacities to increase intracellular Ca2+ or intracellular Ca2+ and cAMP in vitro. Here, we investigated tissue specificity and intracellular signals required for OAMB's function in ovulation. Restricted OAMB expression in the adult oviduct epithelium, but not the nervous system, reinstated ovulation in oamb mutant females, in which either OAMB isoform was sufficient for the rescue. Consistently, strong immunoreactivities for both isoforms were observed in the wild-type oviduct epithelium. To delineate the cellular mechanism by which OAMB regulates ovulation, we explored protein kinases functionally interacting with OAMB by employing a new GAL4 driver with restricted expression in the oviduct epithelium. Conditional inhibition of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), but not protein kinase A or C, in the oviduct epithelium inhibited ovulation. Moreover, constitutively active CaMKII, but not protein kinase A, expressed only in the adult oviduct epithelium fully rescued the oamb female's phenotype, demonstrating CaMKII as a major downstream molecule conveying the OAMB's ovulation signal. This is consistent with the ability of both OAMB isoforms, whose common intracellular signal in vitro is Ca2+, to reinstate ovulation in oamb females. These observations reveal the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. It is conceivable that the OAMB-mediated cellular activities stimulated upon mating are crucial for secretory activities suitable for egg transfer from

  5. Phosphorylation of the Drosophila Transient Receptor Potential Ion Channel Is Regulated by the Phototransduction Cascade and Involves Several Protein Kinases and Phosphatases

    PubMed Central

    Voolstra, Olaf; Bartels, Jonas-Peter; Oberegelsbacher, Claudia; Pfannstiel, Jens; Huber, Armin

    2013-01-01

    Protein phosphorylation plays a cardinal role in regulating cellular processes in eukaryotes. Phosphorylation of proteins is controlled by protein kinases and phosphatases. We previously reported the light-dependent phosphorylation of the Drosophila transient receptor potential (TRP) ion channel at multiple sites. TRP generates the receptor potential upon stimulation of the photoreceptor cell by light. An eye-enriched protein kinase C (eye-PKC) has been implicated in the phosphorylation of TRP by in vitro studies. Other kinases and phosphatases of TRP are elusive. Using phosphospecific antibodies and mass spectrometry, we here show that phosphorylation of most TRP sites depends on the phototransduction cascade and the activity of the TRP ion channel. A candidate screen to identify kinases and phosphatases provided in vivo evidence for an involvement of eye-PKC as well as other kinases and phosphatases in TRP phosphorylation. PMID:24040070

  6. Mitogen-activated protein kinase p38b interaction with delta class glutathione transferases from the fruit fly, Drosophila melanogaster.

    PubMed

    Wongtrakul, Jeerang; Sukittikul, Suchada; Saisawang, Chonticha; Ketterman, Albert J

    2012-01-01

    Glutathione transferases (GSTs) are a family of multifunctional enzymes involved in xenobiotic biotransformation, drug metabolism, and protection against oxidative damage. The p38b mitogen-activated protein kinase is involved in cellular stress response. This study screened interactions between Drosophila melanogaster Meigen (Diptera: Drosophilidae) Delta class glutathione transferases (DmGSTs) and the D. melanogaster p38b MAPK. Therefore, 12 DmGSTs and p38b kinase were obtained as recombinant proteins. The study showed that DmGSTD8 and DmGSTD11b significantly increased p38b activity toward ATF2 and jun, which are transcription factor substrates. DmGSTD3 and DmGSTD5 moderately increased p38b activity for jun. In addition, GST activity in the presence of p38b was also measured. It was found that p38b affected substrate specificity toward CDNB (1-chloro-2,4-dinitrobenzene) and DCNB (1,2-dichloro-4-nitrobenzene) of several GST isoforms, i.e., DmGSTD2, DmGSTD5, DmGSTD8, and DmGSTD11b. The interaction of a GST and p38b can affect the substrate specificity of either enzyme, which suggests induced conformational changes affecting catalysis. Similar interactions do not occur for all the Delta enzymes and p38b, which suggests that these interactions could be specific. PMID:23438069

  7. C-terminal Src Kinase Gates Homeostatic Synaptic Plasticity and Regulates Fasciclin II Expression at the Drosophila Neuromuscular Junction

    PubMed Central

    Spring, Ashlyn M.; Brusich, Douglas J.; Frank, C. Andrew

    2016-01-01

    Forms of homeostatic plasticity stabilize neuronal outputs and promote physiologically favorable synapse function. A well-studied homeostatic system operates at the Drosophila melanogaster larval neuromuscular junction (NMJ). At the NMJ, impairment of postsynaptic glutamate receptor activity is offset by a compensatory increase in presynaptic neurotransmitter release. We aim to elucidate how this process operates on a molecular level and is preserved throughout development. In this study, we identified a tyrosine kinase-driven signaling system that sustains homeostatic control of NMJ function. We identified C-terminal Src Kinase (Csk) as a potential regulator of synaptic homeostasis through an RNAi- and electrophysiology-based genetic screen. We found that Csk loss-of-function mutations impaired the sustained expression of homeostatic plasticity at the NMJ, without drastically altering synapse growth or baseline neurotransmission. Muscle-specific overexpression of Src Family Kinase (SFK) substrates that are negatively regulated by Csk also impaired NMJ homeostasis. Surprisingly, we found that transgenic Csk-YFP can support homeostatic plasticity at the NMJ when expressed either in the muscle or in the nerve. However, only muscle-expressed Csk-YFP was able to localize to NMJ structures. By immunostaining, we found that Csk mutant NMJs had dysregulated expression of the Neural Cell Adhesion Molecule homolog Fasciclin II (FasII). By immunoblotting, we found that levels of a specific isoform of FasII were decreased in homeostatically challenged GluRIIA mutant animals–but markedly increased in Csk mutant animals. Additionally, we found that postsynaptic overexpression of FasII from its endogenous locus was sufficient to impair synaptic homeostasis, and genetically reducing FasII levels in Csk mutants fully restored synaptic homeostasis. Based on these data, we propose that Csk and its SFK substrates impinge upon homeostatic control of NMJ function by regulating

  8. Shaggy/glycogen synthase kinase 3β and phosphorylation of Sarah/regulator of calcineurin are essential for completion of Drosophila female meiosis

    PubMed Central

    Takeo, Satomi; Swanson, Selene K.; Nandanan, Kavyasree; Nakai, Yasuhiro; Aigaki, Toshiro; Washburn, Michael P.; Florens, Laurence; Hawley, R. Scott

    2012-01-01

    The Ca2+/Calmodulin-dependent phosphatase calcineurin is essential for exit from meiotic arrest at metaphases I and II in Drosophila and Xenopus oocytes. We previously found that Sarah, the Drosophila homolog of regulator of calcineurin, acts as a positive regulator of calcineurin and is required to complete anaphase I of female meiosis. Here, we undertook biochemical approaches, including MS and posttranslational modification analyses, to better understand the mechanism by which Sarah regulates calcineurin. A search for phosphorylated residues revealed that Sarah is highly phosphorylated at Ser100, Thr102, and Ser219 in both ovaries and activated eggs and that Ser215 is phosphorylated only in activated eggs. Functional analyses using mutant forms of Sarah showed that phosphorylation at Ser215, a consensus phosphorylation site for glycogen synthase kinase 3β (GSK-3β) and its priming kinase site Ser219, are essential for Sarah function. Furthermore, germ-line clones homozygous for a null allele of shaggy (Drosophila GSK-3β) both fail to complete meiosis and lack phosphorylation of Sarah at Ser215, suggesting that the phosphorylation of Sarah by Shaggy/GSK-3β is required to complete meiosis. Our findings suggest a mechanism in which Shaggy/GSK-3β activates calcineurin through Sarah phosphorylation on egg activation in Drosophila. PMID:22421435

  9. Long Term Expression of Drosophila melanogaster Nucleoside Kinase in Thymidine Kinase 2-deficient Mice with No Lethal Effects Caused by Nucleotide Pool Imbalances*

    PubMed Central

    Krishnan, Shuba; Paredes, João A.; Zhou, Xiaoshan; Kuiper, Raoul V.; Hultenby, Kjell; Curbo, Sophie; Karlsson, Anna

    2014-01-01

    Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2−/−) mice extended the life span of Tk2−/− mice from 3 weeks to at least 20 months. The Dm-dNK+/−Tk2−/− mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK+/−Tk2−/− mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK+/− mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues. PMID:25296759

  10. A microtiter-based assay for protein kinase activity suitable for the analysis of large numbers of samples, and its application to the study of Drosophila learning mutants.

    PubMed

    Buxbaum, J D; Dudai, Y

    1988-02-15

    We have developed a microtiter-based assay for protein kinase activity which depends on the immobilization of substrate proteins to nitrocellulose. The technique makes use of a filtration manifold, allowing as much as a 10-fold increase in efficiency as compared to other protein kinase assays. We have used this assay to measure cAMP-dependent protein kinase (PKA) in Drosophila learning and memory mutants, with exogenous and endogenous substrates. An alteration was found in the affinity of PKA in the mutant turnip. The procedure should be useful for rapid screening of mutants and drugs and could be adapted to additional types of protein kinases as well as protein phosphatases. PMID:3369684

  11. Administration of deoxyribonucleosides or inhibition of their catabolism as a pharmacological approach for mitochondrial DNA depletion syndrome.

    PubMed

    Cámara, Yolanda; González-Vioque, Emiliano; Scarpelli, Mauro; Torres-Torronteras, Javier; Caballero, Andrea; Hirano, Michio; Martí, Ramon

    2014-05-01

    Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is characterized by a reduction in mtDNA copy number and consequent mitochondrial dysfunction in affected tissues. A subgroup of MDS is caused by mutations in genes that disrupt deoxyribonucleotide metabolism, which ultimately leads to limited availability of one or several deoxyribonucleoside triphosphates (dNTPs), and subsequent mtDNA depletion. Here, using in vitro experimental approaches (primary cell culture of deoxyguanosine kinase-deficient cells and thymidine-induced mtDNA depletion in culture as a model of mitochondrial neurogastrointestinal encephalomyopathy, MNGIE), we show that supplements of those deoxyribonucleosides (dNs) involved in each biochemical defect (deoxyguanosine or deoxycytidine, dCtd) prevents mtDNA copy number reduction. Similar effects can be obtained by specific inhibition of dN catabolism using tetrahydrouridine (THU; inhibitor of cytidine deaminase) or immucillin H (inhibitor of purine nucleoside phosphorylase). In addition, using an MNGIE animal model, we provide evidence that mitochondrial dNTP content can be modulated in vivo by systemic administration of dCtd or THU. In spite of the severity associated with diseases due to defects in mtDNA replication, there are currently no effective therapeutic options available. Only in the case of MNGIE, allogeneic hematopoietic stem cell transplantation has proven efficient as a long-term therapeutic strategy. We propose increasing cellular availability of the deficient dNTP precursor by direct administration of the dN or inhibition of its catabolism, as a potential treatment for mtDNA depletion syndrome caused by defects in dNTP metabolism. PMID:24362886

  12. Impaired Coenzyme A metabolism affects histone and tubulin acetylation in Drosophila and human cell models of pantothenate kinase associated neurodegeneration.

    PubMed

    Siudeja, Katarzyna; Srinivasan, Balaji; Xu, Lanjun; Rana, Anil; de Jong, Jannie; Nollen, Ellen A A; Jackowski, Suzanne; Sanford, Lynn; Hayflick, Susan; Sibon, Ody C M

    2011-12-01

    Pantothenate kinase-associated neurodegeneration (PKAN is a neurodegenerative disease with unresolved pathophysiology. Previously, we observed reduced Coenzyme A levels in a Drosophila model for PKAN. Coenzyme A is required for acetyl-Coenzyme A synthesis and acyl groups from the latter are transferred to lysine residues of proteins, in a reaction regulated by acetyltransferases. The tight balance between acetyltransferases and their antagonistic counterparts histone deacetylases is a well-known determining factor for the acetylation status of proteins. However, the influence of Coenzyme A levels on protein acetylation is unknown. Here we investigate whether decreased levels of the central metabolite Coenzyme A induce alterations in protein acetylation and whether this correlates with specific phenotypes of PKAN models. We show that in various organisms proper Coenzyme A metabolism is required for maintenance of histone- and tubulin acetylation, and decreased acetylation of these proteins is associated with an impaired DNA damage response, decreased locomotor function and decreased survival. Decreased protein acetylation and the concurrent phenotypes are partly rescued by pantethine and HDAC inhibitors, suggesting possible directions for future PKAN therapy development. PMID:21998097

  13. Impaired Coenzyme A metabolism affects histone and tubulin acetylation in Drosophila and human cell models of pantothenate kinase associated neurodegeneration

    PubMed Central

    Siudeja, Katarzyna; Srinivasan, Balaji; Xu, Lanjun; Rana, Anil; de Jong, Jannie; Nollen, Ellen A A; Jackowski, Suzanne; Sanford, Lynn; Hayflick, Susan; Sibon, Ody C M

    2011-01-01

    Pantothenate kinase-associated neurodegeneration (PKAN is a neurodegenerative disease with unresolved pathophysiology. Previously, we observed reduced Coenzyme A levels in a Drosophila model for PKAN. Coenzyme A is required for acetyl-Coenzyme A synthesis and acyl groups from the latter are transferred to lysine residues of proteins, in a reaction regulated by acetyltransferases. The tight balance between acetyltransferases and their antagonistic counterparts histone deacetylases is a well-known determining factor for the acetylation status of proteins. However, the influence of Coenzyme A levels on protein acetylation is unknown. Here we investigate whether decreased levels of the central metabolite Coenzyme A induce alterations in protein acetylation and whether this correlates with specific phenotypes of PKAN models. We show that in various organisms proper Coenzyme A metabolism is required for maintenance of histone- and tubulin acetylation, and decreased acetylation of these proteins is associated with an impaired DNA damage response, decreased locomotor function and decreased survival. Decreased protein acetylation and the concurrent phenotypes are partly rescued by pantethine and HDAC inhibitors, suggesting possible directions for future PKAN therapy development. PMID:21998097

  14. Anaplastic Lymphoma Kinase Acts in the Drosophila Mushroom Body to Negatively Regulate Sleep

    PubMed Central

    Bai, Lei; Sehgal, Amita

    2015-01-01

    Though evidence is mounting that a major function of sleep is to maintain brain plasticity and consolidate memory, little is known about the molecular pathways by which learning and sleep processes intercept. Anaplastic lymphoma kinase (Alk), the gene encoding a tyrosine receptor kinase whose inadvertent activation is the cause of many cancers, is implicated in synapse formation and cognitive functions. In particular, Alk genetically interacts with Neurofibromatosis 1 (Nf1) to regulate growth and associative learning in flies. We show that Alk mutants have increased sleep. Using a targeted RNAi screen we localized the negative effects of Alk on sleep to the mushroom body, a structure important for both sleep and memory. We also report that mutations in Nf1 produce a sexually dimorphic short sleep phenotype, and suppress the long sleep phenotype of Alk. Thus Alk and Nf1 interact in both learning and sleep regulation, highlighting a common pathway in these two processes. PMID:26536237

  15. Oxidation of DNA bases, deoxyribonucleosides and homopolymers by peroxyl radicals.

    PubMed Central

    Simandan, T; Sun, J; Dix, T A

    1998-01-01

    DNA base oxidation is considered to be a key event associated with disease initiation and progression in humans. Peroxyl radicals (ROO. ) are important oxidants found in cells whose ability to react with the DNA bases has not been characterized extensively. In this paper, the products resulting from ROO. oxidation of the DNA bases are determined by gas chromatography/MS in comparison with authentic standards. ROO. radicals oxidize adenine and guanine to their 8-hydroxy derivatives, which are considered biomarkers of hydroxyl radical (HO.) oxidations in cells. ROO. radicals also oxidize adenine to its hydroxylamine, a previously unidentified product. ROO. radicals oxidize cytosine and thymine to the monohydroxy and dihydroxy derivatives that are formed by oxidative damage in cells. Identical ROO. oxidation profiles are observed for each base when exposed as deoxyribonucleosides, monohomopolymers and base-paired dihomopolymers. These results have significance for the development, utilization and interpretation of DNA base-derived biomarkers of oxidative damage associated with disease initiation and propagation, and support the idea that the mutagenic potential of N-oxidized bases, when generated in cellular DNA, will require careful evaluation. Adenine hydroxylamine is proposed as a specific molecular probe for the activity of ROO. in cellular systems. PMID:9761719

  16. Hypotonicity Stimulates Potassium Flux through the WNK-SPAK/OSR1 Kinase Cascade and the Ncc69 Sodium-Potassium-2-Chloride Cotransporter in the Drosophila Renal Tubule*

    PubMed Central

    Wu, Yipin; Schellinger, Jeffrey N.; Huang, Chou-Long; Rodan, Aylin R.

    2014-01-01

    The ability to osmoregulate is fundamental to life. Adult Drosophila melanogaster maintain hemolymph osmolarity within a narrow range. Osmolarity modulates transepithelial ion and water flux in the Malpighian (renal) tubules of the fly, which are in direct contact with hemolymph in vivo, but the mechanisms causing increased transepithelial flux in response to hypotonicity are unknown. Fly renal tubules secrete a KCl-rich fluid. We have previously demonstrated a requirement for Ncc69, the fly sodium-potassium-2-chloride cotransporter (NKCC), in tubule K+ secretion. Mammalian NKCCs are regulated by a kinase cascade consisting of the with-no-lysine (WNK) and Ste20-related proline/alanine-rich (SPAK)/oxidative stress response (OSR1) kinases. Here, we show that decreasing Drosophila WNK activity causes a reduction in K+ flux. Similarly, knocking down the SPAK/OSR1 homolog fray also decreases K+ flux. We demonstrate that a hierarchical WNK-Fray signaling cascade regulates K+ flux through Ncc69, because (i) a constitutively active Fray mutant rescues the wnk knockdown phenotype, (ii) Fray directly phosphorylates Ncc69 in vitro, and (iii) the effect of wnk and fray knockdown is abolished in Ncc69 mutants. The stimulatory effect of hypotonicity on K+ flux is absent in wnk, fray, or Ncc69 mutant tubules, suggesting that the Drosophila WNK-SPAK/OSR1-NKCC cascade is an essential molecular pathway for osmoregulation, through its effect on transepithelial ion flux and fluid generation by the renal tubule. PMID:25086033

  17. Exon junction complex subunits are required to splice Drosophila MAP kinase, a large heterochromatic gene

    PubMed Central

    Roignant, Jean-Yves; Treisman, Jessica E.

    2010-01-01

    Summary The exon junction complex (EJC) is assembled on spliced mRNAs upstream of exon-exon junctions, and can regulate their subsequent translation, localization, or degradation. We isolated mutations in Drosophila mago nashi (mago), which encodes a core EJC subunit, based on their unexpectedly specific effects on photoreceptor differentiation. Loss of Mago prevents Epidermal growth factor receptor signaling, due to a large reduction in MAPK mRNA levels. MAPK expression also requires the EJC subunits Y14 and eIF4AIII, and EJC-associated splicing factors. Mago depletion does not affect the transcription or stability of MAPK mRNA, but alters its splicing pattern. MAPK expression from an exogenous promoter requires Mago only when the template includes introns. MAPK is the primary functional target of mago in eye development; in cultured cells, Mago knockdown disproportionately affects other large genes located in heterochromatin. These data support a nuclear role for EJC components in splicing a specific subset of introns. PMID:20946982

  18. Inositol phosphate kinase 2 is required for imaginal disc development in Drosophila

    PubMed Central

    Seeds, Andrew M.; Tsui, Marco M.; Sunu, Christine; Spana, Eric P.; York, John D.

    2015-01-01

    Inositol phosphate kinase 2 (Ipk2), also known as IP multikinase IPMK, is an evolutionarily conserved protein that initiates production of inositol phosphate intracellular messengers (IPs), which are critical for regulating nuclear and cytoplasmic processes. Here we report that Ipk2 kinase activity is required for the development of the adult fruit fly epidermis. Ipk2 mutants show impaired development of their imaginal discs, the primordial tissues that form the adult epidermis. Although disk tissue seems to specify normally during early embryogenesis, loss of Ipk2 activity results in increased apoptosis and impairment of proliferation during larval and pupal development. The proliferation defect is in part attributed to a reduction in JAK/STAT signaling, possibly by controlling production or secretion of the pathway’s activating ligand, Unpaired. Constitutive activation of the JAK/STAT pathway downstream of Unpaired partially rescues the disk growth defects in Ipk2 mutants. Thus, IP production is essential for proliferation of the imaginal discs, in part, by regulating JAK/STAT signaling. Our work demonstrates an essential role for Ipk2 in producing inositide messengers required for imaginal disk tissue maturation and subsequent formation of adult body structures and provides molecular insights to signaling pathways involved in tissue growth and stability during development. PMID:26647185

  19. Inositol phosphate kinase 2 is required for imaginal disc development in Drosophila.

    PubMed

    Seeds, Andrew M; Tsui, Marco M; Sunu, Christine; Spana, Eric P; York, John D

    2015-12-22

    Inositol phosphate kinase 2 (Ipk2), also known as IP multikinase IPMK, is an evolutionarily conserved protein that initiates production of inositol phosphate intracellular messengers (IPs), which are critical for regulating nuclear and cytoplasmic processes. Here we report that Ipk2 kinase activity is required for the development of the adult fruit fly epidermis. Ipk2 mutants show impaired development of their imaginal discs, the primordial tissues that form the adult epidermis. Although disk tissue seems to specify normally during early embryogenesis, loss of Ipk2 activity results in increased apoptosis and impairment of proliferation during larval and pupal development. The proliferation defect is in part attributed to a reduction in JAK/STAT signaling, possibly by controlling production or secretion of the pathway's activating ligand, Unpaired. Constitutive activation of the JAK/STAT pathway downstream of Unpaired partially rescues the disk growth defects in Ipk2 mutants. Thus, IP production is essential for proliferation of the imaginal discs, in part, by regulating JAK/STAT signaling. Our work demonstrates an essential role for Ipk2 in producing inositide messengers required for imaginal disk tissue maturation and subsequent formation of adult body structures and provides molecular insights to signaling pathways involved in tissue growth and stability during development. PMID:26647185

  20. rugose (rg), a Drosophila A kinase anchor protein, is required for retinal pattern formation and interacts genetically with multiple signaling pathways.

    PubMed Central

    Shamloula, Hoda K; Mbogho, Mkajuma P; Pimentel, Angel C; Chrzanowska-Lightowlers, Zosia M A; Hyatt, Vanneta; Okano, Hideyuki; Venkatesh, Tadmiri R

    2002-01-01

    In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the EGFR- and Notch-mediated signaling pathways. Our results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the EGFR and Notch signaling pathways. PMID:12072466

  1. Binding of Drosophila Polo kinase to its regulator Matrimony is noncanonical and involves two separate functional domains

    PubMed Central

    Bonner, Amanda M.; Hughes, Stacie E.; Chisholm, Jennifer A.; Smith, S. Kendall; Slaughter, Brian D.; Unruh, Jay R.; Collins, Kimberly A.; Friederichs, Jennifer M.; Florens, Laurence; Swanson, Selene K.; Pelot, Marissa C.; Miller, Danny E.; Washburn, Michael P.; Jaspersen, Sue L.; Hawley, R. Scott

    2013-01-01

    Drosophila melanogaster Polo kinase physically interacts with, and is repressed by, the Matrimony (Mtrm) protein during oogenesis. Females heterozygous for a deletion of the mtrm gene display defects in chromosome segregation at meiosis I. However, a complete absence of Mtrm results in both meiotic catastrophe and female sterility. We show that three phosphorylated residues in an N-terminal region in Mtrm are required for Mtrm::Polo binding. However, this binding is noncanonical; it does not require either a complete S-pS/pT-P motif in Mtrm or key residues in the Polo-box domain of Polo that allow Polo to bind phosphorylated substrates. By using fluorescence cross-correlation spectroscopy to characterize the Mtrm::Polo interaction in vivo, we show that a sterile α-motif (SAM) domain located at the C terminus of Mtrm increases the stability of Mtrm::Polo binding. Although Mtrm’s C-terminal SAM domain is not required to rescue the chromosome segregation defects observed in mtrm/+ females, it is essential to prevent both meiotic catastrophe and the female sterility observed in mtrm/mtrm females. We propose that Polo’s interaction with the cluster of phosphorylated residues alone is sufficient to rescue the meiosis I defect. However, the strengthening of Mtrm::Polo binding mediated by the SAM domain is necessary to prevent meiotic catastrophe and ensure female fertility. Characterization of the Mtrm::Polo interaction, as well as that of other Polo regulators, may assist in the design of a new class of Polo inhibitors to be used as targeted anticancer therapeutic agents. PMID:23479640

  2. Energy-Dependent Modulation of Glucagon-Like Signaling in Drosophila via the AMP-Activated Protein Kinase

    PubMed Central

    Braco, Jason T.; Gillespie, Emily L.; Alberto, Gregory E.; Brenman, Jay E.; Johnson, Erik C.

    2012-01-01

    Adipokinetic hormone (AKH) is the equivalent of mammalian glucagon, as it is the primary insect hormone that causes energy mobilization. In Drosophila, current knowledge of the mechanisms regulating AKH signaling is limited. Here, we report that AMP-activated protein kinase (AMPK) is critical for normal AKH secretion during periods of metabolic challenges. Reduction of AMPK in AKH cells causes a suite of behavioral and physiological phenotypes resembling AKH cell ablations. Specifically, reduced AMPK function increases life span during starvation and delays starvation-induced hyperactivity. Neither AKH cell survival nor gene expression is significantly impacted by reduced AMPK function. AKH immunolabeling was significantly higher in animals with reduced AMPK function; this result is paralleled by genetic inhibition of synaptic release, suggesting that AMPK promotes AKH secretion. We observed reduced secretion in AKH cells bearing AMPK mutations employing a specific secretion reporter, confirming that AMPK functions in AKH secretion. Live-cell imaging of wild-type AKH neuroendocrine cells shows heightened excitability under reduced sugar levels, and this response was delayed and reduced in AMPK-deficient backgrounds. Furthermore, AMPK activation in AKH cells increases intracellular calcium levels in constant high sugar levels, suggesting that the underlying mechanism of AMPK action is modification of ionic currents. These results demonstrate that AMPK signaling is a critical feature that regulates AKH secretion, and, ultimately, metabolic homeostasis. The significance of these findings is that AMPK is important in the regulation of glucagon signaling, suggesting that the organization of metabolic networks is highly conserved and that AMPK plays a prominent role in these networks. PMID:22798489

  3. Energy-dependent modulation of glucagon-like signaling in Drosophila via the AMP-activated protein kinase.

    PubMed

    Braco, Jason T; Gillespie, Emily L; Alberto, Gregory E; Brenman, Jay E; Johnson, Erik C

    2012-10-01

    Adipokinetic hormone (AKH) is the equivalent of mammalian glucagon, as it is the primary insect hormone that causes energy mobilization. In Drosophila, current knowledge of the mechanisms regulating AKH signaling is limited. Here, we report that AMP-activated protein kinase (AMPK) is critical for normal AKH secretion during periods of metabolic challenges. Reduction of AMPK in AKH cells causes a suite of behavioral and physiological phenotypes resembling AKH cell ablations. Specifically, reduced AMPK function increases life span during starvation and delays starvation-induced hyperactivity. Neither AKH cell survival nor gene expression is significantly impacted by reduced AMPK function. AKH immunolabeling was significantly higher in animals with reduced AMPK function; this result is paralleled by genetic inhibition of synaptic release, suggesting that AMPK promotes AKH secretion. We observed reduced secretion in AKH cells bearing AMPK mutations employing a specific secretion reporter, confirming that AMPK functions in AKH secretion. Live-cell imaging of wild-type AKH neuroendocrine cells shows heightened excitability under reduced sugar levels, and this response was delayed and reduced in AMPK-deficient backgrounds. Furthermore, AMPK activation in AKH cells increases intracellular calcium levels in constant high sugar levels, suggesting that the underlying mechanism of AMPK action is modification of ionic currents. These results demonstrate that AMPK signaling is a critical feature that regulates AKH secretion, and, ultimately, metabolic homeostasis. The significance of these findings is that AMPK is important in the regulation of glucagon signaling, suggesting that the organization of metabolic networks is highly conserved and that AMPK plays a prominent role in these networks. PMID:22798489

  4. Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesis.

    PubMed

    Ferreira, Tânia; Prudêncio, Pedro; Martinho, Rui Gonçalo

    2014-10-15

    Nurse cell dumping is an actin-myosin based process, where 15 nurse cells of a given egg chamber contract and transfer their cytoplasmic content through the ring canals into the growing oocyte. We isolated two mutant alleles of protein kinase N (pkn) and showed that Pkn negatively-regulates activation of the actin-myosin cytoskeleton during the onset of dumping. Using live-cell imaging analysis we observed that nurse cell dumping rates sharply increase during the onset of fast dumping. Such rate increase was severely impaired in pkn mutant nurse cells due to excessive nurse cell actin-myosin activity and/or loss of tissue integrity. Our work demonstrates that the transition between slow and fast dumping is a discrete event, with at least a five to six-fold dumping rate increase. We show that Pkn negatively regulates nurse cell actin-myosin activity. This is likely to be important for directional cytoplasmic flow. We propose Pkn provides a negative feedback loop to help avoid excessive contractility after local activation of Rho GTPase. PMID:25131196

  5. The C-terminal Kinase and ERK-binding Domains of Drosophila S6KII (RSK) Are Required for Phosphorylation of the Protein and Modulation of Circadian Behavior*

    PubMed Central

    Tangredi, Michelle M.; Ng, Fanny S.; Jackson, F. Rob

    2012-01-01

    A detailed structure/function analysis of Drosophila p90 ribosomal S6 kinase (S6KII) or its mammalian homolog RSK has not been performed in the context of neuronal plasticity or behavior. We previously reported that S6KII is required for normal circadian periodicity. Here we report a site-directed mutagenesis of S6KII and analysis of mutants, in vivo, that identifies functional domains and phosphorylation sites critical for the regulation of circadian period. We demonstrate, for the first time, a role for the S6KII C-terminal kinase that is independent of its known role in activation of the N-terminal kinase. Both S6KII C-terminal kinase activity and its ERK-binding domain are required for wild-type circadian period and normal phosphorylation status of the protein. In contrast, the N-terminal kinase of S6KII is dispensable for modulation of circadian period and normal phosphorylation of the protein. We also show that particular sites of S6KII phosphorylation, Ser-515 and Thr-732, are essential for normal circadian behavior. Surprisingly, the phosphorylation of S6KII residues, in vivo, does not follow a strict sequential pattern, as implied by certain cell-based studies of mammalian RSK protein. PMID:22447936

  6. An Amino-Terminal Polo Kinase Interaction Motif Acts in the Regulation of Centrosome Formation and Reveals a Novel Function for centrosomin (cnn) in Drosophila.

    PubMed

    Eisman, Robert C; Phelps, Melissa A S; Kaufman, Thomas

    2015-10-01

    The formation of the pericentriolar matrix (PCM) and a fully functional centrosome in syncytial Drosophila melanogaster embryos requires the rapid transport of Cnn during initiation of the centrosome replication cycle. We show a Cnn and Polo kinase interaction is apparently required during embryogenesis and involves the exon 1A-initiating coding exon, suggesting a subset of Cnn splice variants is regulated by Polo kinase. During PCM formation exon 1A Cnn-Long Form proteins likely bind Polo kinase before phosphorylation by Polo for Cnn transport to the centrosome. Loss of either of these interactions in a portion of the total Cnn protein pool is sufficient to remove native Cnn from the pool, thereby altering the normal localization dynamics of Cnn to the PCM. Additionally, Cnn-Short Form proteins are required for polar body formation, a process known to require Polo kinase after the completion of meiosis. Exon 1A Cnn-LF and Cnn-SF proteins, in conjunction with Polo kinase, are required at the completion of meiosis and for the formation of functional centrosomes during early embryogenesis. PMID:26447129

  7. The kinase Sgg modulates temporal development of macrochaetes in Drosophila by phosphorylation of Scute and Pannier

    PubMed Central

    Yang, Mingyao; Hatton-Ellis, Emma; Simpson, Pat

    2012-01-01

    Evolution of novel structures is often made possible by changes in the timing or spatial expression of genes regulating development. Macrochaetes, large sensory bristles arranged into species-specific stereotypical patterns, are an evolutionary novelty of cyclorraphous flies and are associated with changes in both the temporal and spatial expression of the proneural genes achaete (ac) and scute (sc). Changes in spatial expression are associated with the evolution of cis-regulatory sequences, but it is not known how temporal regulation is achieved. One factor required for ac-sc expression, the expression of which coincides temporally with that of ac-sc in the notum, is Wingless (Wg; also known as Wnt). Wingless downregulates the activity of the serine/threonine kinase Shaggy (Sgg; also known as GSK-3). We demonstrate that Scute is phosphorylated by Sgg on a serine residue and that mutation of this residue results in a form of Sc with heightened proneural activity that can rescue the loss of bristles characteristic of wg mutants. We suggest that the phosphorylated form of Sc has reduced transcriptional activity such that sc is unable to autoregulate, an essential function for the segregation of bristle precursors. Sgg also phosphorylates Pannier, a transcriptional activator of ac-sc, the activity of which is similarly dampened when in the phosphorylated state. Furthermore, we show that Wg signalling does not act directly via a cis-regulatory element of the ac-sc genes. We suggest that temporal control of ac-sc activity in cyclorraphous flies is likely to be regulated by permissive factors and might therefore not be encoded at the level of ac-sc gene sequences. PMID:22159580

  8. Src family kinases are required for WNT5 signaling through the Derailed/RYK receptor in the Drosophila embryonic central nervous system.

    PubMed

    Wouda, Rene R; Bansraj, Monique R K S; de Jong, Anja W M; Noordermeer, Jasprina N; Fradkin, Lee G

    2008-07-01

    Members of the RYK/Derailed family have recently been shown to regulate axon guidance in both Drosophila and mammals by acting as Wnt receptors. Little is known about how the kinase activity-deficient RYKs transduce Wnt signals. Here, we show that the non-receptor Src family tyrosine kinases, SRC64B and SRC42A, are involved in WNT5-mediated signaling through Derailed in the Drosophila embryonic central nervous system. Analysis of animals lacking SRC64B and SRC42A reveals defects in commissure formation similar to those observed in Wnt5 and derailed mutants. Reductions in SRC64B expression levels suppress a Wnt5/derailed-dependent dominant gain-of-function phenotype, and increased levels of either SRC64B or SRC42A enhance Wnt5/derailed-mediated axon commissure switching. Derailed and SRC64B form a complex, which contains catalytically active SRC64B, the formation or stability of which requires SRC64B kinase activity. Furthermore, Derailed is phosphorylated in a SRC64B-dependent manner and coexpression of Derailed and SRC64B results in the activation of SRC64B. The mammalian orthologs of Derailed and SRC64B also form complexes, suggesting that Src roles in RYK signaling are conserved. Finally, we show that coexpression of WNT5 and Derailed has no apparent effect upon TCF/LEF-dependent transcription, suggesting that the WNT5/Derailed signaling pathway is unlikely to directly regulate canonical Wnt pathway targets. Together, these findings indicate that the Src family kinases play novel roles in WNT5/Derailed-mediated signaling. PMID:18539923

  9. Interaction of Omega, Sigma, and Theta Glutathione Transferases with p38b Mitogen-Activated Protein Kinase from the Fruit Fly, Drosophila melanogaster

    PubMed Central

    Wongtrakul, J.; Janphen, K.; Saisawang, C.; Ketterman, A.J.

    2014-01-01

    Glutathione S-transferases (GSTs) are a diverse family of phase II detoxification enzymes found in almost all organisms. Besides playing a major role in the detoxification of xenobiotic and toxic compounds, GSTs are also involved in the regulation of mitogen activated protein (MAP) kinase signal transduction by interaction with proteins in the pathway. An in vitro study was performed for Theta, Omega, Sigma GSTs and their interaction with MAP kinase p38b protein from the fruit fly Drosophila melanogaster Meigen (Diptera: Drosophilidae). The study included the effects of all five Omega class GSTs (DmGSTO1, DmGSTO2a, DmGSTO2b, DmGSTO3, DmGSTO4), all five Theta class GSTs (DmGSTT1, DmGSTT2, DmGSTT3a, DmGSTT3b, DmGSTT4), and one Sigma class glutathione transferase on the activity of Drosophila p38b, including the reciprocal effect of this kinase protein on glutathione transferase activity. It was found that DmGSTT2, DmGSTT3b, DmGSTO1, and DmGSTO3 activated p38b significantly. Substrate specificities of GSTs were also altered after co-incubation with p38b. Although p38b activated DmGSTO1, DmGSTO2a, and DmGSTT2, it inhibited DmGSTT3b and DmGSTO3 activity toward xenobiotic and physiological substrates tested. These results suggest a novel link between Omega and Theta GSTs with the p38b MAP kinase pathway. PMID:25373207

  10. Drosophila homeodomain-interacting protein kinase inhibits the Skp1-Cul1-F-box E3 ligase complex to dually promote Wingless and Hedgehog signaling.

    PubMed

    Swarup, Sharan; Verheyen, Esther M

    2011-06-14

    Drosophila Homeodomain-interacting protein kinase (Hipk) has been shown to regulate in vivo, the stability of Armadillo, the transcriptional effector of Wingless signaling. The Wingless pathway culminates in the stabilization of Armadillo that, in the absence of signaling, is sequentially phosphorylated, polyubiquitinated and degraded. Loss-of-function clones for hipk result in reduced stabilized Armadillo, whereas overexpression of hipk elevates Armadillo levels to promote Wingless-responsive target gene expression. Here, we show that overexpression of hipk can suppress the effects of negative regulators of Armadillo to prevent its degradation in the wing imaginal disc. Hipk acts to stabilize Armadillo by impeding the function of the E3 ubiquitin ligase Skp1-Cul1-F-box (SCF)(Slimb), thereby inhibiting Armadillo ubiquitination and subsequent degradation. Vertebrate Hipk2 displays a similar ability to prevent β-catenin ubiquitination in a functionally conserved mechanism. We find that Hipk's ability to inhibit SCF(Slimb)-mediated ubiquitination is not restricted to Armadillo and extends to other substrates of SCF(Slimb), including the Hedgehog signaling effector Ci. Thus, similar to casein kinase 1 and glycogen synthase kinase 3, Hipk dually regulates both Wingless and Hedgehog signaling by controlling the stability of their respective signaling effectors, but it is the first kinase to our knowledge identified that promotes the stability of both Armadillo and Ci. PMID:21628596

  11. Drosophila casein kinase 2 (CK2) promotes warts protein to suppress Yorkie protein activity for growth control.

    PubMed

    Hu, Lianxin; Huang, Hongling; Li, Jinhui; Yin, Meng-Xin; Lu, Yi; Wu, Wenqing; Zeng, Rong; Jiang, Jin; Zhao, Yun; Zhang, Lei

    2014-11-28

    Drosophila Hippo signaling regulates Wts activity to phosphorylate and inhibit Yki in order to control tissue growth. CK2 is widely expressed and involved in a variety of signaling pathways. In this study we report that Drosophila CK2 promotes Wts activity to phosphorylate and inhibit Yki activity, which is independent of Hpo-induced Wts promotion. In vivo, CK2 overexpression suppresses hpo mutant-induced expanded (Ex) up-regulation and overgrowth phenotype, whereas it cannot affect wts mutant. Consistent with this, knockdown of CK2 up-regulates Hpo pathway target expression. We also found that Drosophila CK2 is essential for tissue growth as a cell death inhibitor as knockdown of CK2 in the developing disc induces severe growth defects as well as caspase3 signals. Taken together, our results uncover a dual role of CK2; although its major role is promoting cell survive, it may potentially be a growth inhibitor as well. PMID:25320084

  12. Drosophila Casein Kinase 2 (CK2) Promotes Warts Protein to Suppress Yorkie Protein Activity for Growth Control*

    PubMed Central

    Hu, Lianxin; Huang, Hongling; Li, Jinhui; Yin, Meng-Xin; Lu, Yi; Wu, Wenqing; Zeng, Rong; Jiang, Jin; Zhao, Yun; Zhang, Lei

    2014-01-01

    Drosophila Hippo signaling regulates Wts activity to phosphorylate and inhibit Yki in order to control tissue growth. CK2 is widely expressed and involved in a variety of signaling pathways. In this study we report that Drosophila CK2 promotes Wts activity to phosphorylate and inhibit Yki activity, which is independent of Hpo-induced Wts promotion. In vivo, CK2 overexpression suppresses hpo mutant-induced expanded (Ex) up-regulation and overgrowth phenotype, whereas it cannot affect wts mutant. Consistent with this, knockdown of CK2 up-regulates Hpo pathway target expression. We also found that Drosophila CK2 is essential for tissue growth as a cell death inhibitor as knockdown of CK2 in the developing disc induces severe growth defects as well as caspase3 signals. Taken together, our results uncover a dual role of CK2; although its major role is promoting cell survive, it may potentially be a growth inhibitor as well. PMID:25320084

  13. skittles, a Drosophila phosphatidylinositol 4-phosphate 5-kinase, is required for cell viability, germline development and bristle morphology, but not for neurotransmitter release.

    PubMed Central

    Hassan, B A; Prokopenko, S N; Breuer, S; Zhang, B; Paululat, A; Bellen, H J

    1998-01-01

    The phosphatidylinositol pathway is implicated in the regulation of numerous cellular functions and responses to extracellular signals. An important branching point in the pathway is the phosphorylation of phosphatidylinositol 4-phosphate by the phosphatidylinositol 4-phosphate 5-kinase (PIP5K) to generate the second messenger phosphatidylinositol 4,5-bis-phosphate (PIP2). PIP5K and PIP2 have been implicated in signal transduction, cytoskeletal regulation, DNA synthesis, and vesicular trafficking. We have cloned and generated mutations in a Drosophila PIP5K type I (skittles). Our analysis indicates that skittles is required for cell viability, germline development, and the proper structural development of sensory bristles. Surprisingly, we found no evidence for PIP5KI involvement in neural secretion. PMID:9832529

  14. Signaling by the Engulfment Receptor Draper: A Screen in Drosophila melanogaster Implicates Cytoskeletal Regulators, Jun N-Terminal Kinase, and Yorkie

    PubMed Central

    Fullard, John F.; Baker, Nicholas E.

    2015-01-01

    Draper, the Drosophila melanogaster homolog of the Ced-1 protein of Caenorhabditis elegans, is a cell-surface receptor required for the recognition and engulfment of apoptotic cells, glial clearance of axon fragments and dendritic pruning, and salivary gland autophagy. To further elucidate mechanisms of Draper signaling, we screened chromosomal deficiencies to identify loci that dominantly modify the phenotype of overexpression of Draper isoform II (suppressed differentiation of the posterior crossvein in the wing). We found evidence for 43 genetic modifiers of Draper II. Twenty-four of the 37 suppressor loci and 3 of the 6 enhancer loci were identified. An additional 5 suppressors and 2 enhancers were identified among mutations in functionally related genes. These studies reveal positive contributions to Drpr signaling for the Jun N-terminal Kinase pathway, supported by genetic interactions with hemipterous, basket, jun, and puckered, and for cytoskeleton regulation as indicated by genetic interactions with rac1, rac2, RhoA, myoblast city, Wiskcott–Aldrich syndrome protein, and the formin CG32138, and for yorkie and expanded. These findings indicate that Jun N-terminal Kinase activation and cytoskeletal remodeling collaborate in Draper signaling. Relationships between Draper signaling and Decapentaplegic signaling, insulin signaling, Salvador/Warts/Hippo signaling, apical-basal cell polarity, and cellular responses to mechanical forces are also discussed. PMID:25395664

  15. Drosophila Aurora-A kinase inhibits neuroblast self-renewal by regulating aPKC/Numb cortical polarity and spindle orientation

    PubMed Central

    Lee, Cheng-Yu; Andersen, Ryan O.; Cabernard, Clemens; Manning, Laurina; Tran, Khoa D.; Lanskey, Marcus J.; Bashirullah, Arash; Doe, Chris Q.

    2006-01-01

    Regulation of stem cell self-renewal versus differentiation is critical for embryonic development and adult tissue homeostasis. Drosophila larval neuroblasts divide asymmetrically to self-renew, and are a model system for studying stem cell self-renewal. Here we identify three mutations showing increased brain neuroblast numbers that map to the aurora-A gene, which encodes a conserved kinase implicated in human cancer. Clonal analysis and time-lapse imaging in aurora-A mutants show single neuroblasts generate multiple neuroblasts (ectopic self-renewal). This phenotype is due to two independent neuroblast defects: abnormal atypical protein kinase C (aPKC)/Numb cortical polarity and failure to align the mitotic spindle with the cortical polarity axis. numb mutant clones have ectopic neuroblasts, and Numb overexpression partially suppresses aurora-A neuroblast overgrowth (but not spindle misalignment). Conversely, mutations that disrupt spindle alignment but not cortical polarity have increased neuroblasts. We conclude that Aurora-A and Numb are novel inhibitors of neuroblast self-renewal and that spindle orientation regulates neuroblast self-renewal. PMID:17182871

  16. Ectopic expression of the Drosophila Cdk1 inhibitory kinases, Wee1 and Myt1, interferes with the second mitotic wave and disrupts pattern formation during eye development.

    PubMed Central

    Price, Donald M; Jin, Zhigang; Rabinovitch, Simon; Campbell, Shelagh D

    2002-01-01

    Wee1 kinases catalyze inhibitory phosphorylation of the mitotic regulator Cdk1, preventing mitosis during S phase and delaying it in response to DNA damage or developmental signals during G2. Unlike yeast, metazoans have two distinct Wee1-like kinases, a nuclear protein (Wee1) and a cytoplasmic protein (Myt1). We have isolated the genes encoding Drosophila Wee1 and Myt1 and are using genetic approaches to dissect their functions during normal development. Overexpression of Dwee1 or Dmyt1 during eye development generates a rough adult eye phenotype. The phenotype can be modified by altering the gene dosage of known regulators of the G2/M transition, suggesting that we could use these transgenic strains in modifier screens to identify potential regulators of Wee1 and Myt1. To confirm this idea, we tested a collection of deletions for loci that can modify the eye overexpression phenotypes and identified several loci as dominant modifiers. Mutations affecting the Delta/Notch signaling pathway strongly enhance a GMR-Dmyt1 eye phenotype but do not affect a GMR-Dwee1 eye phenotype, suggesting that Myt1 is potentially a downstream target for Notch activity during eye development. We also observed interactions with p53, which suggest that Wee1 and Myt1 activity can block apoptosis. PMID:12072468

  17. Polo kinase regulates the localization and activity of the chromosomal passenger complex in meiosis and mitosis in Drosophila melanogaster

    PubMed Central

    Carmena, Mar; Lombardia, Miguel Ortiz; Ogawa, Hiromi; Earnshaw, William C.

    2014-01-01

    Cell cycle progression is regulated by members of the cyclin-dependent kinase (CDK), Polo and Aurora families of protein kinases. The levels of expression and localization of the key regulatory kinases are themselves subject to very tight control. There is increasing evidence that crosstalk between the mitotic kinases provides for an additional level of regulation. We have previously shown that Aurora B activates Polo kinase at the centromere in mitosis, and that the interaction between Polo and the chromosomal passenger complex (CPC) component INCENP is essential in this activation. In this report, we show that Polo kinase is required for the correct localization and activity of the CPC in meiosis and mitosis. Study of the phenotype of different polo allele combinations compared to the effect of chemical inhibition revealed significant differences in the localization and activity of the CPC in diploid tissues. Our results shed new light on the mechanisms that control the activity of Aurora B in meiosis and mitosis. PMID:25376909

  18. Disruption of the protein kinase N gene of Drosophila melanogaster Results in the Recessive delorean Allele (pkndln) With a Negative Impact on Wing Morphogenesis

    PubMed Central

    Sass, Georgette L.; Ostrow, Bruce D.

    2014-01-01

    We describe the delorean mutation of the Drosophila melanogaster protein kinase N gene (pkndln) with defects in wing morphology. Flies homozygous for the recessive pkndln allele have a composite wing phenotype that exhibits changes in relative position and shape of the wing blade as well as loss of specific vein and bristle structures. The pkndln allele is the result of a P-element insertion in the first intron of the pkn locus, and the delorean wing phenotype is contingent upon the interaction of insertion-bearing alleles in trans. The presence of the insertion results in production of a novel transcript that initiates from within the 3′ end of the P-element. The delorean-specific transcript is predicted to produce a wild-type PKN protein. The delorean phenotype is not the result of a reduction in pkn expression, as it could not be recreated using a variety of wing-specific drivers of pkn-RNAi expression. Rather, it is the presence of the delorean-specific transcript that correlates with the mutant phenotype. We consider the delorean wing phenotype to be due to a pairing-dependent, recessive mutation that behaves as a dosage-sensitive, gain of function. Our analysis of genetic interactions with basket and nemo reflects an involvement of pkn and Jun-terminal kinase signaling in common processes during wing differentiation and places PKN as a potential effector of Rho1’s involvement in the Jun-terminal kinase pathway. The delorean phenotype, with its associated defects in wing morphology, provides evidence of a role for PKN in adult morphogenetic processes. PMID:24531729

  19. The Protein Dredd Is an Essential Component of the c-Jun N-terminal Kinase Pathway in the Drosophila Immune Response*

    PubMed Central

    Guntermann, Silvia; Foley, Edan

    2011-01-01

    The Drosophila immune deficiency (IMD) pathway mobilizes c-Jun N-terminal kinase (JNK), caspase, and nuclear factor-κB (NF-κB) modules to counter infection with Gram-negative bacteria. Dredd is an essential caspase in the IMD pathway, and it is widely established that NF-κB activation depends on Dredd. More recent cell culture studies suggested a role for Dredd in the activation of dJNK (Drosophila JNK). However, there are no epistatic or mechanistic data on the involvement of Dredd in dJNK activation. More importantly, there is no in vivo evidence to demonstrate a physiological requirement for Dredd in the IMD/dJNK pathway. We performed a comprehensive analysis of the role of Dredd in the IMD/dJNK pathway, and we demonstrated that Dredd is essential for the activation of IMD/dJNK in cell culture. We positioned Dredd activity at an early point of the IMD/dJNK pathway and uncovered a series of interactions between Dredd and additional proximal IMD pathway molecules. Mechanistically, we showed that the caspase activity inhibitor p35 blocked dJNK activation and the induction of dJNK-dependent genes in cell culture and in vivo. Most importantly, we demonstrated that dredd mutant flies are completely inhibited in their ability to activate dJNK or express dJNK-responsive target genes after bacterial infection in vivo. In conclusion, we established Dredd as an essential component of the IMD pathway required for the full activation of IMD/dJNK in cell culture and in vivo. Our data enhance our appreciation of Dredd-dependent IMD signal transduction events. PMID:21730059

  20. Chromatographic and fluorescence spectroscopic studies of individual 7,12-dimethylbenz(a)anthracene--deoxyribonucleoside adducts

    SciTech Connect

    Moschel, R.C.; Pigott, M.A.; Costantino, N.; Dipple, A.

    1983-09-01

    Compared with standard Sephadex LH-20 column chromatography, a newly developed high pressure liquid chromatographic separation of hydrocarbon deoxyribonucleoside adducts derived from the DNA of mouse embryo cell cultures exposed to 7,12-dimethylbenz(a)anthracene (DMBA) provides markedly superior resolution. Once resolved, the fluorescence spectroscopic properties of the three major DMBA--DNA adducts indicate that the fluorescence exhibited by adducts derived from a bay region syn dihydrodiol epoxide of DMBA differs subtly from that exhibited by adducts derived from the isomeric anti dihydrodiol epoxide.

  1. Enzymatic assay for deoxyribonucleoside triphosphates using synthetic oligonucleotides as template primers

    SciTech Connect

    Sherman, P.A.; Fyfe, J.A. )

    1989-08-01

    The enzymatic assay for deoxyribonucleoside triphosphates has been improved by using synthetic oligonucleotides of a carefully defined sequence as template primers for DNA polymerase. High backgrounds, which limit the sensitivity of the assay when calf thymus DNA or alternating copolymers are used as template primers, were eliminated with these oligonucleotide template primers. Sensitivity was further increased by designing the template primer to incorporate multiple labeled deoxyribonucleotides per limiting unlabeled deoxyribonucleotide. Each of several DNA polymerases exhibited unique reaction characteristics with the oligonucleotide template primers, which was attributed to the differing exonuclease activities associated with these various enzymes. Assay optimization therefore included matching the polymerase with the template primer to obtain the lowest background reaction and highest sensitivity. This modified assay is particularly well suited for keeping cell sample size to a minimum in experimental protocols which generate large numbers of data points or require careful timing of sampling. With this technique, we measured the levels of all four deoxyribonucleoside triphosphates in extracts from as few as 2 x 10(4) cultured cells.

  2. Drosophila Mbm Is a Nucleolar Myc and Casein Kinase 2 Target Required for Ribosome Biogenesis and Cell Growth of Central Brain Neuroblasts

    PubMed Central

    Hovhanyan, Anna; Herter, Eva K.; Pfannstiel, Jens; Gallant, Peter

    2014-01-01

    Proper cell growth is a prerequisite for maintaining repeated cell divisions. Cells need to translate information about intracellular nutrient availability and growth cues from energy-sensing organs into growth-promoting processes, such as sufficient supply with ribosomes for protein synthesis. Mutations in the mushroom body miniature (mbm) gene impair proliferation of neural progenitor cells (neuroblasts) in the central brain of Drosophila melanogaster. Yet the molecular function of Mbm has so far been unknown. Here we show that mbm does not affect the molecular machinery controlling asymmetric cell division of neuroblasts but instead decreases their cell size. Mbm is a nucleolar protein required for small ribosomal subunit biogenesis in neuroblasts. Accordingly, levels of protein synthesis are reduced in mbm neuroblasts. Mbm expression is transcriptionally regulated by Myc, which, among other functions, relays information from nutrient-dependent signaling pathways to ribosomal gene expression. At the posttranslational level, Mbm becomes phosphorylated by casein kinase 2 (CK2), which has an impact on localization of the protein. We conclude that Mbm is a new part of the Myc target network involved in ribosome biogenesis, which, together with CK2-mediated signals, enables neuroblasts to synthesize sufficient amounts of proteins required for proper cell growth. PMID:24615015

  3. Hypertonicity-induced transmitter release at Drosophila neuromuscular junctions is partly mediated by integrins and cAMP/protein kinase A

    NASA Technical Reports Server (NTRS)

    Suzuki, Kazuhiro; Grinnell, Alan D.; Kidokoro, Yoshiaki

    2002-01-01

    The frequency of quantal transmitter release increases upon application of hypertonic solutions. This effect bypasses the Ca(2+) triggering step, but requires the presence of key molecules involved in vesicle fusion, and hence could be a useful tool for dissecting the molecular process of vesicle fusion. We have examined the hypertonicity response at neuromuscular junctions of Drosophila embryos in Ca(2+)-free saline. Relative to wild-type, the response induced by puff application of hypertonic solution was enhanced in a mutant, dunce, in which the cAMP level is elevated, or in wild-type embryos treated with forskolin, an activator of adenylyl cyclase, while protein kinase A (PKA) inhibitors decreased it. The response was also smaller in a mutant, DC0, which lacks the major subunit of PKA. Thus the cAMP/PKA cascade is involved in the hypertonicity response. Peptides containing the sequence Arg-Gly-Asp (RGD), which inhibit binding of integrins to natural ligands, reduced the response, whereas a peptide containing the non-binding sequence Arg-Gly-Glu (RGE) did not. A reduced response persisted in a mutant, myospheroid, which expresses no integrins, and the response in DC0 was unaffected by RGD peptides. These data indicate that there are at lease two components in the hypertonicity response: one that is integrin mediated and involves the cAMP/PKA cascade, and another that is not integrin mediated and does not involve the cAMP/PKA cascade.

  4. Torso, a Drosophila receptor tyrosine kinase, plays a novel role in the larval fat body in regulating insulin signaling and body growth.

    PubMed

    Jun, Jong Woo; Han, Gangsik; Yun, Hyun Myoung; Lee, Gang Jun; Hyun, Seogang

    2016-08-01

    Torso is a receptor tyrosine kinase whose localized activation at the termini of the Drosophila embryo is mediated by its ligand, Trunk. Recent studies have unveiled a second function of Torso in the larval prothoracic gland (PG) as the receptor for the prothoracicotropic hormone, which triggers pupariation. As such, inhibition of Torso in the PG prolongs the larval growth period, thereby increasing the final pupa size. Here, we report that Torso also acts in the larval fat body, regulating body size in a manner opposite from that of Torso in PG. We confirmed the expression of torso mRNA in the larval fat body and its reduction by RNA interference (RNAi). Fat body-specific knockdown of torso, by either of the two independent RNAi transgenes, significantly decreased the final pupal size. We found that torso knockdown suppresses insulin/target of rapamycin (TOR) signaling in the fat body, as confirmed by repression of Akt and S6K. Notably, the decrease in insulin/TOR signaling and decrease of pupal size induced by the knockdown of torso were rescued by the expression of a constitutively active form of the insulin receptor or by the knockdown of FOXO. Our study revealed a novel role for Torso in the fat body with respect to regulation of insulin/TOR signaling and body size. This finding exemplifies the contrasting effects of the same gene expressed in two different organs on organismal physiology. PMID:27126913

  5. The Nonmuscle Myosin Phosphatase PP1β (flapwing) Negatively Regulates Jun N-Terminal Kinase in Wing Imaginal Discs of Drosophila

    PubMed Central

    Kirchner, Jasmin; Gross, Sascha; Bennett, Daimark; Alphey, Luke

    2007-01-01

    Drosophila flapwing (flw) codes for serine/threonine protein phosphatase type 1β (PP1β). Regulation of nonmuscle myosin activity is the single essential flw function that is nonredundant with the three closely related PP1α genes. Flw is thought to dephosphorylate the nonmuscle myosin regulatory light chain, Spaghetti Squash (Sqh); this inactivates the nonmuscle myosin heavy chain, Zipper (Zip). Thus, strong flw mutants lead to hyperphosphorylation of Sqh and hyperactivation of nonmuscle myosin activity. Here, we show genetically that a Jun N-terminal kinase (JNK) mutant suppresses the semilethality of a strong flw allele. Alleles of the JNK phosphatase puckered (puc) genetically enhance the weak allele flw1, leading to severe wing defects. Introducing a mutant of the nonmuscle myosin-binding subunit (Mbs) further enhances this genetic interaction to lethality. We show that puc expression is upregulated in wing imaginal discs mutant for flw1 and pucA251 and that this upregulation is modified by JNK and Zip. The level of phosphorylated (active) JNK is elevated in flw1 enhanced by puc. Together, we show that disruption of nonmuscle myosin activates JNK and puc expression in wing imaginal discs. PMID:17277363

  6. 6-Substituted 2-Aminopurine-2'-deoxyribonucleoside 5'-Triphosphates that Trace Cytosine Methylation.

    PubMed

    von Watzdorf, Janina; Marx, Andreas

    2016-08-17

    Gene expression is extensively regulated by the occurrence and distribution of the epigenetic marker 2'-deoxy 5-methylcytosine (5mC) in genomic DNA. Because of its effects on tumorigenesis there is an important link to human health. In addition, detection of 5mC can serve as an outstanding biomarker for diagnostics as well as for disease therapy. Our previous studies have already shown that, by processing O(6) -alkylated 2'-deoxyguanosine triphosphate (dGTP) analogues, DNA polymerases are able to sense the presence of a single 5mC unit in a template. Here we present the synthesis and evaluation of an extended toolbox of 6-substituted 2-aminopurine-2'-deoxyribonucleoside 5'-triphosphates modified at position 6 with various functionalities. We found that sensing of 5-methylation by this class of nucleotides is more general, not being restricted to O(6) -alkyl modification of dGTP but also applying to other functionalities. PMID:27253512

  7. Spreading depolarization in the brain of Drosophila is induced by inhibition of the Na+/K+-ATPase and mitigated by a decrease in activity of protein kinase G.

    PubMed

    Spong, Kristin E; Rodríguez, Esteban C; Robertson, R Meldrum

    2016-09-01

    Spreading depolarization (SD) is characterized by a massive redistribution of ions accompanied by an arrest in electrical activity that slowly propagates through neural tissue. It has been implicated in numerous human pathologies, including migraine, stroke, and traumatic brain injury, and thus the elucidation of control mechanisms underlying the phenomenon could have many health benefits. Here, we demonstrate the occurrence of SD in the brain of Drosophila melanogaster, providing a model system, whereby cellular mechanisms can be dissected using molecular genetic approaches. Propagating waves of SD were reliably induced by disrupting the extracellular potassium concentration ([K(+)]o), either directly or by inhibition of the Na(+)/K(+)-ATPase with ouabain. The disturbance was monitored by recording the characteristic surges in [K(+)]o using K(+)-sensitive microelectrodes or by monitoring brain activity by measuring direct current potential. With the use of wild-type flies, we show that young adults are more resistant to SD compared with older adults, evidenced by shorter bouts of SD activity and attenuated [K(+)]o disturbances. Furthermore, we show that the susceptibility to SD differs between wild-type flies and w1118 mutants, demonstrating that our ouabain model is influenced by genetic strain. Lastly, flies with low levels of protein kinase G (PKG) had increased latencies to onset of both ouabain-induced SD and anoxic depolarization compared with flies with higher levels. Our findings implicate the PKG pathway as a modulator of SD in the fly brain, and given the conserved nature of the signaling pathway, it could likely play a similar role during SD in the mammalian central nervous system. PMID:27358319

  8. Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism.

    PubMed Central

    Lu, Q; Inouye, M

    1996-01-01

    Nucleoside diphosphate (NDP) kinase is a ubiquitous nonspecific enzyme that evidently is designed to catalyze in vivo ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the corresponding diphosphates. Because Escherichia coli contains only one copy of ndk, the structural gene for this enzyme, we were surprised to find that ndk disruption yields bacteria that are still viable. These mutant cells contain a protein with a small amount NDP kinase activity. The protein responsible for this activity was purified and identified as adenylate kinase. This enzyme, also called myokinase, catalyzes the reversible ATP-dependent synthesis of ADP from AMP. We found that this enzyme from E. coli as well as from higher eukaryotes has a broad substrate specificity displaying dual enzymatic functions. Among the nucleoside monophosphate kinases tested, only adenylate kinase was found to have NDP kinase activity. To our knowledge, this is the first report of NDP kinase activity associated with adenylate kinase. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8650159

  9. Studies on Transcriptional Incorporation of 5’-N-Triphosphates of 5’-Amino-5’-Deoxyribonucleosides

    PubMed Central

    Kotkowiak, Weronika; Pasternak, Anna; Kierzek, Ryszard

    2016-01-01

    In this study, several RNA polymerases were used for the first time to examine the possibility of transcriptional incorporation of 5’-N-triphosphates of 5’-amino-5’-deoxyribonucleosides (5’NH NTPs). The T3, T7, Sp6 and T7 Y639F RNA polymerases were employed to show that the full-length transcript cannot be synthesized. The results suggest that the application of 5’NH NTPs could decrease transcription reaction rates. What is more, the modification of transcription conditions had no influence on the rate of 5’NH NTPs incorporation. Based on experimental data it is postulated that 5’NH NTPs can be used as potential transcription inhibitors. Our findings expand the knowledge on suitable uses of the 5’-N-triphosphates of 5’-amino-5’-deoxyribonucleoside and the exact mechanism of transcriptional inhibition. PMID:26829482

  10. Enzyme Kinetics of the Mitochondrial Deoxyribonucleoside Salvage Pathway Are Not Sufficient to Support Rapid mtDNA Replication

    PubMed Central

    Gandhi, Vishal V.; Samuels, David C.

    2011-01-01

    Using a computational model, we simulated mitochondrial deoxynucleotide metabolism and mitochondrial DNA replication. Our results indicate that the output from the mitochondrial salvage enzymes alone is inadequate to support a mitochondrial DNA replication duration of as long as 10 hours. We find that an external source of deoxyribonucleoside diphosphates or triphosphates (dNTPs), in addition to those supplied by mitochondrial salvage, is essential for the replication of mitochondrial DNA to complete in the experimentally observed duration of approximately 1 to 2 hours. For meeting a relatively fast replication target of 2 hours, almost two-thirds of the dNTP requirements had to be externally supplied as either deoxyribonucleoside di- or triphosphates, at about equal rates for all four dNTPs. Added monophosphates did not suffice. However, for a replication target of 10 hours, mitochondrial salvage was able to provide for most, but not all, of the total substrate requirements. Still, additional dGTPs and dATPs had to be supplied. Our analysis of the enzyme kinetics also revealed that the majority of enzymes of this pathway prefer substrates that are not precursors (canonical deoxyribonucleosides and deoxyribonucleotides) for mitochondrial DNA replication, such as phosphorylated ribonucleotides, instead of the corresponding deoxyribonucleotides. The kinetic constants for reactions between mitochondrial salvage enzymes and deoxyribonucleotide substrates are physiologically unreasonable for achieving efficient catalysis with the expected in situ concentrations of deoxyribonucleotides. PMID:21829339

  11. Strategies for synthesis of adducts of omicron-quinone metabolites of carcinogenic polycyclic aromatic hydrocarbons with 2'-deoxyribonucleosides.

    PubMed

    Ran, Chongzhao; Dai, Qing; Ruan, Qian; Penning, Trevor M; Blair, Ian A; Harvey, Ronald G

    2008-02-01

    Polycyclic aromatic hydrocarbons (PAHs) are major environmental carcinogens produced in the combustion of fossil fuels, tobacco, and other organic matter. Current evidence indicates that PAHs are transformed enzymatically to active metabolites that react with DNA to form adducts that result in mutations. Three activation pathways have been proposed: the diol epoxide path, the radical-cation path, and the quinone path. The latter involves aldo-keto reductase mediated oxidation of PAH dihydrodiol metabolites to catechols that enter into redox cycles with quinones. This results in generation of reactive oxygen species (ROS) that attack DNA, and the PAH quinones also react with DNA to form adducts. Several strategies for synthesis of the stable adducts formed by the o-quinone metabolites of carcinogenic PAHs with 2'-deoxyribonucleosides were investigated and compared. The PAH quinones studied were benz[a]anthracene-3,4-dione and its 7-methyl- and 7,12-dimethyl- derivatives. The parent PAHs represent a range of carcinogenicity from inactive to highly potent. Two synthetic methods were devised that differ in the catalyst employed, Pd(OAc)(2) or CuI. The Pd-mediated method involved coupling a protected amino-catechol PAH derivative with a halo-2'-deoxyribonucleoside. The copper-mediated method entailed reaction of a halo-PAH catechol derivative with a 2'-deoxyribonucleoside. Adducts of benz[a]anthracene-3,4-dione (and its 7-methyl- and 7,12-dimethyl- derivatives) with 2'-deoxyadenosine and 2'-deoxyguanosine were prepared by these methods. Availability of adducts of these types through synthesis makes possible for the first time biological studies to determine the role of these adducts in tumorigenesis. The copper-mediated method offers advantages of economy, adaptability to large-scale preparation, utility for synthesis of (13)C- or (15)N-labeled analogues, and nonformation of bis-adducts as secondary products. PMID:18181642

  12. The Gyc76C Receptor Guanylyl Cyclase and the Foraging cGMP-Dependent Kinase Regulate Extracellular Matrix Organization and BMP Signaling in the Developing Wing of Drosophila melanogaster

    PubMed Central

    Schleede, Justin; Blair, Seth S.

    2015-01-01

    The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, we identify Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. We show that Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. We identify a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases, and show that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. We discuss parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues. PMID:26440503

  13. A review comparing deoxyribonucleoside triphosphate (dNTP) concentrations in the mitochondrial and cytoplasmic compartments of normal and transformed cells

    PubMed Central

    Gandhi, Vishal V.; Samuels, David C.

    2011-01-01

    The deoxyribonucleoside triphosphate (dNTP) pools that support the replication of mitochondrial DNA are physically separated from the rest of the cell by the double membrane of the mitochondria. Perturbed homeostasis of mitochondrial dNTP pools is associated with a set of severe diseases collectively termed mitochondrial DNA depletion syndromes. The degree of interaction of the mitochondrial dNTP pools with the corresponding dNTP pools in the cytoplasm is currently not clear. We reviewed the literature on previously reported simultaneous measurements of mitochondrial and cytoplasmic deoxyribonucleoside triphosphate pools to investigate and quantify the extent of the influence of the cytoplasmic nucleotide metabolism on mitochondrial dNTP pools. We converted the reported measurements to concentrations creating a catalog of paired mitochondrial and cytoplasmic dNTP concentration measurements. Over experiments from multiple laboratories, dNTP concentrations in the mitochondria are highly correlated with dNTP concentrations in the cytoplasm in normal cells in culture (Pearson R = 0.79, p = 3 × 10-7) but not in transformed cells. For dTTP and dATP there was a strong linear relationship between the cytoplasmic and mitochondrial concentrations in normal cells. From this linear model we hypothesize that the salvage pathway within the mitochondrion is only capable of forming a concentration of approximately 2 μM of dTTP and dATP, and that higher concentrations require transport of deoxyribonucleotides from the cytoplasm. PMID:21774628

  14. The wavy Mutation Maps to the Inositol 1,4,5-Trisphosphate 3-Kinase 2 (IP3K2) Gene of Drosophila and Interacts with IP3R to Affect Wing Development

    PubMed Central

    Dean, Derek M.; Maroja, Luana S.; Cottrill, Sarah; Bomkamp, Brent E.; Westervelt, Kathleen A.; Deitcher, David L.

    2015-01-01

    Inositol 1,4,5-trisphosphate (IP3) regulates a host of biological processes from egg activation to cell death. When IP3-specific receptors (IP3Rs) bind to IP3, they release calcium from the ER into the cytoplasm, triggering a variety of cell type- and developmental stage-specific responses. Alternatively, inositol polyphosphate kinases can phosphorylate IP3; this limits IP3R activation by reducing IP3 levels, and also generates new signaling molecules altogether. These divergent pathways draw from the same IP3 pool yet cause very different cellular responses. Therefore, controlling the relative rates of IP3R activation vs. phosphorylation of IP3 is essential for proper cell functioning. Establishing a model system that sensitively reports the net output of IP3 signaling is crucial for identifying the controlling genes. Here we report that mutant alleles of wavy (wy), a classic locus of the fruit fly Drosophila melanogaster, map to IP3 3-kinase 2 (IP3K2), a member of the inositol polyphosphate kinase gene family. Mutations in wy disrupt wing structure in a highly specific pattern. RNAi experiments using GAL4 and GAL80ts indicated that IP3K2 function is required in the wing discs of early pupae for normal wing development. Gradations in the severity of the wy phenotype provide high-resolution readouts of IP3K2 function and of overall IP3 signaling, giving this system strong potential as a model for further study of the IP3 signaling network. In proof of concept, a dominant modifier screen revealed that mutations in IP3R strongly suppress the wy phenotype, suggesting that the wy phenotype results from reduced IP4 levels, and/or excessive IP3R signaling. PMID:26613949

  15. The wavy Mutation Maps to the Inositol 1,4,5-Trisphosphate 3-Kinase 2 (IP3K2) Gene of Drosophila and Interacts with IP3R to Affect Wing Development.

    PubMed

    Dean, Derek M; Maroja, Luana S; Cottrill, Sarah; Bomkamp, Brent E; Westervelt, Kathleen A; Deitcher, David L

    2016-02-01

    Inositol 1,4,5-trisphosphate (IP3) regulates a host of biological processes from egg activation to cell death. When IP3-specific receptors (IP3Rs) bind to IP3, they release calcium from the ER into the cytoplasm, triggering a variety of cell type- and developmental stage-specific responses. Alternatively, inositol polyphosphate kinases can phosphorylate IP3; this limits IP3R activation by reducing IP3 levels, and also generates new signaling molecules altogether. These divergent pathways draw from the same IP3 pool yet cause very different cellular responses. Therefore, controlling the relative rates of IP3R activation vs. phosphorylation of IP3 is essential for proper cell functioning. Establishing a model system that sensitively reports the net output of IP3 signaling is crucial for identifying the controlling genes. Here we report that mutant alleles of wavy (wy), a classic locus of the fruit fly Drosophila melanogaster, map to IP3 3-kinase 2 (IP3K2), a member of the inositol polyphosphate kinase gene family. Mutations in wy disrupt wing structure in a highly specific pattern. RNAi experiments using GAL4 and GAL80(ts) indicated that IP3K2 function is required in the wing discs of early pupae for normal wing development. Gradations in the severity of the wy phenotype provide high-resolution readouts of IP3K2 function and of overall IP3 signaling, giving this system strong potential as a model for further study of the IP3 signaling network. In proof of concept, a dominant modifier screen revealed that mutations in IP3R strongly suppress the wy phenotype, suggesting that the wy phenotype results from reduced IP4 levels, and/or excessive IP3R signaling. PMID:26613949

  16. Vacuolar protein sorting 35 (Vps35) rescues locomotor deficits and shortened lifespan in Drosophila expressing a Parkinson’s disease mutant of Leucine-rich repeat kinase 2 (LRRK2)

    PubMed Central

    2014-01-01

    Background Parkinson’s disease (PD) is the most common movement neurodegenerative movement disorder. An incomplete understanding of the molecular pathways involved in its pathogenesis impedes the development of effective disease-modifying treatments. To address this gap, we have previously generated a Drosophila model of PD that overexpresses PD pathogenic mutant form of the second most common causative gene of PD, Leucine-Rich Repeat Kinase 2 (LRRK2). Findings We employed this model in a genetic modifier screen and identified a gene that encodes for a core subunit of retromer – a complex essential for the sorting and recycling of specific cargo proteins from endosomes to the trans-Golgi network and cell surface. We present evidence that overexpression of the Vps35 or Vps26 component of the cargo-recognition subunit of the retromer complex ameliorates the pathogenic mutant LRRK2 eye phenotype. Furthermore, overexpression of Vps35 or Vps26 significantly protects from the locomotor deficits observed in mutant LRRK2 flies, as assessed by the negative geotaxis assay, and rescues their shortened lifespan. Strikingly, overexpressing Vps35 alone protects from toxicity of rotenone, a neurotoxin commonly used to model parkinsonism, both in terms of lifespan and locomotor activity of the flies, and this protection is sustained and even augmented in the presence of mutant LRRK2. Finally, we demonstrate that knocking down expression of Vps35 in dopaminergic neurons causes a significant locomotor impairment. Conclusions From these results we conclude that LRRK2 plays a role in the retromer pathway and that this pathway is involved in PD pathogenesis. PMID:24915984

  17. Binding of Mn-deoxyribonucleoside Triphosphates to the Active Site of the DNA Polymerase of Bacteriophage T7

    SciTech Connect

    B Akabayov; C Richardson

    2011-12-31

    Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg{sup 2+}, as an example, mediates binding of deoxyribonucleoside 5'-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg{sup 2+} to an active site because Mg{sup 2+} is spectroscopically silent and Mg{sup 2+} binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg{sup 2+} with Mn{sup 2+}:Mn{sup 2+} that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstrate that the majority of Mn{sup 2+} is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn{sup 2+} that is free in solution and Mn{sup 2+} bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.

  18. Trichomonas vaginalis thymidine kinase: purification, characterization and search for inhibitors.

    PubMed Central

    Strosselli, S; Spadari, S; Walker, R T; Basnak, I; Focher, F

    1998-01-01

    We report that a thymidine kinase (TK) activity is present in Trichomonas vaginalis and can be separated from the deoxyribonucleoside phosphotransferase. T. vaginalis TK, purified 11200-fold to apparent homogeneity, has a molecular mass of 31500 Da. It phosphorylates not only thymidine (Km 0.18 microM) but also deoxycytidine (Km 0.88 microM) and deoxyuridine (Km 0.14 microM). In contrast with T. vaginalis deoxyribonucleoside phosphotransferase, the TK activity is strongly inhibited by novel deoxyuridine analogues such as 5-methyl-4'-thio-2'-deoxyuridine (MTdU) (Ki 20 nM) and 5-iodo-4'-thio-2'-deoxyuridine (ITdU) (Ki 24 nM). MTdU and ITdU are phosphorylated by T. vaginalis TK in vitro. In vivo they inhibit [3H]thymidine incorporation in T. vaginalis cultured cells and T. vaginalis growth (IC50 7.5 and 24 microM respectively; minimal lethal dose 100 microM). Thus the TK inhibitors described here demonstrate the key role of T. vaginalis TK for protozoal growth and viability and indicate TK as a new target for the design of antitrichomonal drugs. PMID:9693096

  19. Insulin receptor in Drosophila melanogaster

    SciTech Connect

    Petruzzelli, L.; Herrera, R.; Rosen, O.

    1986-05-01

    A specific, high affinity insulin receptor is present in both adult Drosophila and in Drosophila embryos. Wheat germ lectin-enriched extracts of detergent-solubilized membranes from embryos and adults bind insulin with a K/sub d/ of 15 nM. Binding is specific for insulin; micromolar concentrations of proinsulin, IGFI, and IGFII are required to displace bound /sup 125/I-insulin. Insulin-dependent protein tyrosine kinase activity appears during embryogenesis. It is evident between 6 and 12 hours of development, peaks between 12 and 18 hours and falls in the adult. During 0-6 hours of embryogenesis, and in the adult, a specific protein band (Mr = 135,000) is crosslinked to /sup 125/I-insulin. During 6-12 and 12-18 hours of embryogenesis stages in which insulin-dependent protein tyrosine kinase is high, an additional band (Mr = 100,000) becomes crosslinked to /sup 125/I-insulin. Isolation and DNA sequence analysis of genomic clones encoding the Drosophila insulin receptor will be presented as will the characterization of insulin receptor mRNA's during development.

  20. Drosophila Photoreceptors and Signaling Mechanisms

    PubMed Central

    Katz, Ben; Minke, Baruch

    2009-01-01

    Fly eyes have been a useful biological system in which fundamental principles of sensory signaling have been elucidated. The physiological optics of the fly compound eye, which was discovered in the Musca, Calliphora and Drosophila flies, has been widely exploited in pioneering genetic and developmental studies. The detailed photochemical cycle of bistable photopigments has been elucidated in Drosophila using the genetic approach. Studies of Drosophila phototransduction using the genetic approach have led to the discovery of novel proteins crucial to many biological processes. A notable example is the discovery of the inactivation no afterpotential D scaffold protein, which binds the light-activated channel, its activator the phospholipase C and it regulator protein kinase C. An additional protein discovered in the Drosophila eye is the light-activated channel transient receptor potential (TRP), the founding member of the diverse and widely spread TRP channel superfamily. The fly eye has thus played a major role in the molecular identification of processes and proteins with prime importance. PMID:19623243

  1. Drosophila spermiogenesis

    PubMed Central

    Fabian, Lacramioara; Brill, Julie A.

    2012-01-01

    Drosophila melanogaster spermatids undergo dramatic morphological changes as they differentiate from small round cells approximately 12 μm in diameter into highly polarized, 1.8 mm long, motile sperm capable of participating in fertilization. During spermiogenesis, syncytial cysts of 64 haploid spermatids undergo synchronous differentiation. Numerous changes occur at a subcellular level, including remodeling of existing organelles (mitochondria, nuclei), formation of new organelles (flagellar axonemes, acrosomes), polarization of elongating cysts and plasma membrane addition. At the end of spermatid morphogenesis, organelles, mitochondrial DNA and cytoplasmic components not needed in mature sperm are stripped away in a caspase-dependent process called individualization that results in formation of individual sperm. Here, we review the stages of Drosophila spermiogenesis and examine our current understanding of the cellular and molecular mechanisms involved in shaping male germ cell-specific organelles and forming mature, fertile sperm. PMID:23087837

  2. Drosophila myogenesis.

    PubMed

    Bothe, Ingo; Baylies, Mary K

    2016-09-12

    The skeletal muscle system is the largest organ in motile animals, constituting between 35 and 55% of the human body mass, and up to 75% of the body mass in flying organisms like Drosophila. The flight muscles alone in flying insects comprise up to 65% of total body mass. Not only is the musculature the largest organ system, it is also exquisitely complex, with single muscles existing in different shapes and sizes. These different morphologies allow for such different functions as the high-frequency beating of a wing in a hummingbird, the dilation of the pupil in a human eye, or the maintenance of posture in a giraffe's neck. PMID:27623256

  3. Yeast Dun1 Kinase Regulates Ribonucleotide Reductase Inhibitor Sml1 in Response to Iron Deficiency

    PubMed Central

    Sanvisens, Nerea; Romero, Antonia M.; An, Xiuxiang; Zhang, Caiguo; de Llanos, Rosa; Martínez-Pastor, María Teresa; Bañó, M. Carmen

    2014-01-01

    Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox-active cofactor in many biological processes, including DNA replication and repair. Eukaryotic ribonucleotide reductases (RNRs) are Fe-dependent enzymes that catalyze deoxyribonucleoside diphosphate (dNDP) synthesis. We show here that the levels of the Sml1 protein, a yeast RNR large-subunit inhibitor, specifically decrease in response to both nutritional and genetic Fe deficiencies in a Dun1-dependent but Mec1/Rad53- and Aft1-independent manner. The decline of Sml1 protein levels upon Fe starvation depends on Dun1 forkhead-associated and kinase domains, the 26S proteasome, and the vacuolar proteolytic pathway. Depletion of core components of the mitochondrial iron-sulfur cluster assembly leads to a Dun1-dependent diminution of Sml1 protein levels. The physiological relevance of Sml1 downregulation by Dun1 under low-Fe conditions is highlighted by the synthetic growth defect observed between dun1Δ and fet3Δ fet4Δ mutants, which is rescued by SML1 deletion. Consistent with an increase in RNR function, Rnr1 protein levels are upregulated upon Fe deficiency. Finally, dun1Δ mutants display defects in deoxyribonucleoside triphosphate (dNTP) biosynthesis under low-Fe conditions. Taken together, these results reveal that the Dun1 checkpoint kinase promotes RNR function in response to Fe starvation by stimulating Sml1 protein degradation. PMID:24958100

  4. Visual Pattern Memory Requires "Foraging" Function in the Central Complex of "Drosophila"

    ERIC Educational Resources Information Center

    Wang, Zhipeng; Pan, Yufeng; Li, Weizhe; Jiang, Huoqing; Chatzimanolis, Lazaros; Chang, Jianhong; Gong, Zhefeng; Liu, Li

    2008-01-01

    The role of the "foraging" ("for)" gene, which encodes a cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG), in food-search behavior in "Drosophila" has been intensively studied. However, its functions in other complex behaviors have not been well-characterized. Here, we show experimentally in "Drosophila" that the "for"…

  5. MST kinases in development and disease

    PubMed Central

    2015-01-01

    The mammalian MST kinase family, which is related to the Hippo kinase in Drosophila melanogaster, includes five related proteins: MST1 (also called STK4), MST2 (also called STK3), MST3 (also called STK24), MST4, and YSK1 (also called STK25 or SOK1). MST kinases are emerging as key signaling molecules that influence cell proliferation, organ size, cell migration, and cell polarity. Here we review the regulation and function of these kinases in normal physiology and pathologies, including cancer, endothelial malformations, and autoimmune disease. PMID:26370497

  6. Assays To Detect the Formation of Triphosphates of Unnatural Nucleotides: Application to Escherichia coli Nucleoside Diphosphate Kinase.

    PubMed

    Matsuura, Mariko F; Shaw, Ryan W; Moses, Jennifer D; Kim, Hyo-Joong; Kim, Myong-Jung; Kim, Myong-Sang; Hoshika, Shuichi; Karalkar, Nilesh; Benner, Steven A

    2016-03-18

    One frontier in synthetic biology seeks to move artificially expanded genetic information systems (AEGIS) into natural living cells and to arrange the metabolism of those cells to allow them to replicate plasmids built from these unnatural genetic systems. In addition to requiring polymerases that replicate AEGIS oligonucleotides, such cells require metabolic pathways that biosynthesize the triphosphates of AEGIS nucleosides, the substrates for those polymerases. Such pathways generally require nucleoside and nucleotide kinases to phosphorylate AEGIS nucleosides and nucleotides on the path to these triphosphates. Thus, constructing such pathways focuses on engineering natural nucleoside and nucleotide kinases, which often do not accept the unnatural AEGIS biosynthetic intermediates. This, in turn, requires assays that allow the enzyme engineer to follow the kinase reaction, assays that are easily confused by ATPase and other spurious activities that might arise through "site-directed damage" of the natural kinases being engineered. This article introduces three assays that can detect the formation of both natural and unnatural deoxyribonucleoside triphosphates, assessing their value as polymerase substrates at the same time as monitoring the progress of kinase engineering. Here, we focus on two complementary AEGIS nucleoside diphosphates, 6-amino-5-nitro-3-(1'-β-D-2'-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one. These assays provide new ways to detect the formation of unnatural deoxyribonucleoside triphosphates in vitro and to confirm their incorporation into DNA. Thus, these assays can be used with other unnatural nucleotides. PMID:26829203

  7. The Drosophila EKC/KEOPS complex

    PubMed Central

    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 (t6A) 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 t6A-modified tRNA availability with translational rates, which are ultimately reflected in animal growth. PMID:23823807

  8. Bromovinyl-deoxyuridine: A selective substrate for mitochondrial thymidine kinase in cell extracts

    SciTech Connect

    Franzolin, Elisa; Rampazzo, Chiara; Perez-Perez, Maria-Jesus; Hernandez, Ana-Isabel; Balzarini, Jan; Bianchi, Vera . E-mail: vbianchi@mail.bio.unipd.it

    2006-05-26

    Cellular models of mitochondrial thymidine kinase (TK2) deficiency require a reliable method to measure TK2 activity in whole cell extracts containing two interfering deoxyribonucleoside kinases, thymidine kinase 1 (TK1) and deoxycytidine kinase. We tested the value of the thymidine analog (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) as a TK2-specific substrate. With extracts of OSTTK1{sup -} cells containing TK2 as the only thymidine kinase and a highly specific TK2 inhibitor we established conditions to detect the low TK2 activity commonly present in cells. With extracts of TK1-proficient osteosarcoma cells and normal human fibroblasts we showed that BVDU, but not 1-({beta}-D-arabinofuranosyl)thymine (Ara-T), discriminates TK2 activity even in the presence of 100-fold excess TK1. A comparison with current procedures based on TK2 inhibition demonstrated the better performance of the new TK2 assay. When cultured human fibroblasts passed from proliferation to quiescence TK2 activity increased by 3-fold, stressing the importance of TK2 function in the absence of TK1.

  9. The Drosophila visual system

    PubMed Central

    Zhu, Yan

    2013-01-01

    A compact genome and a tiny brain make Drosophila the prime model to understand the neural substrate of behavior. The neurogenetic efforts to reveal neural circuits underlying Drosophila vision started about half a century ago, and now the field is booming with sophisticated genetic tools, rich behavioral assays, and importantly, a greater number of scientists joining from different backgrounds. This review will briefly cover the structural anatomy of the Drosophila visual system, the animal’s visual behaviors, the genes involved in assembling these circuits, the new and powerful techniques, and the challenges ahead for ultimately identifying the general principles of biological computation in the brain.   A typical brain utilizes a great many compact neural circuits to collect and process information from the internal biological and external environmental worlds and generates motor commands for observable behaviors. The fruit fly Drosophila melanogaster, despite of its miniature body and tiny brain, can survive in almost any corner of the world.1 It can find food, court mate, fight rival conspecific, avoid predators, and amazingly fly without crashing into trees. Drosophila vision and its underlying neuronal machinery has been a key research model for at least half century for neurogeneticists.2 Given the efforts invested on the visual system, this animal model is likely to offer the first full understanding of how visual information is computed by a multi-cellular organism. Furthermore, research in Drosophila has revealed many genes that play crucial roles in the formation of functional brains across species. The architectural similarities between the visual systems of Drosophila and vertebrate at the molecular, cellular, and network levels suggest new principles discovered at the circuit level on the relationship between neurons and behavior in Drosophila shall also contribute greatly to our understanding of the general principles for how bigger brains work.3

  10. Drosophila Blastorderm Analysis Software

    SciTech Connect

    2006-10-25

    PointCloudMake analyzes 3D fluorescent images of whole Drosophila embryo and produces a table-style "PointCloud" file which contains the coordinates and volumes of all the nuclei, cells, their associated relative gene expression levels along with morphological features of the embryo. See: Luengo Hendrix et at 2006 3D Morphology and Gene Expression in the Drosophila Blastoderm at Cellular Resolution manuscript submitted LBNL # LBNL-60178 Knowles DW, Keranen SVE, Biggin M. Sudar S (2002) Mapping organism expression levels at cellular resolution in developing Drosophila. In: Conchello JA, Cogswell CJ, Wilson T, editors. Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing IX. pp. 57-64

  11. Pyrimidine and purine salvage deoxyribonucleoside metabolism in hepatic and renal homogenates from rats pretreated with propylthiouracil or L-thyroxine.

    PubMed

    Karbownik, Malgorzata; Modrzejewska, Hanna; Reiter, Russel J.; Zasada, Krzysztof; Greger, Janusz; Lewinski, Andrzej

    2000-01-01

    OBJECTIVES: In vitro activities of thymidine kinase (TK, EC 2.7.1.21), adenosine kinase (AK, EC 2.7.1.20) and deoxycytidine kinase (dCK, EC 2.7.1.74) enzymes involved in the salvage pathway of DNA precursor synthesis, in homogenates of the rat liver and kidney, were examined. Type I iodothyronine-5'-deiodinase (5'D-I) is the main enzyme responsible for peripheral metabolism of thyroid hormones. This occurs especially in the liver, kidney and muscle. The activity of 5'D-I is inhibited bypropylthiouracil (PTU), an antithyroid drug. METHODS: The liver and kidney were collected from rats pretreated in vivo with either a 0.1% solution of PTU in drinking water for 2 weeks or injected with levothyroxine (L-T(4), 50 &mgr;g/kg BW, daily) for 2 weeks. The enzyme activities were measured by ascending chromatography and expressed asthe amounts of radioactive reaction products of the phosphorylation of dThd (for TK), ofdAdo (for AK and dCK) and of dGuo (for dCK). RESULTS: In liver homogenates, PTU-pretreatment decreased the activities of the three enzymes when compared to control values and those of L-T(4)-treated animals; also L-T(4) injections decreased the AK and dCK activities in the liver homogenates. PTU-pretreatment increased TK activity and the rate of dGuo phosphorylation in kidney homogenates, when compared to controls and to the L-T(4)-pretreated animals. Conversely, both PTU- and L-T(4)-pretreatment reduced the rate of dAdo phosphorylation in kidney homogenates. CONCLUSION: Changes in the activities of examined enzymes which participate inpyrimidine orpurine metabolism of the salvage pathway of DNA synthesis in the liver afterPTU-pretreatment (as shown herein) are similar to the changes of the 5'D-I activity after PTU-treatment (as reported by others). Thus, the observations suggest a role of the salvage pathway of DNA synthesis in the peripheral metabolism of thyroid hormones. PMID:11455332

  12. 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

  13. In focus: spotted wing drosophila, Drosophila suzukii, across perspectives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An effective response to the invasion of spotted wing Drosophila (SWD), Drosophila suzukii, requires proper taxonomic identification at the initial phase, understanding its basic biology and phenology, developing management tools, transferring information and technology quickly to user groups, and e...

  14. Immunohistological techniques for studying the Drosophila male germline stem cell.

    PubMed

    Singh, Shree Ram; Hou, Steven X

    2008-01-01

    Stem cells are undifferentiated cells that have a remarkable ability to self-renew and produce differentiated cells that support normal development and tissue homeostasis. This unique capacity makes stem cells a powerful tool for future regenerative medicine and gene therapy. Accumulative evidence suggests that stem cell self-renewal or differentiation is controlled by both intrinsic and extrinsic factors, and that deregulation of stem cell behavior results in cancer formation, tissue degeneration, and premature aging. The Drosophila testis provides an excellent in vivo model for studying and understanding the fundamental cellular and molecular mechanisms controlling stem cell behavior and the relationship between niches and stem cells. At the tip of the Drosophila testes, germline stem cells (GSCs) and somatic stem cells (SSCs) contact each other and share common niches (known as a hub) to maintain spermatogenesis. Signaling pathways, such as the Janus kinase (JAK)/signal transducer and activator of transcription (STAT), bone morphogenetic protein (BMP), ras-associated protein-guanine nucleotide exchange factor for small GTPase (Rap-GEF), and epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK), are known to regulate self-renewal or differentiation of Drosophila male germline stem cells. We describe the detailed in vivo immunohistological protocols that mark GSCs, SSCs, and their progeny in Drosophila testes. PMID:18370050

  15. Heterochromatin remodeling by CDK12 contributes to learning in Drosophila

    PubMed Central

    Pan, Lixia; Xie, Wenbing; Li, Kai-Le; Yang, Zhihao; Xu, Jiang; Zhang, Wenhao; Liu, Lu-Ping; Ren, Xingjie; He, Zhimin; Wu, Junyu; Sun, Jin; Wei, Hui-Min; Wang, Daliang; Xie, Wei; Li, Wei; Ni, Jian-Quan; Sun, Fang-Lin

    2015-01-01

    Dynamic regulation of chromatin structure is required to modulate the transcription of genes in eukaryotes. However, the factors that contribute to the plasticity of heterochromatin structure are elusive. Here, we report that cyclin-dependent kinase 12 (CDK12), a transcription elongation-associated RNA polymerase II (RNAPII) kinase, antagonizes heterochromatin enrichment in Drosophila chromosomes. Notably, loss of CDK12 induces the ectopic accumulation of heterochromatin protein 1 (HP1) on euchromatic arms, with a prominent enrichment on the X chromosome. Furthermore, ChIP and sequencing analysis reveals that the heterochromatin enrichment on the X chromosome mainly occurs within long genes involved in neuronal functions. Consequently, heterochromatin enrichment reduces the transcription of neuronal genes in the adult brain and results in a defect in Drosophila courtship learning. Taken together, these results define a previously unidentified role of CDK12 in controlling the epigenetic transition between euchromatin and heterochromatin and suggest a chromatin regulatory mechanism in neuronal behaviors. PMID:26508632

  16. Heterochromatin remodeling by CDK12 contributes to learning in Drosophila.

    PubMed

    Pan, Lixia; Xie, Wenbing; Li, Kai-Le; Yang, Zhihao; Xu, Jiang; Zhang, Wenhao; Liu, Lu-Ping; Ren, Xingjie; He, Zhimin; Wu, Junyu; Sun, Jin; Wei, Hui-Min; Wang, Daliang; Xie, Wei; Li, Wei; Ni, Jian-Quan; Sun, Fang-Lin

    2015-11-10

    Dynamic regulation of chromatin structure is required to modulate the transcription of genes in eukaryotes. However, the factors that contribute to the plasticity of heterochromatin structure are elusive. Here, we report that cyclin-dependent kinase 12 (CDK12), a transcription elongation-associated RNA polymerase II (RNAPII) kinase, antagonizes heterochromatin enrichment in Drosophila chromosomes. Notably, loss of CDK12 induces the ectopic accumulation of heterochromatin protein 1 (HP1) on euchromatic arms, with a prominent enrichment on the X chromosome. Furthermore, ChIP and sequencing analysis reveals that the heterochromatin enrichment on the X chromosome mainly occurs within long genes involved in neuronal functions. Consequently, heterochromatin enrichment reduces the transcription of neuronal genes in the adult brain and results in a defect in Drosophila courtship learning. Taken together, these results define a previously unidentified role of CDK12 in controlling the epigenetic transition between euchromatin and heterochromatin and suggest a chromatin regulatory mechanism in neuronal behaviors. PMID:26508632

  17. Heterologous expression of mammalian Plk1 in Drosophila reveals divergence from Polo during late mitosis

    SciTech Connect

    Pearson, John . E-mail: jrobpea@upo.es; Godinho, Susana A.; Tavares, Alvaro; Glover, David M.

    2006-04-01

    Drosophila Polo kinase is the founder member of a conserved kinase family required for multiple stages of mitosis. We assessed the ability of mouse Polo-like kinase 1 (Plk1) to perform the multiple mitotic functions of Polo kinase, by expressing a Plk1-GFP fusion in Drosophila. Consistent with the previously reported localization of Polo kinase, Plk1-GFP was strongly localized to centrosomes and recruited to the centromeric regions of condensing chromosomes during early mitosis. However, in contrast to a functional Polo-GFP fusion, Plk1-GFP failed to localize to the central spindle midzone in both syncytial embryo mitosis and the conventional mitoses of cellularized embryos and S2 cells. Moreover, unlike endogenous Polo kinase and Polo-GFP, Plk1-GFP failed to associate with the contractile ring. Expression of Plk1-GFP enhanced the lethality of hypomorphic polo mutants and disrupted the organization of the actinomyosin cytoskeleton in a dominant-negative manner. Taken together, our results suggest that endogenous Polo kinase has specific roles in regulating actinomyosin rearrangements during Drosophila mitoses that its mammalian counterpart, Plk1, cannot fulfill. Consistent with this hypothesis, we observed defects in the cortical recruitment of myosin and myosin regulatory light chain in Polo deficient cells.

  18. Drosophila Blastorderm Analysis Software

    Energy Science and Technology Software Center (ESTSC)

    2006-10-25

    PointCloudMake analyzes 3D fluorescent images of whole Drosophila embryo and produces a table-style "PointCloud" file which contains the coordinates and volumes of all the nuclei, cells, their associated relative gene expression levels along with morphological features of the embryo. See: Luengo Hendrix et at 2006 3D Morphology and Gene Expression in the Drosophila Blastoderm at Cellular Resolution manuscript submitted LBNL # LBNL-60178 Knowles DW, Keranen SVE, Biggin M. Sudar S (2002) Mapping organism expression levelsmore » at cellular resolution in developing Drosophila. In: Conchello JA, Cogswell CJ, Wilson T, editors. Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing IX. pp. 57-64« less

  19. Genome-wide analysis of regulation of gene expression and H3K9me2 distribution by JIL-1 kinase mediated histone H3S10 phosphorylation in Drosophila

    PubMed Central

    Cai, Weili; Wang, Chao; Li, Yeran; Yao, Changfu; Shen, Lu; Liu, Sanzhen; Bao, Xiaomin; Schnable, Patrick S.; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M.

    2014-01-01

    In this study we have determined the genome-wide relationship of JIL-1 kinase mediated H3S10 phosphorylation with gene expression and the distribution of the epigenetic H3K9me2 mark. We show in wild-type salivary gland cells that the H3S10ph mark is predominantly enriched at active genes whereas the H3K9me2 mark is largely associated with inactive genes. Comparison of global transcription profiles in salivary glands from wild-type and JIL-1 null mutant larvae revealed that the expression levels of 1539 genes changed at least 2-fold in the mutant and that a substantial number (49%) of these genes were upregulated whereas 51% were downregulated. Furthermore, the results showed that downregulation of genes in the mutant was correlated with higher levels or acquisition of the H3K9me2 mark whereas upregulation of a gene was correlated with loss of or diminished H3K9 dimethylation. These results are compatible with a model where gene expression levels are modulated by the levels of the H3K9me2 mark independent of the state of the H3S10ph mark, which is not required for either transcription or gene activation to occur. Rather, H3S10 phosphorylation functions to indirectly maintain active transcription by counteracting H3K9 dimethylation and gene silencing. PMID:24598257

  20. Heritable Endosymbionts of Drosophila

    PubMed Central

    Mateos, Mariana; Castrezana, Sergio J.; Nankivell, Becky J.; Estes, Anne M.; Markow, Therese A.; Moran, Nancy A.

    2006-01-01

    Although heritable microorganisms are increasingly recognized as widespread in insects, no systematic screens for such symbionts have been conducted in Drosophila species (the primary insect genetic models for studies of evolution, development, and innate immunity). Previous efforts screened relatively few Drosophila lineages, mainly for Wolbachia. We conducted an extensive survey of potentially heritable endosymbionts from any bacterial lineage via PCR screens of mature ovaries in 181 recently collected fly strains representing 35 species from 11 species groups. Due to our fly sampling methods, however, we are likely to have missed fly strains infected with sex ratio-distorting endosymbionts. Only Wolbachia and Spiroplasma, both widespread in insects, were confirmed as symbionts. These findings indicate that in contrast to some other insect groups, other heritable symbionts are uncommon in Drosophila species, possibly reflecting a robust innate immune response that eliminates many bacteria. A more extensive survey targeted these two symbiont types through diagnostic PCR in 1225 strains representing 225 species from 32 species groups. Of these, 19 species were infected by Wolbachia while only 3 species had Spiroplasma. Several new strains of Wolbachia and Spiroplasma were discovered, including ones divergent from any reported to date. The phylogenetic distribution of Wolbachia and Spiroplasma in Drosophila is discussed. PMID:16783009

  1. The drosophila T-box transcription factor midline functions within Insulin/Akt and c-Jun-N terminal kinase stress-reactive signaling pathways to regulate interommatial bristle formation and cell survival

    PubMed Central

    Chen, Q. Brent; Das, Sudeshna; Visic, Petra; Buford, Kendrick D.; Zong, Yan; Buti, Wisam; Odom, Kelly R.; Lee, Hannah; Leal, Sandra M.

    2015-01-01

    We recently reported that the T-box transcription factor midline (mid) functions within the Notch-Delta signaling pathway to specify sensory organ precursor (SOP) cell fates in early-staged pupal eye imaginal discs and to suppress apoptosis (Das et al.). From genetic and allelic modifier screens, we now report that mid interacts with genes downstream of the insulin receptor(InR)/Akt, c-Jun-N-terminal kinase (JNK) and Notch signaling pathways to regulate interommatidial bristle (IOB) formation and cell survival. One of the most significant mid-interacting genes identified from the modifier screen is dFOXO, a transcription factor exhibiting a nucleocytoplasmic subcellular distribution pattern. In common with dFOXO, we show that Mid exhibits a nucleocytoplasmic distribution pattern within WT third-instar larval (3°L) tissue homogenates. Because dFOXO is a stress-responsive factor, we assayed the effects of either oxidative or metabolic stress responses on modifying the mid mutant phenotype which is characterized by a 50% loss of IOBs within the adult compound eye. While metabolic starvation stress does not affect the mid mutant phenotype, either 1 mM paraquat or 20% coconut oil, oxidative stress inducers, partially suppresses the mid mutant phenotype resulting in a significant recovery of IOBs. Another significant mid-interacting gene we identified is groucho (gro). Mid and Gro are predicted to act as corepressors of the enhancer-of-split gene complex downstream of Notch. Immunolabeling WT and dFOXO null 3°L eye-antennal imaginal discs with anti-Mid and anti-Engrailed (En) antibodies indicate that dFOXO is required to activate Mid and En expression within photoreceptor neurons of the eye disc. Taken together, these studies show that Mid and dFOXO serve as critical effectors of cell fate specification and survival within integrated Notch, InR/dAkt, and JNK signaling pathways during 3°L and pupal eye imaginal disc development. PMID:25748605

  2. The drosophila T-box transcription factor midline functions within Insulin/Akt and c-Jun-N terminal kinase stress-reactive signaling pathways to regulate interommatial bristle formation and cell survival.

    PubMed

    Chen, Q Brent; Das, Sudeshna; Visic, Petra; Buford, Kendrick D; Zong, Yan; Buti, Wisam; Odom, Kelly R; Lee, Hannah; Leal, Sandra M

    2015-05-01

    We recently reported that the T-box transcription factor midline (mid) functions within the Notch-Delta signaling pathway to specify sensory organ precursor (SOP) cell fates in early-staged pupal eye imaginal discs and to suppress apoptosis (Das et al.). From genetic and allelic modifier screens, we now report that mid interacts with genes downstream of the insulin receptor(InR)/Akt, c-Jun-N-terminal kinase (JNK) and Notch signaling pathways to regulate interommatidial bristle (IOB) formation and cell survival. One of the most significant mid-interacting genes identified from the modifier screen is dFOXO, a transcription factor exhibiting a nucleocytoplasmic subcellular distribution pattern. In common with dFOXO, we show that Mid exhibits a nucleocytoplasmic distribution pattern within WT third-instar larval (3(o)L) tissue homogenates. Because dFOXO is a stress-responsive factor, we assayed the effects of either oxidative or metabolic stress responses on modifying the mid mutant phenotype which is characterized by a 50% loss of IOBs within the adult compound eye. While metabolic starvation stress does not affect the mid mutant phenotype, either 1 mM paraquat or 20% coconut oil, oxidative stress inducers, partially suppresses the mid mutant phenotype resulting in a significant recovery of IOBs. Another significant mid-interacting gene we identified is groucho (gro). Mid and Gro are predicted to act as corepressors of the enhancer-of-split gene complex downstream of Notch. Immunolabeling WT and dFOXO null 3(o)L eye-antennal imaginal discs with anti-Mid and anti-Engrailed (En) antibodies indicate that dFOXO is required to activate Mid and En expression within photoreceptor neurons of the eye disc. Taken together, these studies show that Mid and dFOXO serve as critical effectors of cell fate specification and survival within integrated Notch, InR/dAkt, and JNK signaling pathways during 3(o)L and pupal eye imaginal disc development. PMID:25748605

  3. Aging Studies in Drosophila melanogaster

    PubMed Central

    Sun, Yaning; Yolitz, Jason; Wang, Cecilia; Spangler, Edward; Zhan, Ming; Zou, Sige

    2015-01-01

    Summary Drosophila is a genetically tractable system ideal for investigating the mechanisms of aging and developing interventions for promoting healthy aging. Here we describe methods commonly used in Drosophila aging research. These include basic approaches for preparation of diets and measurements of lifespan, food intake and reproductive output. We also describe some commonly used assays to measure changes in physiological and behavioral functions of Drosophila in aging, such as stress resistance and locomotor activity. PMID:23929099

  4. A Drosophila ABC transporter regulates lifespan.

    PubMed

    Huang, He; Lu-Bo, Ying; Haddad, Gabriel G

    2014-12-01

    MRP4 (multidrug resistance-associated protein 4) is a member of the MRP/ABCC subfamily of ATP-binding cassette (ABC) transporters that are essential for many cellular processes requiring the transport of substrates across cell membranes. Although MRP4 has been implicated as a detoxification protein by transport of structurally diverse endogenous and xenobiotic compounds, including antivirus and anticancer drugs, that usually induce oxidative stress in cells, its in vivo biological function remains unknown. In this study, we investigate the biological functions of a Drosophila homolog of human MRP4, dMRP4. We show that dMRP4 expression is elevated in response to oxidative stress (paraquat, hydrogen peroxide and hyperoxia) in Drosophila. Flies lacking dMRP4 have a shortened lifespan under both oxidative and normal conditions. Overexpression of dMRP4, on the other hand, is sufficient to increase oxidative stress resistance and extend lifespan. By genetic manipulations, we demonstrate that dMRP4 is required for JNK (c-Jun NH2-terminal kinase) activation during paraquat challenge and for basal transcription of some JNK target genes under normal condition. We show that impaired JNK signaling is an important cause for major defects associated with dMRP4 mutations, suggesting that dMRP4 regulates lifespan by modulating the expression of a set of genes related to both oxidative resistance and aging, at least in part, through JNK signaling. PMID:25474322

  5. Drosophila by the dozen

    SciTech Connect

    Celniker, Susan E.; Hoskins, Roger A.

    2007-07-13

    This year's conference on Drosophila research illustratedwell the current focus of Drosophila genomics on the comprehensiveidentification of functional elements in the genome sequence, includingmRNA transcripts arising from multiple alternative start sites and splicesites, a multiplicity of noncoding transcripts and small RNAs,identification of binding sites for transcription factors, sequenceconservation in related species and sequence variation within species.Resources and technologies for genetics and functional genomics aresteadily being improved, including the building of collections oftransposon insertion mutants and hairpin constructs for RNA interference(RNAi). The conference also highlighted progress in the use of genomicinformation by many laboratories to study diverse aspects of biology andmodels of human disease. Here we will review a few highlights of especialinterest to readers of Genome Biology.

  6. The Drosophila Auditory System

    PubMed Central

    Boekhoff-Falk, Grace; Eberl, Daniel F.

    2013-01-01

    Development of a functional auditory system in Drosophila requires specification and differentiation of the chordotonal sensilla of Johnston’s organ (JO) in the antenna, correct axonal targeting to the antennal mechanosensory and motor center (AMMC) in the brain, and synaptic connections to neurons in the downstream circuit. Chordotonal development in JO is functionally complicated by structural, molecular and functional diversity that is not yet fully understood, and construction of the auditory neural circuitry is only beginning to unfold. Here we describe our current understanding of developmental and molecular mechanisms that generate the exquisite functions of the Drosophila auditory system, emphasizing recent progress and highlighting important new questions arising from research on this remarkable sensory system. PMID:24719289

  7. Data on the phosphorylation of p38MAPK and JNK induced by chlorpyrifos in Drosophila melanogaster.

    PubMed

    Batista, J E S; Sousa, L R; Martins, I K; Rodrigues, N R; Posser, T; Franco, J L

    2016-12-01

    Exposure to organophosphate compounds, such as chlorpyrifos, has been linked to disturbances on cell signaling pathways. Mitogen activated protein kinases (MAPK) are a family of protein kinases involved in a range of cellular processes, including stress response, apoptosis and survival. Therefore, changes in the activation state of these kinases may characterize key mechanisms of toxicity elicited by xenobiotics. Here we report data on the phosphorylation of p38MAPK and JNK, members of the MAPK family, in Drosophila melanogaster exposed to chlorpyrifos, as characterized by western blotting assays. PMID:27626050

  8. Pantethine rescues a Drosophila model for pantothenate kinase–associated neurodegeneration

    PubMed Central

    Rana, Anil; Seinen, Erwin; Siudeja, Katarzyna; Muntendam, Remco; Srinivasan, Balaji; van der Want, Johannes J.; Hayflick, Susan; Reijngoud, Dirk-Jan; Kayser, Oliver; Sibon, Ody C. M.

    2010-01-01

    Pantothenate kinase–associated neurodegeneration (PKAN), a progressive neurodegenerative disorder, is associated with impairment of pantothenate kinase function. Pantothenate kinase is the first enzyme required for de novo synthesis of CoA, an essential metabolic cofactor. The pathophysiology of PKAN is not understood, and there is no cure to halt or reverse the symptoms of this devastating disease. Recently, we and others presented a PKAN Drosophila model, and we demonstrated that impaired function of pantothenate kinase induces a neurodegenerative phenotype and a reduced lifespan. We have explored this Drosophila model further and have demonstrated that impairment of pantothenate kinase is associated with decreased levels of CoA, mitochondrial dysfunction, and increased protein oxidation. Furthermore, we searched for compounds that can rescue pertinent phenotypes of the Drosophila PKAN model and identified pantethine. Pantethine feeding restores CoA levels, improves mitochondrial function, rescues brain degeneration, enhances locomotor abilities, and increases lifespan. We show evidence for the presence of a de novo CoA biosynthesis pathway in which pantethine is used as a precursor compound. Importantly, this pathway is effective in the presence of disrupted pantothenate kinase function. Our data suggest that pantethine may serve as a starting point to develop a possible treatment for PKAN. PMID:20351285

  9. Oncoprotein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2001-02-27

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  10. Drosophila Cancer Models Identify Functional Differences between Ret Fusions.

    PubMed

    Levinson, Sarah; Cagan, Ross L

    2016-09-13

    We generated and compared Drosophila models of RET fusions CCDC6-RET and NCOA4-RET. Both RET fusions directed cells to migrate, delaminate, and undergo EMT, and both resulted in lethality when broadly expressed. In all phenotypes examined, NCOA4-RET was more severe than CCDC6-RET, mirroring their effects on patients. A functional screen against the Drosophila kinome and a library of cancer drugs found that CCDC6-RET and NCOA4-RET acted through different signaling networks and displayed distinct drug sensitivities. Combining data from the kinome and drug screens identified the WEE1 inhibitor AZD1775 plus the multi-kinase inhibitor sorafenib as a synergistic drug combination that is specific for NCOA4-RET. Our work emphasizes the importance of identifying and tailoring a patient's treatment to their specific RET fusion isoform and identifies a multi-targeted therapy that may prove effective against tumors containing the NCOA4-RET fusion. PMID:27626672

  11. Genomic structure and chromosomal localization of the human deoxycytidine kinase gene

    SciTech Connect

    Song, J.J.; Walker, S.; Gribbin, T. ); Chen, E. Univ. of North Carolina, Chapel Hill ); Johnson, E.E.; Spychala, J.; Mitchell, B.S. )

    1993-01-15

    Deoxycytidine kinase (NTP:deoxycytidine 5[prime]-phosphotransferase, EC 2.7.1.74) is an enzyme that catalyzes phosphorylation of deoxyribonucleosides and a number of nucleoside analogs that are important in antiviral and cancer chemotherapy. Deficiency of this enzyme activity is associated with resistance to these agents, whereas increased enzyme activity is associated with increased activation of such compounds to cytotoxic nucleoside triphosphate derivatives. To characterize the regulation of expression of this gene, we have isolated genomic clones encompassing its entire coding and 5[prime] flanking regions and delinated all the exon/intron boundaries. The gene extends over more than 34 kilobases on chromosome 4 and the coding region is composed of 7 exons ranging in size from 90 to 1544 base pairs (bp). The 5[prime] flanking region is highly G+C-rich and contains four regions that are potential Sp1 binding sites. A 697-bp fragment encompassing 386 bp of 5[prime] upstream region, the 250-bp first exon, and 61 bp of the first intron was demonstrated to promote chloramphenicol acetyltransferase activity in a T-lymphoblast cell line and to have >6-fold greater activity in a Jurkat T-lymphoblast than in a Raji B-lymphoblast cell line. Our data suggest that these 5[prime] sequences may contain elements that are important for the tissue-specific differences in deoxycytidine kinase expression. 32 refs., 4 figs., 2 tabs.

  12. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways

    PubMed Central

    Clemens, James C.; Worby, Carolyn A.; Simonson-Leff, Nancy; Muda, Marco; Maehama, Tomohiko; Hemmings, Brian A.; Dixon, Jack E.

    2000-01-01

    We demonstrate the efficacy of double-stranded RNA-mediated interference (RNAi) of gene expression in generating “knock-out” phenotypes for specific proteins in several Drosophila cell lines. We prove the applicability of this technique for studying signaling cascades by dissecting the well-characterized insulin signal transduction pathway. Specifically, we demonstrate that inhibiting the expression of the DSOR1 (mitogen-activated protein kinase kinase, MAPKK) prevents the activation of the downstream ERK-A (MAPK). In contrast, blocking ERK-A expression results in increased activation of DSOR1. We also show that Drosophila AKT (DAKT) activation depends on the insulin receptor substrate, CHICO (IRS1–4). Finally, we demonstrate that blocking the expression of Drosophila PTEN results in the activation of DAKT. In all cases, the interference of the biochemical cascade by RNAi is consistent with the known steps in the pathway. We extend this powerful technique to study two proteins, DSH3PX1 and Drosophila ACK (DACK). DSH3PX1 is an SH3, phox homology domain-containing protein, and DACK is homologous to the mammalian activated Cdc42 tyrosine kinase, ACK. Using RNAi, we demonstrate that DACK is upstream of DSH3PX1 phosphorylation, making DSH3PX1 an identified downstream target/substrate of ACK-like tyrosine kinases. These experiments highlight the usefulness of RNAi in dissecting complex biochemical signaling cascades and provide a highly effective method for determining the function of the identified genes arising from the Drosophila genome sequencing project. PMID:10823906

  13. The Drosophila anatomy ontology

    PubMed Central

    2013-01-01

    Background Anatomy ontologies are query-able classifications of anatomical structures. They provide a widely-used means for standardising the annotation of phenotypes and expression in both human-readable and programmatically accessible forms. They are also frequently used to group annotations in biologically meaningful ways. Accurate annotation requires clear textual definitions for terms, ideally accompanied by images. Accurate grouping and fruitful programmatic usage requires high-quality formal definitions that can be used to automate classification and check for errors. The Drosophila anatomy ontology (DAO) consists of over 8000 classes with broad coverage of Drosophila anatomy. It has been used extensively for annotation by a range of resources, but until recently it was poorly formalised and had few textual definitions. Results We have transformed the DAO into an ontology rich in formal and textual definitions in which the majority of classifications are automated and extensive error checking ensures quality. Here we present an overview of the content of the DAO, the patterns used in its formalisation, and the various uses it has been put to. Conclusions As a result of the work described here, the DAO provides a high-quality, queryable reference for the wild-type anatomy of Drosophila melanogaster and a set of terms to annotate data related to that anatomy. Extensive, well referenced textual definitions make it both a reliable and useful reference and ensure accurate use in annotation. Wide use of formal axioms allows a large proportion of classification to be automated and the use of consistency checking to eliminate errors. This increased formalisation has resulted in significant improvements to the completeness and accuracy of classification. The broad use of both formal and informal definitions make further development of the ontology sustainable and scalable. The patterns of formalisation used in the DAO are likely to be useful to developers of other

  14. Sexual circuitry in Drosophila.

    PubMed

    Auer, Thomas O; Benton, Richard

    2016-06-01

    The sexual behavior of Drosophila melanogaster is an outstanding paradigm to understand the molecular and neuronal basis of sophisticated animal actions. We discuss recent advances in our knowledge of the genetic hardwiring of the underlying neuronal circuitry, and how pertinent sensory cues are differentially detected and integrated in the male and female brain. We also consider how experience influences these circuits over short timescales, and the evolution of these pathways over longer timescales to endow species-specific sexual displays and responses. PMID:26851712

  15. Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

    NASA Technical Reports Server (NTRS)

    Li, H.; Roux, S. J.

    1992-01-01

    Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.

  16. Identification of murine homologues of the Drosophila son of sevenless gene: potential activators of ras.

    PubMed Central

    Bowtell, D; Fu, P; Simon, M; Senior, P

    1992-01-01

    Several findings suggest that signals from tyrosine kinases are transduced, at least in part, through ras proteins. These findings include (i) blockage of the transforming activity of constitutively active tyrosine kinases by inhibiting ras function and (ii) genetic screens in Caenorhabditis elegans and in Drosophila that identified ras genes as downstream effectors of tyrosine kinases. The recently isolated Drosophila gene Son of sevenless (Sos) is postulated to act as a positive regulatory link between tyrosine kinase and ras proteins by catalyzing exchange of GDP for GTP on ras protein. Such exchange proteins have been reported in extracts of mammalian cells but have not been previously characterized at a molecular level. As Sos appears to function in this role in Drosophila, we sought to isolate a vertebrate counterpart(s). We have characterized two widely expressed murine genes with a high degree of homology to Sos. Hybridization with human DNA and RNA indicates a high degree of conservation of these genes in other vertebrates. Images PMID:1631150

  17. Myoblast fusion in Drosophila

    SciTech Connect

    Haralalka, Shruti; Abmayr, Susan M.

    2010-11-01

    The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process . With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.

  18. Initial neurogenesis in Drosophila

    PubMed Central

    Hartenstein, Volker; Wodarz, Andreas

    2014-01-01

    Early neurogenesis comprises the phase of nervous system development during which neural progenitor cells are born. In early development, the embryonic ectoderm is subdivided by a conserved signaling mechanism into two main domains, the epidermal ectoderm and the neurectoderm. Subsequently, cells of the neurectoderm are internalized and form a cell layer of proliferating neural progenitors. In vertebrates, the entire neurectoderm folds into the embryo to give rise to the neural tube. In Drosophila and many other invertebrates, a subset of neurectodermal cells, called neuroblasts (NBs), delaminates and forms the neural primordium inside the embryo where they divide in an asymmetric, stem cell-like mode. The remainder of the neuroectodermal cells that stay behind at the surface loose their neurogenic potential and later give rise to the ventral part of the epidermis. The genetic and molecular analysis of the mechanisms controlling specification and proliferation of NBs in the Drosophila embryo, which played a significant part in pioneering the field of modern developmental neurobiology, represents the topic of this review. PMID:24014455

  19. Initial neurogenesis in Drosophila.

    PubMed

    Hartenstein, Volker; Wodarz, Andreas

    2013-01-01

    Early neurogenesis comprises the phase of nervous system development during which neural progenitor cells are born. In early development, the embryonic ectoderm is subdivided by a conserved signaling mechanism into two main domains, the epidermal ectoderm and the neurectoderm. Subsequently, cells of the neurectoderm are internalized and form a cell layer of proliferating neural progenitors. In vertebrates, the entire neurectoderm folds into the embryo to give rise to the neural tube. In Drosophila and many other invertebrates, a subset of neurectodermal cells, called neuroblasts (NBs), delaminates and forms the neural primordium inside the embryo where they divide in an asymmetric, stem cell-like mode. The remainder of the neurectodermal cells that stay behind at the surface loose their neurogenic potential and later give rise to the ventral part of the epidermis. The genetic and molecular analysis of the mechanisms controlling specification and proliferation of NBs in the Drosophila embryo, which played a significant part in pioneering the field of modern developmental neurobiology, represents the topic of this review. PMID:24014455

  20. Microbiota-Dependent Priming of Antiviral Intestinal Immunity in Drosophila.

    PubMed

    Sansone, Christine L; Cohen, Jonathan; Yasunaga, Ari; Xu, Jie; Osborn, Greg; Subramanian, Harry; Gold, Beth; Buchon, Nicolas; Cherry, Sara

    2015-11-11

    Enteric pathogens must overcome intestinal defenses to establish infection. In Drosophila, the ERK signaling pathway inhibits enteric virus infection. The intestinal microflora also impacts immunity but its role in enteric viral infection is unknown. Here we show that two signals are required to activate antiviral ERK signaling in the intestinal epithelium. One signal depends on recognition of peptidoglycan from the microbiota, particularly from the commensal Acetobacter pomorum, which primes the NF-kB-dependent induction of a secreted factor, Pvf2. However, the microbiota is not sufficient to induce this pathway; a second virus-initiated signaling event involving release of transcriptional paused genes mediated by the kinase Cdk9 is also required for Pvf2 production. Pvf2 stimulates antiviral immunity by binding to the receptor tyrosine kinase PVR, which is necessary and sufficient for intestinal ERK responses. These findings demonstrate that sensing of specific commensals primes inflammatory signaling required for epithelial responses that restrict enteric viral infections. PMID:26567510

  1. A spindle-independent cleavage pathway controls germ cell formation in Drosophila.

    PubMed

    Cinalli, Ryan M; Lehmann, Ruth

    2013-07-01

    The primordial germ cells (PGCs) are the first cells to form during Drosophila melanogaster embryogenesis. Whereas the process of somatic cell formation has been studied in detail, the mechanics of PGC formation are poorly understood. Here, using four-dimensional multi-photon imaging combined with genetic and pharmacological manipulations, we find that PGC formation requires an anaphase spindle-independent cleavage pathway. In addition to using core regulators of cleavage, including the small GTPase RhoA (Drosophila rho1) and the Rho-associated kinase, ROCK (Drosophila drok), we show that this pathway requires Germ cell-less (GCL), a conserved BTB-domain protein not previously implicated in cleavage mechanics. This alternative form of cell formation suggests that organisms have evolved multiple molecular strategies for regulating the cytoskeleton during cleavage. PMID:23728423

  2. Review: Thermal preference in Drosophila

    PubMed Central

    Dillon, Michael E.; Wang, George; Garrity, Paul A.; Huey, Raymond B.

    2009-01-01

    Environmental temperature strongly affects physiology of ectotherms. Small ectotherms, like Drosophila, cannot endogenously regulate body temperature so must rely on behavior to maintain body temperature within a physiologically permissive range. Here we review what is known about Drosophila thermal preference. Work on thermal behavior in this group is particularly exciting because it provides the opportunity to connect genes to neuromolecular mechanisms to behavior to fitness in the wild. PMID:20161211

  3. Feeding and Fasting Signals Converge on the LKB1-SIK3 Pathway to Regulate Lipid Metabolism in Drosophila.

    PubMed

    Choi, Sekyu; Lim, Dae-Sik; Chung, Jongkyeong

    2015-05-01

    LKB1 plays important roles in governing energy homeostasis by regulating AMP-activated protein kinase (AMPK) and other AMPK-related kinases, including the salt-inducible kinases (SIKs). However, the roles and regulation of LKB1 in lipid metabolism are poorly understood. Here we show that Drosophila LKB1 mutants display decreased lipid storage and increased gene expression of brummer, the Drosophila homolog of adipose triglyceride lipase (ATGL). These phenotypes are consistent with those of SIK3 mutants and are rescued by expression of constitutively active SIK3 in the fat body, suggesting that SIK3 is a key downstream kinase of LKB1. Using genetic and biochemical analyses, we identify HDAC4, a class IIa histone deacetylase, as a lipolytic target of the LKB1-SIK3 pathway. Interestingly, we found that the LKB1-SIK3-HDAC4 signaling axis is modulated by dietary conditions. In short-term fasting, the adipokinetic hormone (AKH) pathway, related to the mammalian glucagon pathway, inhibits the kinase activity of LKB1 as shown by decreased SIK3 Thr196 phosphorylation, and consequently induces HDAC4 nuclear localization and brummer gene expression. However, under prolonged fasting conditions, AKH-independent signaling decreases the activity of the LKB1-SIK3 pathway to induce lipolytic responses. We also identify that the Drosophila insulin-like peptides (DILPs) pathway, related to mammalian insulin pathway, regulates SIK3 activity in feeding conditions independently of increasing LKB1 kinase activity. Overall, these data suggest that fasting stimuli specifically control the kinase activity of LKB1 and establish the LKB1-SIK3 pathway as a converging point between feeding and fasting signals to control lipid homeostasis in Drosophila. PMID:25996931

  4. Axonal injury and regeneration in the adult brain of Drosophila

    PubMed Central

    Ayaz, Derya; Leyssen, Maarten; Koch, Marta; Yan, Jiekun; Srahna, Mohammed; Sheeba, Vasu; Fogle, Keri J.; Holmes, Todd C.; Hassan, Bassem A.

    2009-01-01

    Drosophila melanogaster is a leading genetic model system in nervous system development and disease research. Using the power of fly genetics in traumatic axonal injury research will significantly speed up the characterization of molecular processes that control axonal regeneration in the Central Nervous System (CNS). We developed a versatile and physiologically robust preparation for the long-term culture of the whole Drosophila brain. We use this method to develop a novel Drosophila model for CNS axonal injury and regeneration. We first show that, similar to mammalian CNS axons, injured adult wild type fly CNS axons fail to regenerate, whereas adult-specific enhancement of Protein Kinase A activity increases the regenerative capacity of lesioned neurons. Combined, these observations suggest conservation of neuronal regeneration mechanisms following injury. We next exploit this model to explore pathways that induce robust regeneration and find that adult-specific activation of JNK signalling is sufficient for de novo CNS axonal regeneration after injury, including the growth of new axons past the lesion site and into the normal target area. PMID:18524906

  5. The Hydra FGFR, Kringelchen, partially replaces the Drosophila Heartless FGFR.

    PubMed

    Rudolf, Anja; Hübinger, Christine; Hüsken, Katrin; Vogt, Angelika; Rebscher, Nicole; Onel, Susanne-Filiz; Renkawitz-Pohl, Renate; Hassel, Monika

    2013-05-01

    Fibroblast growth factor receptors (FGFR) are highly conserved receptor tyrosine kinases, and evolved early in metazoan evolution. In order to investigate their functional conservation, we asked whether the Kringelchen FGFR in the freshwater polyp Hydra vulgaris, is able to functionally replace FGFR in fly embryos. In Drosophila, two endogenous FGFR, Breathless (Btl) and Heartless (Htl), ensure formation of the tracheal system and mesodermal cell migration as well as formation of the heart. Using UAS-kringelchen-5xmyc transgenic flies and targeted expression, we show that Kringelchen is integrated correctly into the cell membrane of mesodermal and tracheal cells in Drosophila. Nevertheless, Kringelchen expression driven in tracheal cells failed to rescue the btl (LG19) mutant. The Hydra FGFR was able to substitute for Heartless in the htl (AB42) null mutant; however, this occurred only during early mesodermal cell migration. Our data provide evidence for functional conservation of this early-diverged FGFR across these distantly related phyla, but also selectivity for the Htl FGFR in the Drosophila system. PMID:23111653

  6. Optogenetics in Drosophila Neuroscience.

    PubMed

    Riemensperger, Thomas; Kittel, Robert J; Fiala, André

    2016-01-01

    Optogenetic techniques enable one to target specific neurons with light-sensitive proteins, e.g., ion channels, ion pumps, or enzymes, and to manipulate their physiological state through illumination. Such artificial interference with selected elements of complex neuronal circuits can help to determine causal relationships between neuronal activity and the effect on the functioning of neuronal circuits controlling animal behavior. The advantages of optogenetics can best be exploited in genetically tractable animals whose nervous systems are, on the one hand, small enough in terms of cell numbers and to a certain degree stereotypically organized, such that distinct and identifiable neurons can be targeted reproducibly. On the other hand, the neuronal circuitry and the behavioral repertoire should be complex enough to enable one to address interesting questions. The fruit fly Drosophila melanogaster is a favorable model organism in this regard. However, the application of optogenetic tools to depolarize or hyperpolarize neurons through light-induced ionic currents has been difficult in adult flies. Only recently, several variants of Channelrhodopsin-2 (ChR2) have been introduced that provide sufficient light sensitivity, expression, and stability to depolarize central brain neurons efficiently in adult Drosophila. Here, we focus on the version currently providing highest photostimulation efficiency, ChR2-XXL. We exemplify the use of this optogenetic tool by applying it to a widely used aversive olfactory learning paradigm. Optogenetic activation of a population of dopamine-releasing neurons mimics the reinforcing properties of a punitive electric shock typically used as an unconditioned stimulus. In temporal coincidence with an odor stimulus this artificially induced neuronal activity causes learning of the odor signal, thereby creating a light-induced memory. PMID:26965122

  7. Purification of Drosophila snRNPs and characterization of two populations of functional U1 particles.

    PubMed Central

    Labourier, E; Rio, D C

    2001-01-01

    U1 snRNP is required at an early stage during assembly of the spliceosome, the dynamic ribonucleoprotein (RNP) complex that performs nuclear pre-mRNA splicing. Here, we report the purification of U1 snRNP particles from Drosophila nuclear extracts and the characterization of their biochemical properties, polypeptide contents, and splicing activities. On the basis of their antigenicity, apparent molecular weight, and by peptide sequencing, the Drosophila 70K, SNF, B, U1-C, D1, D2, D3, E, F, and G proteins are shown to be integral components of these particles. Sequence database searches revealed that both the U1-specific and the Sm proteins are extensively conserved between human and Drosophila snRNPs. Furthermore, both species possess a conserved intrinsic U1-associated kinase activity with identical substrate specificity in vitro. Finally, our results demonstrate that a second type of functional U1 particle, completely lacking the U1/U2-specific protein SNF and the associated protein kinase activity, can be isolated from cultured Kc cell or Canton S embryonic nuclear extracts. This work describes the first characterization of a purified Drosophila snRNP particle and reinforces the view that their activity and composition, with the exception of the atypical bifunctional U1-A/U2-B" SNF protein, are highly conserved in metazoans. PMID:11333025

  8. The PIKE Homolog Centaurin gamma Regulates Developmental Timing in Drosophila

    PubMed Central

    Sendscheid, Oliver; Aberle, Hermann; Hoch, Michael

    2014-01-01

    Phosphoinositide-3-kinase enhancer (PIKE) proteins encoded by the PIKE/CENTG1 gene are members of the gamma subgroup of the Centaurin superfamily of small GTPases. They are characterized by their chimeric protein domain architecture consisting of a pleckstrin homology (PH) domain, a GTPase-activating (GAP) domain, Ankyrin repeats as well as an intrinsic GTPase domain. In mammals, three PIKE isoforms with variations in protein structure and subcellular localization are encoded by the PIKE locus. PIKE inactivation in mice results in a broad range of defects, including neuronal cell death during brain development and misregulation of mammary gland development. PIKE -/- mutant mice are smaller, contain less white adipose tissue, and show insulin resistance due to misregulation of AMP-activated protein kinase (AMPK) and insulin receptor/Akt signaling. here, we have studied the role of PIKE proteins in metabolic regulation in the fly. We show that the Drosophila PIKE homolog, ceng1A, encodes functional GTPases whose internal GAP domains catalyze their GTPase activity. To elucidate the biological function of ceng1A in flies, we introduced a deletion in the ceng1A gene by homologous recombination that removes all predicted functional PIKE domains. We found that homozygous ceng1A mutant animals survive to adulthood. In contrast to PIKE -/- mouse mutants, genetic ablation of Drosophila ceng1A does not result in growth defects or weight reduction. Although metabolic pathways such as insulin signaling, sensitivity towards starvation and mobilization of lipids under high fed conditions are not perturbed in ceng1A mutants, homozygous ceng1A mutants show a prolonged development in second instar larval stage, leading to a late onset of pupariation. In line with these results we found that expression of ecdysone inducible genes is reduced in ceng1A mutants. Together, we propose a novel role for Drosophila Ceng1A in regulating ecdysone signaling-dependent second to third instar

  9. cAMP signalling in mushroom bodies modulates temperature preference behaviour in Drosophila.

    PubMed

    Hong, Sung-Tae; Bang, Sunhoe; Hyun, Seogang; Kang, Jongkyun; Jeong, Kyunghwa; Paik, Donggi; Chung, Jongkyeong; Kim, Jaeseob

    2008-08-01

    Homoiotherms, for example mammals, regulate their body temperature with physiological responses such as a change of metabolic rate and sweating. In contrast, the body temperature of poikilotherms, for example Drosophila, is the result of heat exchange with the surrounding environment as a result of the large ratio of surface area to volume of their bodies. Accordingly, these animals must instinctively move to places with an environmental temperature as close as possible to their genetically determined desired temperature. The temperature that Drosophila instinctively prefers has a function equivalent to the 'set point' temperature in mammals. Although various temperature-gated TRP channels have been discovered, molecular and cellular components in Drosophila brain responsible for determining the desired temperature remain unknown. We identified these components by performing a large-scale genetic screen of temperature preference behaviour (TPB) in Drosophila. In parallel, we mapped areas of the Drosophila brain controlling TPB by targeted inactivation of neurons with tetanus toxin and a potassium channel (Kir2.1) driven with various brain-specific GAL4s. Here we show that mushroom bodies (MBs) and the cyclic AMP-cAMP-dependent protein kinase A (cAMP-PKA) pathway are essential for controlling TPB. Furthermore, targeted expression of cAMP-PKA pathway components in only the MB was sufficient to rescue abnormal TPB of the corresponding mutants. Preferred temperatures were affected by the level of cAMP and PKA activity in the MBs in various PKA pathway mutants. PMID:18594510

  10. [COMPARATIVE STUDY OF Hrs AND OTHER ENDOSOMAL MARKERS CELLULAR LOCALIZATION IN DROSOPHILA MELANOGASTER SPERMATOGENESIS BY GFP-CHIMERICAL PROTEIN APPROACH].

    PubMed

    Marilovtseva, E V; Dubatolova, T D; Galimova, J A; Kopyl, S A; Omelyanchuk, L V

    2015-01-01

    Acrosome is a special organelle in spermatozoids necessary for fertilizing oocyte and originates, according to various theories, either from Golgi apparatus, or from endosomes and lysosomes. One of the proteins, found at mammalian acrosome, is Hgs, a homologue of Drosophila melanogaster Hrs (Hepatocyte growth factor regulated tyrosine kinase substrate), a known marker of multivesicular bodies (MVBs). However, although Drosophila acrosome was extensively studied, it is yet unknown whether Hrs localizes at acrosome similar to Hgs and, more generally, whether the spectrum of acrosomal proteins in Drosophila is the same as in mammals. Hrs (hepatocyte growth factor regulated tyrosine kinase substrate) is the multidomain vesicular protein participating in the endosome-lysosome protein sorting. We demonstrated that two protein variants of the Drosophila Hrs are expressed in testes: a longer isoform B, and a shorter isoform A, which lacks VHS and FYVE domains that are necessary for anchoring Hrs in endosomes. We found that Hrs isoform B is concentrated at fusoma of spermatocytes in contrast to mammalian Hrs. This localization requires the C-terminus of the protein, starting from the aminoacid residue 383. In situ hybridization of hrs RNA probe showed that the gene is expressed early in spermatogenesis consistently with Hrs localization in early fusoma. Additionally, we demonstrated that Hrs is dispensable for cytokinesis. Finally, it was found that although Drosophila Hrs does not localize at acrosome, the other endosomal markers--Rab4, Rab7, and Rab11--are detected at the organelle. PMID:26591063

  11. Deoxycytidine Kinase Augments ATM-Mediated DNA Repair and Contributes to Radiation Resistance

    PubMed Central

    Bunimovich, Yuri L.; Nair-Gill, Evan; Riedinger, Mireille; McCracken, Melissa N.; Cheng, Donghui; McLaughlin, Jami; Radu, Caius G.; Witte, Owen N.

    2014-01-01

    Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [18F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy. PMID:25101980

  12. Why Drosophila to Study Phototransduction?

    PubMed Central

    Pak, William L.

    2010-01-01

    This review recounts the early history of Drosophila phototransduction genetics, covering the period between approximately 1966 to 1979. Early in this period, the author felt that there was an urgent need for a new approach in phototransduction research. Through inputs from a number of colleagues, he was led to consider isolating Drosophila mutants that are defective in the electroretinogram. Thanks to the efforts of dedicated associates and technical staff, by the end of this period, he was able to accumulate a large number of such mutants. Particularly important in this effort was the use of the mutant assay protocol based on the “prolonged depolarizing afterpotential.” This collection of mutants formed the basis of the subsequent intensive investigations of the Drosophila phototransduction cascade by many investigators. PMID:20536286

  13. Micromechanics of Drosophila Audition

    NASA Astrophysics Data System (ADS)

    Göpfert, M. C.; Robert, D.

    2003-02-01

    An analysis is presented of the auditory micromechanics of the fruit fly Drosophila melanogaster. In this animal, the distal part of the antenna constitutes a resonantly tuned sound receiver, the vibrations of which are transduced by a chordotonal sense organ in the antenna's base. Analyzing the mechanical behavior of the antennal receiver by means of microscanning laser Doppler vibrometry, we show that the auditory system of wild-type flies exhibits a hardening stiffness nonlinearity and spontaneously generates oscillations in the absence of external stimuli. According to the deprivation of these mechanical properties in mechanosensory mutants, the receiver's nonlinearity and oscillation activity are introduced by chordotonal auditory neurons. Requiring the mechanoreceptor-specific extracellular linker protein No-mechanoreceptor-potential-A (NompA), NompC mechanosensory transduction channels, Beethoven (Btv), and Touch-insensitive-larva-B (TilB), nonlinearity and oscillation activity of the fly's antennal receiver depend on prominent components of the auditory transduction machinery and seem to originate from motility of auditory receptor cilia.

  14. Retinal differentiation in Drosophila.

    PubMed

    Treisman, Jessica E

    2013-07-01

    Drosophila eye development has been extensively studied, due to the ease of genetic screens for mutations disrupting this process. The eye imaginal disc is specified during embryonic and larval development by the Pax6 homolog Eyeless and a network of downstream transcription factors. Expression of these factors is regulated by signaling molecules and also indirectly by growth of the eye disc. Differentiation of photoreceptor clusters initiates in the third larval instar at the posterior of the eye disc and progresses anteriorly, driven by the secreted protein Hedgehog. Within each cluster, the combined activities of Hedgehog signaling and Notch-mediated lateral inhibition induce and refine the expression of the transcription factor Atonal, which specifies the founding R8 photoreceptor of each ommatidium. Seven additional photoreceptors, followed by cone and pigment cells, are successively recruited by the signaling molecules Spitz, Delta, and Bride of sevenless. Combinations of these signals and of intrinsic transcription factors give each ommatidial cell its specific identity. During the pupal stages, rhodopsins are expressed, and the photoreceptors and accessory cells take on their final positions and morphologies to form the adult retina. Over the past few decades, the genetic analysis of this small number of cell types arranged in a repetitive structure has allowed a remarkably detailed understanding of the basic mechanisms controlling cell differentiation and morphological rearrangement. PMID:24014422

  15. Collective synchronization of divisions in Drosophila development

    NASA Astrophysics Data System (ADS)

    Vergassola, Massimo

    Mitoses in the early development of most metazoans are rapid and synchronized across the entire embryo. While diffusion is too slow, in vitro experiments have shown that waves of the cell-cycle regulator Cdk1 can transfer information rapidly across hundreds of microns. However, the signaling dynamics and the physical properties of chemical waves during embryonic development remain unclear. We develop FRET biosensors for the activity of Cdk1 and the checkpoint kinase Chk1 in Drosophila embryos and exploit them to measure waves in vivo. We demonstrate that Cdk1 chemical waves control mitotic waves and that their speed is regulated by the activity of Cdk1 during the S-phase (and not mitosis). We quantify the progressive slowdown of the waves with developmental cycles and identify its underlying control mechanism by the DNA replication checkpoint through the Chk1/Wee1 pathway. The global dynamics of the mitotic signaling network illustrates a novel control principle: the S-phase activity of Cdk1 regulates the speed of the mitotic wave, while the Cdk1 positive feedback ensures an invariantly rapid onset of mitosis. Mathematical modeling captures the speed of the waves and predicts a fundamental distinction between the S-phase Cdk1 trigger waves and the mitotic phase waves, which is illustrated by embryonic ablation experiments. In collaboration with Victoria Deneke1, Anna Melbinger2, and Stefano Di Talia1 1 Department of Cell Biology, Duke University Medical Center 2 Department of Physics, University of California San Diego.

  16. Pantothenate kinase-associated neurodegeneration.

    PubMed

    Hartig, Monika B; Prokisch, Holger; Meitinger, Thomas; Klopstock, Thomas

    2012-08-01

    Pantothenate kinase-associated neurodegeneration (PKAN) is a hereditary progressive disorder and the most frequent form of neurodegeneration with brain iron accumulation (NBIA). PKAN patients present with a progressive movement disorder, dysarthria, cognitive impairment and retinitis pigmentosa. In magnetic resonance imaging, PKAN patients exhibit the pathognonomic "eye of the tiger" sign in the globus pallidus which corresponds to iron accumulation and gliosis as shown in neuropathological examinations. The discovery of the disease causing mutations in PANK2 has linked the disorder to coenzyme A (CoA) metabolism. PANK2 is the only one out of four PANK genes encoding an isoform which localizes to mitochondria. At least two other NBIA genes (PLA2G6, C19orf12) encode proteins that share with PANK2 a mitochondrial localization and all are suggested to play a role in lipid homeostasis. With no causal therapy available for PKAN until now, only symptomatic treatment is possible. A multi-centre retrospective study with bilateral pallidal deep brain stimulation in patients with NBIA revealed a significant improvement of dystonia. Recently, studies in the PANK Drosophila model "fumble" revealed improvement by the compound pantethine which is hypothesized to feed an alternate CoA biosynthesis pathway. In addition, pilot studies with the iron chelator deferiprone that crosses the blood brain barrier showed a good safety profile and some indication of efficacy. An adequately powered randomized clinical trial will start in 2012. This review summarizes clinical presentation, neuropathology and pathogenesis of PKAN. PMID:22515741

  17. The Roles of NDR Protein Kinases in Hippo Signalling

    PubMed Central

    Hergovich, Alexander

    2016-01-01

    The Hippo tumour suppressor pathway has emerged as a critical regulator of tissue growth through controlling cellular processes such as cell proliferation, death, differentiation and stemness. Traditionally, the core cassette of the Hippo pathway includes the MST1/2 protein kinases, the LATS1/2 protein kinases, and the MOB1 scaffold signal transducer, which together regulate the transcriptional co-activator functions of the proto-oncoproteins YAP and TAZ through LATS1/2-mediated phosphorylation of YAP/TAZ. Recent research has identified additional kinases, such as NDR1/2 (also known as STK38/STK38L) and MAP4Ks, which should be considered as novel members of the Hippo core cassette. While these efforts helped to expand our understanding of Hippo core signalling, they also began to provide insights into the complexity and redundancy of the Hippo signalling network. Here, we focus on summarising our current knowledge of the regulation and functions of mammalian NDR kinases, discussing parallels between the NDR pathways in Drosophila and mammals. Initially, we provide a general overview of the cellular functions of NDR kinases in cell cycle progression, centrosome biology, apoptosis, autophagy, DNA damage signalling, immunology and neurobiology. Finally, we put particular emphasis on discussing NDR1/2 as YAP kinases downstream of MST1/2 and MOB1 signalling in Hippo signalling. PMID:27213455

  18. Cyclin G Functions as a Positive Regulator of Growth and Metabolism in Drosophila.

    PubMed

    Fischer, Patrick; La Rosa, Martina K; Schulz, Adriana; Preiss, Anette; Nagel, Anja C

    2015-08-01

    In multicellular organisms, growth and proliferation is adjusted to nutritional conditions by a complex signaling network. The Insulin receptor/target of rapamycin (InR/TOR) signaling cascade plays a pivotal role in nutrient dependent growth regulation in Drosophila and mammals alike. Here we identify Cyclin G (CycG) as a regulator of growth and metabolism in Drosophila. CycG mutants have a reduced body size and weight and show signs of starvation accompanied by a disturbed fat metabolism. InR/TOR signaling activity is impaired in cycG mutants, combined with a reduced phosphorylation status of the kinase Akt1 and the downstream factors S6-kinase and eukaryotic translation initiation factor 4E binding protein (4E-BP). Moreover, the expression and accumulation of Drosophila insulin like peptides (dILPs) is disturbed in cycG mutant brains. Using a reporter assay, we show that the activity of one of the first effectors of InR signaling, Phosphoinositide 3-kinase (PI3K92E), is unaffected in cycG mutants. However, the metabolic defects and weight loss in cycG mutants were rescued by overexpression of Akt1 specifically in the fat body and by mutants in widerborst (wdb), the B'-subunit of the phosphatase PP2A, known to downregulate Akt1 by dephosphorylation. Together, our data suggest that CycG acts at the level of Akt1 to regulate growth and metabolism via PP2A in Drosophila. PMID:26274446

  19. Dtrk, a Drosophila gene related to the trk family of neurotrophin receptors, encodes a novel class of neural cell adhesion molecule.

    PubMed Central

    Pulido, D; Campuzano, S; Koda, T; Modolell, J; Barbacid, M

    1992-01-01

    We report the identification and molecular characterization of Dtrk, a Drosophila gene encoding a receptor tyrosine kinase highly related to the trk family of mammalian neurotrophin receptors. The product of the Dtrk gene, gp160Dtrk, is dynamically expressed during Drosophila embryogenesis in several areas of the developing nervous system, including neurons and fasciculating axons. gp160Dtrk has structural homology with neural cell adhesion molecules of the immunoglobulin superfamily and promotes cell adhesion in a homophilic, Ca2+ independent manner. More importantly, this adhesion process specifically activates its tyrosine protein kinase activity. These findings suggest that gp160Dtrk represents a new class of neural cell adhesion molecules that may regulate neuronal recognition and axonal guidance during the development of the Drosophila nervous system. Images PMID:1371458

  20. MUTAGENESIS SCREENING OF PESTICIDES 'DROSOPHILA'

    EPA Science Inventory

    Drosophila melanogaster males were exposed by feeding (plus contact and possibly inhalation). The genetic test found most sensitive and appropriate was the sex-linked recessive lethal test. For this, males of the Canton-S wild type stock were exposed. They were mated individually...

  1. A DNA Virus of Drosophila

    PubMed Central

    Unckless, Robert L.

    2011-01-01

    Little is known about the viruses infecting most species. Even in groups as well-studied as Drosophila, only a handful of viruses have been well-characterized. A viral metagenomic approach was used to explore viral diversity in 83 wild-caught Drosophila innubila, a mushroom feeding member of the quinaria group. A single fly that was injected with, and died from, Drosophila C Virus (DCV) was added to the sample as a control. Two-thirds of reads in the infected sample had DCV as the best BLAST hit, suggesting that the protocol developed is highly sensitive. In addition to the DCV hits, several sequences had Oryctes rhinoceros Nudivirus, a double-stranded DNA virus, as a best BLAST hit. The virus associated with these sequences was termed Drosophila innubila Nudivirus (DiNV). PCR screens of natural populations showed that DiNV was both common and widespread taxonomically and geographically. Electron microscopy confirms the presence of virions in fly fecal material similar in structure to other described Nudiviruses. In 2 species, D. innubila and D. falleni, the virus is associated with a severe (∼80–90%) loss of fecundity and significantly decreased lifespan. PMID:22053195

  2. Antibody Staining in Drosophila Germaria.

    PubMed

    Lie-Jensen, Anette; Haglund, Kaisa

    2016-01-01

    Drosophila oogenesis is a powerful model for studying a wide spectrum of cellular and developmental processes in vivo. Oogenesis starts in a specialized structure called the germarium, which harbors the stem cells for both germ and somatic cells. The germarium produces egg chambers, each of which will develop into an egg. Active areas of research in Drosophila germaria include stem cell self-renewal, division, and maintenance, cell cycle control and differentiation, oocyte specification, intercellular communication, and signaling, among others. The solid knowledge base, the genetic tractability of the Drosophila model, as well as the availability and fast development of tools and imaging techniques for oogenesis research ensure that studies in this model will keep being instrumental for novel discoveries within cell and developmental biology also in the future. This chapter focuses on antibody staining in Drosophila germaria and provides a protocol for immunostaining as well as an overview of commonly used antibodies for visualization of different cell types and cellular structures. The protocol is well-suited for subsequent confocal microscopy analyses, and in addition we present key adaptations of the protocol that are useful when performing structured illumination microscopy (SIM) super-resolution imaging. PMID:27557571

  3. Iron Absorption in Drosophila melanogaster

    PubMed Central

    Mandilaras, Konstantinos; Pathmanathan, Tharse; Missirlis, Fanis

    2013-01-01

    The way in which Drosophila melanogaster acquires iron from the diet remains poorly understood despite iron absorption being of vital significance for larval growth. To describe the process of organismal iron absorption, consideration needs to be given to cellular iron import, storage, export and how intestinal epithelial cells sense and respond to iron availability. Here we review studies on the Divalent Metal Transporter-1 homolog Malvolio (iron import), the recent discovery that Multicopper Oxidase-1 has ferroxidase activity (iron export) and the role of ferritin in the process of iron acquisition (iron storage). We also describe what is known about iron regulation in insect cells. We then draw upon knowledge from mammalian iron homeostasis to identify candidate genes in flies. Questions arise from the lack of conservation in Drosophila for key mammalian players, such as ferroportin, hepcidin and all the components of the hemochromatosis-related pathway. Drosophila and other insects also lack erythropoiesis. Thus, systemic iron regulation is likely to be conveyed by different signaling pathways and tissue requirements. The significance of regulating intestinal iron uptake is inferred from reports linking Drosophila developmental, immune, heat-shock and behavioral responses to iron sequestration. PMID:23686013

  4. Iron absorption in Drosophila melanogaster.

    PubMed

    Mandilaras, Konstantinos; Pathmanathan, Tharse; Missirlis, Fanis

    2013-05-01

    The way in which Drosophila melanogaster acquires iron from the diet remains poorly understood despite iron absorption being of vital significance for larval growth. To describe the process of organismal iron absorption, consideration needs to be given to cellular iron import, storage, export and how intestinal epithelial cells sense and respond to iron availability. Here we review studies on the Divalent Metal Transporter-1 homolog Malvolio (iron import), the recent discovery that Multicopper Oxidase-1 has ferroxidase activity (iron export) and the role of ferritin in the process of iron acquisition (iron storage). We also describe what is known about iron regulation in insect cells. We then draw upon knowledge from mammalian iron homeostasis to identify candidate genes in flies. Questions arise from the lack of conservation in Drosophila for key mammalian players, such as ferroportin, hepcidin and all the components of the hemochromatosis-related pathway. Drosophila and other insects also lack erythropoiesis. Thus, systemic iron regulation is likely to be conveyed by different signaling pathways and tissue requirements. The significance of regulating intestinal iron uptake is inferred from reports linking Drosophila developmental, immune, heat-shock and behavioral responses to iron sequestration. PMID:23686013

  5. Methods to assay Drosophila behavior.

    PubMed

    Nichols, Charles D; Becnel, Jaime; Pandey, Udai B

    2012-01-01

    Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases(1). We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials(2-4). The rapid iterative negative geotaxis (RING) assay(5) has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously

  6. Circular DNA Molecules in the Genus Drosophila

    PubMed Central

    Travaglini, E. C.; Schultz, J.

    1972-01-01

    The satellite DNA's from the embryos of five species of Drosophila (D. melanogaster, D. simulans, D. nasuta, D. virilis and D. hydei) have been analyzed for the presence of closed circular duplex DNA molecules, as determined by CsCl-EBr gradients. Circular DNA molecules were found in every species but D. melanogaster. Analyses of cell fractions from adult Drosophila and organ fractions from Drosophila larvae show that fractions containing mitochondria are highly enriched in these molecules. PMID:4643820

  7. Prokaryotic Diacylglycerol Kinase and Undecaprenol Kinase

    PubMed Central

    Van Horn, Wade D.; Sanders, Charles R.

    2013-01-01

    Prokaryotic diacylglycerol kinase (DAGK) and undecaprenol kinase (UDPK) are the lone members of a family of multispan membrane enzymes that are very small, lack relationships to any other family of proteins—including water soluble kinases, and that exhibit an unusual structure and active site architecture. Escherichia coli DAGK plays an important role in recycling diacylglycerol produced as a byproduct of biosynthesis of molecules located in the periplasmic space. UDPK seems to play an analogous role in Gram-positive bacteria, where its importance is evident by the fact that UDPK is essential for biofilm formation by the oral pathogen Streptococcus mutans. DAGK has also long served as a model system for studies of membrane protein biocatalysis, folding, stability, and structure. This review explores our current understanding of the microbial physiology, enzymology, structural biology, and folding of the prokaryotic diacylglycerol kinase family, which is based on over 40 years of studies. PMID:22224599

  8. kuzbanian-mediated cleavage of Drosophila Notch

    PubMed Central

    Lieber, Toby; Kidd, Simon; Young, Michael W.

    2002-01-01

    Loss of Kuzbanian, a member of the ADAM family of metalloproteases, produces neurogenic phenotypes in Drosophila. It has been suggested that this results from a requirement for kuzbanian-mediated cleavage of the Notch ligand Delta. Using transgenic Drosophila expressing transmembrane Notch proteins, we show that kuzbanian, independent of any role in Delta processing, is required for the cleavage of Notch. We show that Kuzbanian can physically associate with Notch and that removal of kuzbanian activity by RNA-mediated interference in Drosophila tissue culture cells eliminates processing of ligand-independent transmembrane Notch molecules. Our data suggest that in Drosophila, kuzbanian can mediate S2 cleavage of Notch. PMID:11799064

  9. Akap200 suppresses the effects of Dv-cbl expression in the Drosophila eye.

    PubMed

    Sannang, Rowena T; Robertson, Hannah; Siddall, Nicole A; Hime, Gary R

    2012-10-01

    The Drosophila melanogaster orthologue of the c-Cbl proto-oncogene acts to downregulate signalling from receptor tyrosine kinases by enhancing endocytosis of activated receptors. Expression of an analogue of the C-terminally truncated v-Cbl oncogene, Dv-cbl, in the developing Drosophila eye conversely leads to excess signalling and disruption to the well-ordered adult compound eye. Co-expression of activated Ras with Dv-cbl leads to a severe disruption of eye development. We have used a transposon-based inducible expression system to screen for molecules that can suppress the Dv-cbl phenotype and have identified an allele that upregulates the A-kinase anchoring protein, Akap200. Overexpression of Akap200 not only suppresses the phenotype caused by Dv-cbl expression, but also the severe disruption to eye development caused by the combined expression of Dv-cbl and activated Ras. Akap200 is also endogenously expressed in the developing Drosophila eye at a level that modulates the effects of excessive signalling caused by expression of Dv-cbl. PMID:22773306

  10. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  11. Novel Drosophila receptor that binds multiple growth factors

    SciTech Connect

    Rosner, M.R.; Thompson, K.L.; Garcia, V.; Decker, S.J.

    1986-05-01

    The authors have recently reported the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100 kDa protein is also antigenically related to the cytoplasmic region of the mammalian EGF receptor-tyrosine kinase. They now report that this protein binds to mammalian nerve growth factor and human transforming growth factor alpha as well as insulin and EGF with apparent dissociation constants ranging from 10/sup -6/ to 10/sup -8/ M. The 100 kDa protein can be affinity-labeled with these /sup 125/I-labeled growth factors after immunoprecipitation with anti-EGF receptor antiserum. These four growth factors appear to share a common binding site, as evidenced by their ability to block affinity labelling by /sup 125/I-insulin. No significant binding to the 100 kDa protein was observed with platelet-derived growth factor, transforming growth factor beta, or glucagon. The 100 kDa Drosophila protein has a unique ligand-binding spectrum with no direct counterpart in mammalian cells and may represent an evolutionary precursor of the mammalian receptors for these growth factors.

  12. Ehrlichia chaffeensis replication sites in adult Drosophila melanogaster

    PubMed Central

    Drolia, Rishi; Von Ohlen, Tonia; Chapes, Stephen K.

    2012-01-01

    Ehrlichia chaffeensis is a Gram-negative, obligate intracellular bacterium which causes the tick-borne disease human monocytic ehrlichiosis. In vertebrates, E. chaffeensis replicates in monocytes and macrophages. However, no clear cell or tissue tropism has been defined in arthropods. Our group identified two host genes that control E. chaffeensis replication and infection in vivo in Drosophila, Uridine cytidine kinase and separation anxiety. Using the UAS-GAL4 RNAi system, we generated F1 flies (UAS-gene of interest RNAi x tissue-GAL4 flies) that have Uck2 or san silenced in ubiquitous or tissue-specific fashion. When Uck2 or san were suppressed in the hemocytes or in the fat body, E. chaffeensis replicated poorly and caused significantly less severe infections. Silencing of these genes in the eyes, wings, or the salivary glands did not impact fly susceptibility or bacterial replication. Our data suggest that in Drosophila, E. chaffeensis replicates within the hemocytes, the insect homolog of mammalian macrophages, and in the fat body, the liver homolog of mammals. PMID:23306065

  13. A Drosophila complementary DNA resource

    SciTech Connect

    Rubin, Gerald M.; Hong, Ling; Brokstein, Peter; Evans-Holm, Martha; Frise, Erwin; Stapleton, Mark; Harvey, Damon A.

    2000-03-24

    Collections of nonredundant, full-length complementary DNA (cDNA) clones for each of the model organisms and humans will be important resources for studies of gene structure and function. We describe a general strategy for producing such collections and its implementation, which so far has generated a set of cDNAs corresponding to over 40% of the genes in the fruit fly Drosophila melanogaster.

  14. Optogenetic pacing in Drosophila melanogaster

    PubMed Central

    Alex, Aneesh; Li, Airong; Tanzi, Rudolph E.; Zhou, Chao

    2015-01-01

    Electrical stimulation is currently the gold standard for cardiac pacing. However, it is invasive and nonspecific for cardiac tissues. We recently developed a noninvasive cardiac pacing technique using optogenetic tools, which are widely used in neuroscience. Optogenetic pacing of the heart provides high spatial and temporal precisions, is specific for cardiac tissues, avoids artifacts associated with electrical stimulation, and therefore promises to be a powerful tool in basic cardiac research. We demonstrated optogenetic control of heart rhythm in a well-established model organism, Drosophila melanogaster. We developed transgenic flies expressing a light-gated cation channel, channelrhodopsin-2 (ChR2), specifically in their hearts and demonstrated successful optogenetic pacing of ChR2-expressing Drosophila at different developmental stages, including the larva, pupa, and adult stages. A high-speed and ultrahigh-resolution optical coherence microscopy imaging system that is capable of providing images at a rate of 130 frames/s with axial and transverse resolutions of 1.5 and 3.9 μm, respectively, was used to noninvasively monitor Drosophila cardiac function and its response to pacing stimulation. The development of a noninvasive integrated optical pacing and imaging system provides a novel platform for performing research studies in developmental cardiology. PMID:26601299

  15. Leigh Syndrome in Drosophila melanogaster

    PubMed Central

    Da-Rè, Caterina; von Stockum, Sophia; Biscontin, Alberto; Millino, Caterina; Cisotto, Paola; Zordan, Mauro A.; Zeviani, Massimo; Bernardi, Paolo; De Pittà, Cristiano; Costa, Rodolfo

    2014-01-01

    Leigh Syndrome (LS) is the most common early-onset, progressive mitochondrial encephalopathy usually leading to early death. The single most prevalent cause of LS is occurrence of mutations in the SURF1 gene, and LSSurf1 patients show a ubiquitous and specific decrease in the activity of mitochondrial respiratory chain complex IV (cytochrome c oxidase, COX). SURF1 encodes an inner membrane mitochondrial protein involved in COX assembly. We established a Drosophila melanogaster model of LS based on the post-transcriptional silencing of CG9943, the Drosophila homolog of SURF1. Knockdown of Surf1 was induced ubiquitously in larvae and adults, which led to lethality; in the mesodermal derivatives, which led to pupal lethality; or in the central nervous system, which allowed survival. A biochemical characterization was carried out in knockdown individuals, which revealed that larvae unexpectedly displayed defects in all complexes of the mitochondrial respiratory chain and in the F-ATP synthase, while adults had a COX-selective impairment. Silencing of Surf1 expression in Drosophila S2R+ cells led to selective loss of COX activity associated with decreased oxygen consumption and respiratory reserve. We conclude that Surf1 is essential for COX activity and mitochondrial function in D. melanogaster, thus providing a new tool that may help clarify the pathogenic mechanisms of LS. PMID:25164807

  16. 'Peer pressure' in larval Drosophila?

    PubMed

    Niewalda, Thomas; Jeske, Ines; Michels, Birgit; Gerber, Bertram

    2014-01-01

    Understanding social behaviour requires a study case that is simple enough to be tractable, yet complex enough to remain interesting. Do larval Drosophila meet these requirements? In a broad sense, this question can refer to effects of the mere presence of other larvae on the behaviour of a target individual. Here we focused in a more strict sense on 'peer pressure', that is on the question of whether the behaviour of a target individual larva is affected by what a surrounding group of larvae is doing. We found that innate olfactory preference of a target individual was neither affected (i) by the level of innate olfactory preference in the surrounding group nor (ii) by the expression of learned olfactory preference in the group. Likewise, learned olfactory preference of a target individual was neither affected (iii) by the level of innate olfactory preference of the surrounding group nor (iv) by the learned olfactory preference the group was expressing. We conclude that larval Drosophila thus do not take note of specifically what surrounding larvae are doing. This implies that in a strict sense, and to the extent tested, there is no social interaction between larvae. These results validate widely used en mass approaches to the behaviour of larval Drosophila. PMID:24907371

  17. Mutations in the Catalytic Loop HRD Motif Alter the Activity and Function of Drosophila Src64

    PubMed Central

    Strong, Taylor C.; Kaur, Gurvinder; Thomas, Jeffrey H.

    2011-01-01

    The catalytic loop HRD motif is found in most protein kinases and these amino acids are predicted to perform functions in catalysis, transition to, and stabilization of the active conformation of the kinase domain. We have identified mutations in a Drosophila src gene, src64, that alter the three HRD amino acids. We have analyzed the mutants for both biochemical activity and biological function during development. Mutation of the aspartate to asparagine eliminates biological function in cytoskeletal processes and severely reduces fertility, supporting the amino acid's critical role in enzymatic activity. The arginine to cysteine mutation has little to no effect on kinase activity or cytoskeletal reorganization, suggesting that the HRD arginine may not be critical for coordinating phosphotyrosine in the active conformation. The histidine to leucine mutant retains some kinase activity and biological function, suggesting that this amino acid may have a biochemical function in the active kinase that is independent of its side chain hydrogen bonding interactions in the active site. We also describe the phenotypic effects of other mutations in the SH2 and tyrosine kinase domains of src64, and we compare them to the phenotypic effects of the src64 null allele. PMID:22132220

  18. KEA: kinase enrichment analysis

    PubMed Central

    Lachmann, Alexander; Ma'ayan, Avi

    2009-01-01

    Motivation: Multivariate experiments applied to mammalian cells often produce lists of proteins/genes altered under treatment versus control conditions. Such lists can be projected onto prior knowledge of kinase–substrate interactions to infer the list of kinases associated with a specific protein list. By computing how the proportion of kinases, associated with a specific list of proteins/genes, deviates from an expected distribution, we can rank kinases and kinase families based on the likelihood that these kinases are functionally associated with regulating the cell under specific experimental conditions. Such analysis can assist in producing hypotheses that can explain how the kinome is involved in the maintenance of different cellular states and can be manipulated to modulate cells towards a desired phenotype. Summary: Kinase enrichment analysis (KEA) is a web-based tool with an underlying database providing users with the ability to link lists of mammalian proteins/genes with the kinases that phosphorylate them. The system draws from several available kinase–substrate databases to compute kinase enrichment probability based on the distribution of kinase–substrate proportions in the background kinase–substrate database compared with kinases found to be associated with an input list of genes/proteins. Availability: The KEA system is freely available at http://amp.pharm.mssm.edu/lib/kea.jsp Contact: avi.maayan@mssm.edu PMID:19176546

  19. Aurora Kinases and Potential Medical Applications of Aurora Kinase Inhibitors: A Review

    PubMed Central

    Gavriilidis, Paschalis; Giakoustidis, Alexandros; Giakoustidis, Dimitrios

    2015-01-01

    Aurora kinases (AKs) represent a novel group of serine/threonine kinases. They were originally described in 1995 by David Glover in the course of studies of mutant alleles characterized with unusual spindle pole configuration in Drosophila melanogaster. Thus far, three AKs A, B, and C have been discovered in human healthy and neoplastic cells. Each one locates in different subcellular locations and they are all nuclear proteins. AKs are playing an essential role in mitotic events such as monitoring of the mitotic checkpoint, creation of bipolar mitotic spindle and alignment of centrosomes on it, also regulating centrosome separation, bio-orientation of chromosomes and cytokinesis. Any inactivation of them can have catastrophic consequences on mitotic events of spindle formation, alignment of centrosomes and cytokinesis, resulting in apoptosis. Overexpression of AKs has been detected in diverse solid and hematological cancers and has been linked with a dismal prognosis. After discovery and identification of the first aurora kinase inhibitor (AKI) ZM447439 as a potential drug for targeted therapy in cancer treatment, approximately 30 AKIs have been introduced in cancer treatment. PMID:26345296

  20. Changes in Drosophila melanogaster midgut proteins in response to dietary Bowman-Birk inhibitor.

    PubMed

    Li, H-M; Margam, V; Muir, W M; Murdock, L L; Pittendrigh, B R

    2007-10-01

    The midgut proteome of Drosophila melanogaster was compared in larvae fed dietary Bowman-Birk inhibitor (BBI) vs. larvae fed a control diet. By using two-dimensional gel electrophoresis, nine differentially expressed proteins were observed, which were associated with enzymes or transport functions such as sterol carrier protein X (SCPX), ubiquitin-conjugating enzyme, endopeptidase, receptor signalling protein kinase, ATP-dependent RNA helicase and alpha-tocopherol transport. Quantitative real-time PCR verified differential expression of transcripts coding for six of the proteins observed from the proteomic analysis. BBI evidently affects expression of proteins associated with protein degradation, transport and fatty acid catabolism. We then tested the hypothesis that SCPX was critical for the Drosophila third instars' response to BBI treatment. Inhibition of SCPX caused the third instars to become more susceptible to dietary BBI. PMID:17725801

  1. The Hippo signalling pathway maintains quiescence in Drosophila neural stem cells

    PubMed Central

    Ding, Rouven; Weynans, Kevin; Bossing, Torsten; Barros, Claudia S.; Berger, Christian

    2016-01-01

    Stem cells control their mitotic activity to decide whether to proliferate or to stay in quiescence. Drosophila neural stem cells (NSCs) are quiescent at early larval stages, when they are reactivated in response to metabolic changes. Here we report that cell-contact inhibition of growth through the canonical Hippo signalling pathway maintains NSC quiescence. Loss of the core kinases hippo or warts leads to premature nuclear localization of the transcriptional co-activator Yorkie and initiation of growth and proliferation in NSCs. Yorkie is necessary and sufficient for NSC reactivation, growth and proliferation. The Hippo pathway activity is modulated via inter-cellular transmembrane proteins Crumbs and Echinoid that are both expressed in a nutrient-dependent way in niche glial cells and NSCs. Loss of crumbs or echinoid in the niche only is sufficient to reactivate NSCs. Finally, we provide evidence that the Hippo pathway activity discriminates quiescent from non-quiescent NSCs in the Drosophila nervous system. PMID:26821647

  2. Localization and posttranslational modifications of otefin, a protein required for vesicle attachment to chromatin, during Drosophila melanogaster development.

    PubMed Central

    Ashery-Padan, R; Ulitzur, N; Arbel, A; Goldberg, M; Weiss, A M; Maus, N; Fisher, P A; Gruenbaum, Y

    1997-01-01

    Otefin is a peripheral protein of the inner nuclear membrane in Drosophila melanogaster. Here we show that during nuclear assembly in vitro, it is required for the attachment of membrane vesicles to chromatin. With the exception of sperm cells, otefin colocalizes with lamin Dm0 derivatives in situ and presumably in vivo and is present in all somatic cells examined during the different stages of Drosophila development. In the egg chamber, otefin accumulates in the cytoplasm, in the nuclear periphery, and within the nucleoplasm of the oocyte, in a pattern similar to that of lamin Dm0 derivatives. There is a relatively large nonnuclear pool of otefin present from stages 6 to 7 of egg chamber maturation through 6 to 8 h of embryonic development at 25 degrees C. In this pool, otefin is peripherally associated with a fraction containing the membrane vesicles. This association is biochemically different from the association of otefin with the nuclear envelope. Otefin is a phosphoprotein in vivo and is a substrate for in vitro phosphorylation by cdc2 kinase and cyclic AMP-dependent protein kinase. A major site for cdc2 kinase phosphorylation in vitro was mapped to serine 36 of otefin. Together, our data suggest an essential role for otefin in the assembly of the Drosophila nuclear envelope. PMID:9199347

  3. Biology and physiology of Drosophila suzukii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Spotted wing drosophila, Drosophila suzukii, quickly emerged as a devastating invasive pest of small and stone fruits in the Americas and Europe. To better understand the population dynamics of D. suzukii, we reviewed recent work on juvenile development, adult reproduction, and seasonal variation in...

  4. Drosophila and Beer: An Experimental Laboratory Exercise

    ERIC Educational Resources Information Center

    Kurvink, Karen

    2004-01-01

    Drosophila melanogaster is a popular organism for studying genetics and development. Maintaining Drosophila on medium prepared with varying concentrations of beer and evaluating the effects on reproduction, life cycle stages and other factors is one of the exercises that is versatile and applicable to many student levels.

  5. Mitotic activation of the kinase Aurora-A requires its binding partner Bora.

    PubMed

    Hutterer, Andrea; Berdnik, Daniela; Wirtz-Peitz, Frederik; Zigman, Mihaela; Schleiffer, Alexander; Knoblich, Juergen A

    2006-08-01

    The protein kinase Aurora-A is required for centrosome maturation, spindle assembly, and asymmetric protein localization during mitosis. Here, we describe the identification of Bora, a conserved protein that is required for the activation of Aurora-A at the onset of mitosis. In the Drosophila peripheral nervous system, bora mutants have defects during asymmetric cell division identical to those observed in aurora-A. Furthermore, overexpression of bora can rescue defects caused by mutations in aurora-A. Bora is conserved in vertebrates, and both Drosophila and human Bora can bind to Aurora-A and activate the kinase in vitro. In interphase cells, Bora is a nuclear protein, but upon entry into mitosis, Bora is excluded from the nucleus and translocates into the cytoplasm in a Cdc2-dependent manner. We propose a model in which activation of Cdc2 initiates the release of Bora into the cytoplasm where it can bind and activate Aurora-A. PMID:16890155

  6. Aurora A inhibition by MNL8054 promotes centriole elongation during Drosophila male meiosis

    PubMed Central

    Gottardo, Marco; Callaini, Giuliano; Riparbelli, Maria G

    2015-01-01

    Aurora A kinase plays an important role in several aspects of cell division, including centrosome maturation and separation, a crucial step for the correct organization of the bipolar spindle. Although it has long been showed that this kinase accumulates at the centrosome throughout mitosis its precise contribution to centriole biogenesis and structure has until now not been reported. It is not surprising that so little is known, due to the small size of somatic centrioles, where only dramatic structural changes may be identified by careful electron microscopy analysis. Conversely, centrioles of Drosophila primary spermatocytes increase tenfold in length during the first prophase, thus making any change easily detectable. Therefore, we examined the consequence of the pharmacological inhibition of Aurora A by MLN8054 on centriole biogenesis during early Drosophila gametogenesis. Here, we show that depletion of this kinase results in longer centrioles, mainly during transition from prophase to prometaphase of the first meiosis. We also found abnormal ciliogenesis characterized by irregularly growing axonemal doublets. Our results represent the first documentation of a potential requirement of Aurora A in centriole integrity and elongation. PMID:25785740

  7. From Phosphosites to Kinases.

    PubMed

    Munk, Stephanie; Refsgaard, Jan C; Olsen, Jesper V; Jensen, Lars J

    2016-01-01

    Kinases play a pivotal role in propagating the phosphorylation-mediated signaling networks in living cells. With the overwhelming quantities of phosphoproteomics data being generated, the number of identified phosphorylation sites (phosphosites) is ever increasing. Often, proteomics investigations aim to understand the global signaling modulation that takes place in different biological conditions investigated. For phosphoproteomics data, identifying the kinases central to mediating this response is key. This has prompted several efforts to catalogue the immense amounts of phosphorylation data and known or predicted kinases responsible for the modifications. However, barely 20 % of the known phosphosites are assigned to a kinase, initiating various bioinformatics efforts that attempt to predict the responsible kinases. These algorithms employ different approaches to predict kinase consensus sequence motifs, mostly based on large scale in vivo and in vitro experiments. The context of the kinase and the phosphorylated proteins in a biological system is equally important for predicting association between the enzymes and substrates, an aspect that is also being tackled with available bioinformatics tools. This chapter summarizes the use of the larger phosphorylation databases, and approaches that can be applied to predict kinases that phosphorylate individual sites or that are globally modulated in phosphoproteomics datasets. PMID:26584935

  8. Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

    NASA Technical Reports Server (NTRS)

    Li, H.; Roux, S. J.

    1992-01-01

    A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

  9. Slik and the receptor tyrosine kinase Breathless mediate localized activation of Moesin in terminal tracheal cells.

    PubMed

    Ukken, Fiona Paul; Aprill, Imola; JayaNandanan, N; Leptin, Maria

    2014-01-01

    A key element in the regulation of subcellular branching and tube morphogenesis of the Drosophila tracheal system is the organization of the actin cytoskeleton by the ERM protein Moesin. Activation of Moesin within specific subdomains of cells, critical for its interaction with actin, is a tightly controlled process and involves regulatory inputs from membrane proteins, kinases and phosphatases. The kinases that activate Moesin in tracheal cells are not known. Here we show that the Sterile-20 like kinase Slik, enriched at the luminal membrane, is necessary for the activation of Moesin at the luminal membrane and regulates branching and subcellular tube morphogenesis of terminal cells. Our results reveal the FGF-receptor Breathless as an additional necessary cue for the activation of Moesin in terminal cells. Breathless-mediated activation of Moesin is independent of the canonical MAP kinase pathway. PMID:25061859

  10. Recent advances in the development of Aurora kinases inhibitors in hematological malignancies

    PubMed Central

    Choudary, Iqra; Barr, Paul M.; Friedberg, Jonathan

    2015-01-01

    Over the last two decades, since the discovery of Drosophila mutants in 1995, much effort has been made to understand Aurora kinase biology. Three mammalian subtypes have been identified thus far which include the Aurora A, B and C kinases. These regulatory proteins specifically work at the cytoskeleton and chromosomal structures between the kinetochores and have vital functions in the early phases of the mitotic cell cycle. Today, there are multiple phase I and phase II clinical trials as well as numerous preclinical studies taking place looking at Aurora kinase inhibitors in both hematologic and solid malignancies. This review focuses on the preclinical and clinical development of Aurora kinase inhibitors in hematological malignancy and discusses their therapeutic potential. PMID:26622997

  11. Functions of the Nonsense-Mediated mRNA Decay Pathway in Drosophila Development

    PubMed Central

    Metzstein, Mark M; Krasnow, Mark A

    2006-01-01

    Nonsense-mediated mRNA decay (NMD) is a cellular surveillance mechanism that degrades transcripts containing premature translation termination codons, and it also influences expression of certain wild-type transcripts. Although the biochemical mechanisms of NMD have been studied intensively, its developmental functions and importance are less clear. Here, we describe the isolation and characterization of Drosophila “photoshop” mutations, which increase expression of green fluorescent protein and other transgenes. Mapping and molecular analyses show that photoshop mutations are loss-of-function mutations in the Drosophila homologs of NMD genes Upf1, Upf2, and Smg1. We find that Upf1 and Upf2 are broadly active during development, and they are required for NMD as well as for proper expression of dozens of wild-type genes during development and for larval viability. Genetic mosaic analysis shows that Upf1 and Upf2 are required for growth and/or survival of imaginal cell clones, but this defect can be overcome if surrounding wild-type cells are eliminated. By contrast, we find that the PI3K-related kinase Smg1 potentiates but is not required for NMD or for viability, implying that the Upf1 phosphorylation cycle that is required for mammalian and Caenorhabditis elegans NMD has a more limited role during Drosophila development. Finally, we show that the SV40 3′ UTR, present in many Drosophila transgenes, targets the transgenes for regulation by the NMD pathway. The results establish that the Drosophila NMD pathway is broadly active and essential for development, and one critical function of the pathway is to endow proliferating imaginal cells with a competitive growth advantage that prevents them from being overtaken by other proliferating cells. PMID:17196039

  12. Phosphorylation of the insect immunophilin FKBP46 by the Spodoptera frugiperda homolog of casein kinase II.

    PubMed

    Steplewski, A; Ebel, W; Planey, S L; Alnemri, E S; Robertson, N M; Litwack, G

    2000-04-01

    Immunophilins are a family of conserved proteins found in both prokaryotes and eukaryotes, that exhibit peptidylprolyl isomerase (PPIase) activity. Members of this family bind to immunosuppressive drugs and on this basis are divided into two classes: FKBPs bind to FK506 and rapamycin, while cyclophilins bind to cyclosporin A. In this paper, we report on insect immunophilin FKBP46 and its associated kinase. The insect FKBP46 belongs to the high-molecular-weight immunophilins and shares many characteristic features with its mammalian counterparts, but its functional role remains unclear. Here, we show that FKBP46 is phosphorylated by a protein kinase present in the nucleus of both insect Spodoptera frugiperda (Sf9) and human Jurkat cells. This protein kinase is immunoreactive with polyclonal antiserum raised against Drosophila melanogaster casein kinase II (CKII). We have cloned, overexpressed and characterized a new member of the CKII family derived from Spodoptera frugiperda cells. Recombinant Sf9 CKII alpha subunit shares 75% identity to human, chicken and Drosophila melanogaster homologs, whereas the Sf9 CKII beta subunit is 77% identical to rat, chicken and human. Moreover, we demonstrate that the insect immunophilin FKBP46 can be phosphorylated by human and Sf9 casein kinase II. Finally, we show that FKBP46 interacts with DNA, and this interaction is not prevented by phosphorylation. PMID:10767538

  13. Receptor Tyrosine Kinase and Tyrosine Kinase Inhibitors

    PubMed Central

    Mirshafiey, Abbas; Ghalamfarsa, Ghasem; Asghari, Babak

    2014-01-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication and their function as relay points for signaling pathways. They have a key role in numerous processes that control cellular proliferation and differentiation, regulate cell growth and cellular metabolism, and promote cell survival and apoptosis. Recently, the role of RTKs including TCR, FLT-3, c-Kit, c-Fms, PDGFR, ephrin, neurotrophin receptor, and TAM receptor in autoimmune disorder, especially rheumatoid arthritis and multiple sclerosis has been suggested. In multiple sclerosis pathogenesis, RTKs and their tyrosine kinase enzymes are selective important targets for tyrosine kinase inhibitor (TKI) agents. TKIs, compete with the ATP binding site of the catalytic domain of several tyrosine kinases, and act as small molecules that have a favorable safety profile in disease treatment. Up to now, the efficacy of TKIs in numerous animal models of MS has been demonstrated, but application of these drugs in human diseases should be tested in future clinical trials. PMID:25337443

  14. A Drosophila Model of Multiple Endocrine Neoplasia Type 2

    PubMed Central

    Read, Renee D.; Goodfellow, Paul J.; Mardis, Elaine R.; Novak, Nancy; Armstrong, Jon R.; Cagan, Ross L.

    2005-01-01

    Dominant mutations in the Ret receptor tyrosine kinase lead to the familial cancer syndrome multiple endocrine neoplasia type 2 (MEN2). Mammalian tissue culture studies suggest that RetMEN2 mutations significantly alter Ret-signaling properties, but the precise mechanisms by which RetMEN2 promotes tumorigenesis remain poorly understood. To determine the signal transduction pathways required for RetMEN2 activity, we analyzed analogous mutations in the Drosophila Ret ortholog dRet. Overexpressed dRetMEN2 isoforms targeted to the developing retina led to aberrant cell proliferation, inappropriate cell fate specification, and excessive Ras pathway activation. Genetic analysis indicated that dRetMEN2 acts through the Ras-ERK, Src, and Jun kinase pathways. A genetic screen for mutations that dominantly suppress or enhance dRetMEN2 phenotypes identified new genes that are required for the phenotypic outcomes of dRetMEN2 activity. Finally, we identified human orthologs for many of these genes and examined their status in human tumors. Two of these loci showed loss of heterozygosity (LOH) within both sporadic and MEN2-associated pheochromocytomas, suggesting that they may contribute to Ret-dependent oncogenesis. PMID:15965261

  15. Distinct tissue distributions and subcellular localizations of differently phosphorylated forms of the myosin regulatory light chain in Drosophila.

    PubMed

    Zhang, Liang; Ward, Robert E

    2011-01-01

    Nonmuscle myosin II (myosin hereafter) has well-established roles in generating contractile force on actin filaments during morphogenetic processes in all metazoans. Myosin activation is regulated by phosphorylation of the myosin regulatory light chain (MRLC, encoded by spaghettisquash or sqh in Drosophila) first on Ser21 and subsequently on Thr20. These phosphorylation events are positively controlled by a variety of kinases including myosin light chain kinase, Rho kinase, citron kinase, and AMP kinase and are negatively regulated by myosin phosphatase. The activation of myosin is thus highly regulated and likely developmentally controlled. In order to monitor the activity of myosin during development, we have generated antibodies against the monophosphorylated (Sqh1P) and diphosphorylated (Sqh2P) forms of Sqh. We first show that the antibodies are highly specific. We then used these antibodies to monitor myosin activation in wild type Drosophila tissues. Interestingly, Sqh1P and Sqh2P show distinct patterns of expression in embryos. Sqh1P is expressed nearly ubiquitously and outlines cells consistent with a junctional localization, whereas Sqh2P is strongly expressed on the apical surfaces and in filopodia of tissues undergoing extensive cell shape change or cell movements including the invaginating fore- and hindgut, the invaginating tracheal system, the dorsal pouch and the dorsal most row of epidermal (DME) cells during dorsal closure. In imaginal discs, Sqh1P predominantly localizes in the adherens junction, whereas Sqh2P locates to the apical domain. These antibodies thus have the potential to be very useful in monitoring myosin activation for functional studies of morphogenesis in Drosophila. PMID:20920606

  16. Cryobiological preservation of Drosophila embryos

    SciTech Connect

    Mazur, P.; Schreuders, P.D.; Cole, K.W.; Hall, J.W. ); Mahowald, A.P. )

    1992-12-18

    The inability to cryobiologically preserve the fruit fly Drosophila melanogaster has required that fly stocks be maintained by frequent transfer of adults. This method is costly in terms of time and can lead to loss of stocks. Traditional slow freezing methods do not succeed because the embryos are highly sensitive to chilling. With the procedures described here, 68 percent of precisely staged 15-hour Oregon R (wild-type) embryos hatch after vitrification at -205[degree]C, and 40 percent of the resulting larvae develop into normal adult flies. These embryos are among the most complex organisms successfully preserved by cryobiology.

  17. Taste processing in Drosophila larvae

    PubMed Central

    Apostolopoulou, Anthi A.; Rist, Anna; Thum, Andreas S.

    2015-01-01

    The sense of taste allows animals to detect chemical substances in their environment to initiate appropriate behaviors: to find food or a mate, to avoid hostile environments and predators. Drosophila larvae are a promising model organism to study gustation. Their simple nervous system triggers stereotypic behavioral responses, and the coding of taste can be studied by genetic tools at the single cell level. This review briefly summarizes recent progress on how taste information is sensed and processed by larval cephalic and pharyngeal sense organs. The focus lies on several studies, which revealed cellular and molecular mechanisms required to process sugar, salt, and bitter substances. PMID:26528147

  18. Geotaxis baseline data for Drosophila

    NASA Technical Reports Server (NTRS)

    Schnebel, E. M.; Bhargava, R.; Grossfield, J.

    1987-01-01

    Geotaxis profiles for 20 Drosophila species and semispecies at different ages have been examined using a calibrated, adjustable slant board device. Measurements were taken at 5 deg intervals ranging from 0 deg to 85 deg. Clear strain and species differences are observed, with some groups tending to move upward (- geotaxis) with increasing angles, while others move downward (+ geotaxis). Geotactic responses change with age in some, but not all experimental groups. Sample geotaxis profiles are presented and their application to ecological and aging studies are discussed. Data provide a baseline for future evaluations of the biological effects of microgravity.

  19. Macrophages and cellular immunity in Drosophila melanogaster.

    PubMed

    Gold, Katrina S; Brückner, Katja

    2015-12-01

    The invertebrate Drosophila melanogaster has been a powerful model for understanding blood cell development and immunity. Drosophila is a holometabolous insect, which transitions through a series of life stages from embryo, larva and pupa to adulthood. In spite of this, remarkable parallels exist between Drosophila and vertebrate macrophages, both in terms of development and function. More than 90% of Drosophila blood cells (hemocytes) are macrophages (plasmatocytes), making this highly tractable genetic system attractive for studying a variety of questions in macrophage biology. In vertebrates, recent findings revealed that macrophages have two independent origins: self-renewing macrophages, which reside and proliferate in local microenvironments in a variety of tissues, and macrophages of the monocyte lineage, which derive from hematopoietic stem or progenitor cells. Like vertebrates, Drosophila possesses two macrophage lineages with a conserved dual ontogeny. These parallels allow us to take advantage of the Drosophila model when investigating macrophage lineage specification, maintenance and amplification, and the induction of macrophages and their progenitors by local microenvironments and systemic cues. Beyond macrophage development, Drosophila further serves as a paradigm for understanding the mechanisms underlying macrophage function and cellular immunity in infection, tissue homeostasis and cancer, throughout development and adult life. PMID:27117654

  20. Timing the Drosophila Mid-Blastula Transition: A Cell Cycle-Centered View.

    PubMed

    Yuan, Kai; Seller, Charles A; Shermoen, Antony W; O'Farrell, Patrick H

    2016-08-01

    At the mid-blastula transition (MBT), externally developing embryos refocus from increasing cell number to elaboration of the body plan. Studies in Drosophila reveal a sequence of changes in regulators of Cyclin:Cdk1 that increasingly restricts the activity of this cell cycle kinase to slow cell cycles during early embryogenesis. By reviewing these events, we provide an outline of the mechanisms slowing the cell cycle at and around the time of MBT. The perspectives developed should provide a guiding paradigm for the study of other MBT changes as the embryo transits from maternal control to a regulatory program centered on the expression of zygotic genes. PMID:27339317

  1. Complete mitochondrial genome of Drosophila albomicans.

    PubMed

    Kang, Xiongbin; Luo, Xiao; Zhang, Zhi; Zhang, Zhen; Yang, Junqing; Bi, Guiqi

    2016-09-01

    Drosophila albomicans has been widely used as an important animal model for chromosome evolution. In this study, the mitochondrial genome sequence of this species is determined and described for the first time. The mitochondrial genome (15 849 bp) encompasses two rRNA, 22 tRNA, and 13 protein-coding genes. Genome content and structure are similar to those reported from other Drosophila mitochondrial genomes. Phylogeny analysis indicates that D. albomicans have a closer genetic relationship with Drosophil aincompta and Drosophil alittoralis. This mitochondrial genome is potentially important for studying molecular evolution and conservation genetics in Drosophila genus. PMID:26358579

  2. Cellular immune defenses of Drosophila melanogaster.

    PubMed

    Parsons, Brendon; Foley, Edan

    2016-05-01

    Drosophila melanogaster is a widely used model for the characterization of blood cell development and function, with an array of protocols for the manipulation and visualization of fixed or live cells in vitro or in vivo. Researchers have deployed these techniques to reveal Drosophila hemocytes as a remarkably versatile cell type that engulfs apoptotic corpses; neutralizes invading parasites; seals epithelial wounds; and deposits extracellular matrix proteins. In this review, we will discuss the key features of Drosophila hemocyte development and function, and identify similarities with vertebrate counterparts. PMID:26748247

  3. Drosophila Modeling of Heritable Neurodevelopmental Disorders

    PubMed Central

    Gatto, Cheryl L.; Broadie, Kendal

    2011-01-01

    Heritable neurodevelopmental disorders are multifaceted disease conditions encompassing a wide range of symptoms including intellectual disability, cognitive dysfunction, autism and myriad other behavioral impairments. In cases where single, causative genetic defects have been identified, such as Angelman syndrome, Rett syndrome, Neurofibromatosis Type 1 and Fragile X syndrome, the classical Drosophila genetic system has provided fruitful disease models. Recent Drosophila studies have advanced our understanding of UBE3A, MECP2, NF1 and FMR1 function, respectively, in genetic, biochemical, anatomical, physiological and behavioral contexts. Investigations in Drosophila continue to provide the essential mechanistic understanding required to facilitate the conception of rational therapeutic treatments. PMID:21596554

  4. Protein kinase C mediated phosphorylation blocks juvenile hormone action.

    PubMed

    Kethidi, Damu R; Li, Yiping; Palli, Subba R

    2006-03-01

    Juvenile hormones (JH) regulate a wide variety of developmental and physiological processes in insects. Although the biological actions of JH are well documented, the molecular mechanisms underlying JH action are poorly understood. We studied the molecular basis of JH action using a JH response element (JHRE) identified in the promoter region of JH esterase gene cloned from Choristoneura fumiferana, which is responsive to JH and 20-hydroxyecdysone (20E). In Drosophila melanogaster L57 cells, the JHRE-regulated reporter gene was induced by JH I, JH III, methoprene, and hydroprene. Nuclear proteins isolated from L57 cells bound to the JHRE and exposure of these proteins to ATP resulted in a reduction in their DNA binding. Either JH III or calf intestinal alkaline phosphatase (CIAP) was able to restore the binding of nuclear proteins to the DNA. In addition, protein kinase C inhibitors increased and protein kinase C activators reduced the binding of nuclear proteins to the JHRE. In transactivation assays, protein kinase C inhibitors induced the luciferase gene placed under the control of a minimal promoter and the JHRE. These data suggest that protein kinase C mediated phosphorylation prevents binding of nuclear proteins to juvenile hormone responsive promoters resulting in suppression of JH action. PMID:16448742

  5. Automated Tracking of Drosophila Specimens

    PubMed Central

    Chao, Rubén; Macía-Vázquez, Germán; Zalama, Eduardo; Gómez-García-Bermejo, Jaime; Perán, José-Ramón

    2015-01-01

    The fruit fly Drosophila Melanogaster has become a model organism in the study of neurobiology and behavior patterns. The analysis of the way the fly moves and its behavior is of great scientific interest for research on aspects such as drug tolerance, aggression or ageing in humans. In this article, a procedure for detecting, identifying and tracking numerous specimens of Drosophila by means of computer vision-based sensing systems is presented. This procedure allows dynamic information about each specimen to be collected at each moment, and then for its behavior to be quantitatively characterized. The proposed algorithm operates in three main steps: a pre-processing step, a detection and segmentation step, and tracking shape. The pre-processing and segmentation steps allow some limits of the image acquisition system and some visual artifacts (such as shadows and reflections) to be dealt with. The improvements introduced in the tracking step allow the problems corresponding to identity loss and swaps, caused by the interaction between individual flies, to be solved efficiently. Thus, a robust method that compares favorably to other existing methods is obtained. PMID:26258779

  6. Automated Tracking of Drosophila Specimens.

    PubMed

    Chao, Rubén; Macía-Vázquez, Germán; Zalama, Eduardo; Gómez-García-Bermejo, Jaime; Perán, José-Ramón

    2015-01-01

    The fruit fly Drosophila Melanogaster has become a model organism in the study of neurobiology and behavior patterns. The analysis of the way the fly moves and its behavior is of great scientific interest for research on aspects such as drug tolerance, aggression or ageing in humans. In this article, a procedure for detecting, identifying and tracking numerous specimens of Drosophila by means of computer vision-based sensing systems is presented. This procedure allows dynamic information about each specimen to be collected at each moment, and then for its behavior to be quantitatively characterized. The proposed algorithm operates in three main steps: a pre-processing step, a detection and segmentation step, and tracking shape. The pre-processing and segmentation steps allow some limits of the image acquisition system and some visual artifacts (such as shadows and reflections) to be dealt with. The improvements introduced in the tracking step allow the problems corresponding to identity loss and swaps, caused by the interaction between individual flies, to be solved efficiently. Thus, a robust method that compares favorably to other existing methods is obtained. PMID:26258779

  7. Drosophila Models of Cardiac Disease

    PubMed Central

    Piazza, Nicole; Wessells, R.J.

    2013-01-01

    The fruit fly Drosophila melanogaster has emerged as a useful model for cardiac diseases, both developmental abnormalities and adult functional impairment. Using the tools of both classical and molecular genetics, the study of the developing fly heart has been instrumental in identifying the major signaling events of cardiac field formation, cardiomyocyte specification, and the formation of the functioning heart tube. The larval stage of fly cardiac development has become an important model system for testing isolated preparations of living hearts for the effects of biological and pharmacological compounds on cardiac activity. Meanwhile, the recent development of effective techniques to study adult cardiac performance in the fly has opened new uses for the Drosophila model system. The fly system is now being used to study long-term alterations in adult performance caused by factors such as diet, exercise, and normal aging. The fly is a unique and valuable system for the study of such complex, long-term interactions, as it is the only invertebrate genetic model system with a working heart developmentally homologous to the vertebrate heart. Thus, the fly model combines the advantages of invertebrate genetics (such as large populations, facile molecular genetic techniques, and short lifespan) with physiological measurement techniques that allow meaningful comparisons with data from vertebrate model systems. As such, the fly model is well situated to make important contributions to the understanding of complicated interactions between environmental factors and genetics in the long-term regulation of cardiac performance. PMID:21377627

  8. Monoamines and sleep in Drosophila.

    PubMed

    Nall, Aleksandra; Sehgal, Amita

    2014-06-01

    Sleep is an important physiological state, but its function and regulation remain elusive. Drosophila melanogaster is a useful model organism for studying sleep because it has a well-established diurnal activity pattern, including consolidated periods of quiescence that share many characteristics with human sleep. Sleep behavior is regulated by circadian and homeostatic processes and is modulated by environmental and physiological context cues. These cues are communicated to sleep circuits by neurohormones and neuromodulators. A major class of neuromodulators, monoamines, has been found to be essential in various aspects of sleep regulation. Dopamine promotes arousal and sleep-dependent memory formation as well as daily activity. Octopamine, the insect homolog of norepinephrine, promotes wake and may play a role in circadian clock-dependent sleep and arousal. Serotonin promotes sleep and modulates circadian entrainment to light. The different monoamines each signal through multiple receptors in various brain regions in response to different conditions. How these separate circuits integrate their inputs into a single program of behavior is an open field of study for which Drosophila will continue to be a useful model. Monoamine biosynthetic pathways and receptors are conserved between flies and humans, and, thus far, their roles in modulating sleep also appear to be conserved. PMID:24886188

  9. Pyruvate kinase blood test

    MedlinePlus

    ... break down faster than normal, a condition called hemolytic anemia . This test helps diagnose pyruvate kinase deficiency (PKD) . ... Pa: Elsevier Saunders; 2011:chap 32. Gallagher PG. Hemolytic anemias: red cell membrane and metabolic defects In: Goldman ...

  10. Comparative analysis of the human and zebrafish kinomes: focus on the development of kinase inhibitors

    PubMed Central

    Wlodarchak, Nathan; Tariq, Rehan; Striker, Rob

    2016-01-01

    Targeting kinases with semi-selective kinase inhibitors is one of the most successful drug development strategies of the 21st century. Zebrafish have become an increasingly useful model for pharmaceutical development. Water-soluble compounds can be screened for zebrafish phenotypes in a high throughput format against a living vertebrate, and cell-signaling events can be imaged in transparent living fish. Despite zebrafish being a more relevant model than more distantly related systems such as the well-annotated kinome of yeast and drosophila, there is no comparative analysis of the human and zebrafish kinome. Furthermore most approved kinase inhibitors, often called ‘DFG in’ ATP competitive inhibitors, act on conserved active site residues in the kinase. Since the active site residues can be identified by examining the primary sequence, primary sequence identity can be a rough guide as to whether a particular inhibitor will have activity against another kinase. There is a need to evaluate the utility of zebrafish as a drug development model for active site inhibitors of kinases. Here we offer a systematic comparison of the catalytic domains of classical human kinases with the catalytic domains of all annotated zebrafish kinases. We found a high degree of identity between the catalytic domains of most human kinases and their zebrafish homologs, and we ranked 504 human kinase catalytic domains by order of similarity. We found only 23 human kinases with no easily recognizable homologous zebrafish catalytic domain. On the other hand we found 78 zebrafish kinase catalytic domains with no close human counterpart. These ‘additional kinase active sites’ could represent potential mediators of zebrafish toxicity that may not be relevant to human kinase inhibitors. We used two clinically approved human kinase inhibitors, one targeting a highly homologous target and one targeting a lesser homologous target, and we compared the known human kinase target structures with

  11. Gene Regulation Networks for Modeling Drosophila Development

    NASA Technical Reports Server (NTRS)

    Mjolsness, E.

    1999-01-01

    This chapter will very briefly introduce and review some computational experiments in using trainable gene regulation network models to simulate and understand selected episodes in the development of the fruit fly, Drosophila Melanogaster.

  12. Drosophila RNAi screening in a postgenomic world

    PubMed Central

    2011-01-01

    Drosophila melanogaster has a long history as a model organism with several unique features that make it an ideal research tool for the study of the relationship between genotype and phenotype. Importantly fundamental genetic principles as well as key human disease genes have been uncovered through the use of Drosophila. The contribution of the fruit fly to science and medicine continues in the postgenomic era as cell-based Drosophila RNAi screens are a cost-effective and scalable enabling technology that can be used to quantify the contribution of different genes to diverse cellular processes. Drosophila high-throughput screens can also be used as integral part of systems-level approaches to describe the architecture and dynamics of cellular networks. PMID:21752787

  13. Drosophila Cajal bodies: accessories not included

    PubMed Central

    Matera, A. Gregory

    2006-01-01

    Cajal bodies are nuclear sites of small ribonucleoprotein (RNP) remodeling and maturation. A recent study describes the discovery of the Drosophila Cajal body, revealing some interesting insights into the subnuclear organization of RNA processing machineries among different species. PMID:16533940

  14. Targeted genome engineering techniques in Drosophila

    PubMed Central

    Beumer, Kelly J.; Carroll, Dana

    2014-01-01

    For a century, Drosophila has been a favored organism for genetic research. However, the array of materials and methods available to the Drosophila worker has expanded dramatically in the last decade. The most common gene targeting tools, zinc finger nucleases, TALENs, and RNA-guided CRISPR/Cas9, have all been adapted for use in Drosophila, both for simple mutagenesis and for gene editing via homologous recombination. For each tool, there exist a number of web sites, design applications, and delivery methods. The successful application of any of these tools also requires an understanding of methods for detecting successful genome modifications. This article provides an overview of the available gene targeting tools and their application in Drosophila. In lieu of simply providing a protocol for gene targeting, we direct the researcher to resources that will allow access to the latest research in this rapidly evolving field. PMID:24412316

  15. Circadian light-input pathways in Drosophila.

    PubMed

    Yoshii, Taishi; Hermann-Luibl, Christiane; Helfrich-Förster, Charlotte

    2016-01-01

    Light is the most important environmental cue to entrain the circadian clock in most animals. In the fruit fly Drosophila melanogaster, the light entrainment mechanisms of the clock have been well-studied. The Drosophila brain contains approximately 150 neurons that rhythmically express circadian clock genes. These neurons are called "clock neurons" and control behavioral activity rhythms. Many clock neurons express the Cryptochrome (CRY) protein, which is sensitive to UV and blue light, and thus enables clock neurons deep in the brain to directly perceive light. In addition to the CRY protein, external photoreceptors in the Drosophila eyes play an important role in circadian light-input pathways. Recent studies have provided new insights into the mechanisms that integrate these light inputs into the circadian network of the brain. In this review, we will summarize the current knowledge on the light entrainment pathways in the Drosophila circadian clock. PMID:27066180

  16. Ecdysteroid receptors in Drosophila melanogaster adult females

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ecdysteroid receptors were identified and partially characterized from total cell extracts of whole animals and dissected tissues from Drosophila melanogaster adult females. Binding studies indicated the presence of two ecdysteroid binding components having high affinity and specificity consistent w...

  17. Colour vision: parallel pathways intersect in Drosophila.

    PubMed

    Kelber, Almut; Henze, Miriam J

    2013-12-01

    In the last one hundred years, colour vision has been demonstrated in bees and many other insects. But the underlying neural wiring remained elusive. A new study on Drosophila melanogaster combining behavioural and genetic tools yields surprising insights. PMID:24309280

  18. Drosophila PI4KIIIalpha is required in follicle cells for oocyte polarization and Hippo signaling

    PubMed Central

    Yan, Yan; Denef, Natalie; Tang, Charm; Schüpbach, Trudi

    2011-01-01

    In a genetic screen we isolated mutations in CG10260, which encodes a phosphatidylinositol 4-kinase (PI4KIIIalpha), and found that PI4KIIIalpha is required for Hippo signaling in Drosophila ovarian follicle cells. PI4KIIIalpha mutations in the posterior follicle cells lead to oocyte polarization defects similar to those caused by mutations in the Hippo signaling pathway. PI4KIIIalpha mutations also cause misexpression of well-established Hippo signaling targets. The Merlin-Expanded-Kibra complex is required at the apical membrane for Hippo activity. In PI4KIIIalpha mutant follicle cells, Merlin fails to localize to the apical domain. Our analysis of PI4KIIIalpha mutants provides a new link in Hippo signal transduction from the cell membrane to its core kinase cascade. PMID:21429988

  19. Rhabdomere biogenesis in Drosophila photoreceptors is acutely sensitive to phosphatidic acid levels

    PubMed Central

    Coessens, Elise; Manifava, Maria; Georgiev, Plamen; Pettitt, Trevor; Wood, Eleanor; Garcia-Murillas, Isaac; Okkenhaug, Hanneke; Trivedi, Deepti; Zhang, Qifeng; Razzaq, Azam; Zaid, Ola; Wakelam, Michael; O'Kane, Cahir J; Ktistakis, Nicholas

    2009-01-01

    Phosphatidic acid (PA) is postulated to have both structural and signaling functions during membrane dynamics in animal cells. In this study, we show that before a critical time period during rhabdomere biogenesis in Drosophila melanogaster photoreceptors, elevated levels of PA disrupt membrane transport to the apical domain. Lipidomic analysis shows that this effect is associated with an increase in the abundance of a single, relatively minor molecular species of PA. These transport defects are dependent on the activation state of Arf1. Transport defects via PA generated by phospholipase D require the activity of type I phosphatidylinositol (PI) 4 phosphate 5 kinase, are phenocopied by knockdown of PI 4 kinase, and are associated with normal endoplasmic reticulum to Golgi transport. We propose that PA levels are critical for apical membrane transport events required for rhabdomere biogenesis. PMID:19349583

  20. The bHLH transcription factor Hand is regulated by Alk in the Drosophila embryonic gut

    SciTech Connect

    Varshney, Gaurav K.; Palmer, Ruth H. . E-mail: Ruth.Palmer@ucmp.umu.se

    2006-12-29

    During embryonic development the midgut visceral muscle is formed by fusion of cells within the visceral mesoderm, a process initiated by the specification of a specialised cell type, the founder cell, within this tissue. Activation of the receptor tyrosine kinase Anaplastic lymphoma kinase (Alk) in the developing visceral muscle of Drosophila melanogaster initiates a signal transduction pathway required for muscle fusion. In this paper, we have investigated downstream components which are regulated by this novel signalling pathway. Here we show that Alk-mediated signal transduction drives the expression of the bHLH transcription factor Hand in vivo. Loss of Alk function results in a complete lack of Hand expression in this tissue, whereas Alk gain of function results in an expansion of Hand expression. Finally, we have investigated the process of muscle fusion in the gut of Hand mutant animals and can find no obvious defects in this process, suggesting that Hand is not critical for visceral muscle fusion per se.

  1. The Development of Germline Stem Cells in Drosophila

    PubMed Central

    Dansereau, David A.; Lasko, Paul

    2009-01-01

    Summary Germline stem cells (GSCs) in Drosophila are a valuable model to explore of how adult stem cells are regulated in vivo. Genetic dissection of this system has shown that stem cell fate is determined and maintained by the stem cell’s somatic microenvironment or niche. In Drosophila gonads, the stem cell niche—the cap cell cluster in females and the hub in males—acts as a signaling center to recruit GSCs from among a small population of undifferentiated primordial germ cells (PGCs). Short-range signals from the niche specify and regulate stem cell fate by maintaining the undifferentiated state of the PGCs next to the niche. Germline cells that do not receive the niche signals because of their location assume the default fate and differentiate. Once GSCs are specified, adherens junctions maintain close association between the stem cells and their niche and help to orient stem cell division so that one daughter is displaced from the niche and differentiates. In females, stem cell fate depends on bone morphogenetic protein (BMP) signals from the cap cells; in males, hub cells express the cytokine-like ligand Unpaired, which activates the Janus kinase-signal transducers and activators of transcription (Jak-Stat) pathway in stem cells. Although the signaling pathways operating between the niche and stem cells are different, there are common general features in both males and females, including the arrangement of cell types, many of the genes used, and the logic of the system that maintains stem cell fate. PMID:18370048

  2. Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila

    PubMed Central

    Buchon, Nicolas; Broderick, Nichole A.; Chakrabarti, Sveta; Lemaitre, Bruno

    2009-01-01

    Gut homeostasis is controlled by both immune and developmental mechanisms, and its disruption can lead to inflammatory disorders or cancerous lesions of the intestine. While the impact of bacteria on the mucosal immune system is beginning to be precisely understood, little is known about the effects of bacteria on gut epithelium renewal. Here, we addressed how both infectious and indigenous bacteria modulate stem cell activity in Drosophila. We show that the increased epithelium renewal observed upon some bacterial infections is a consequence of the oxidative burst, a major defense of the Drosophila gut. Additionally, we provide evidence that the JAK–STAT (Janus kinase–signal transducers and activators of transcription) and JNK (c-Jun NH2 terminal kinase) pathways are both required for bacteria-induced stem cell proliferation. Similarly, we demonstrate that indigenous gut microbiota activate the same, albeit reduced, program at basal levels. Altered control of gut microbiota in immune-deficient or aged flies correlates with increased epithelium renewal. Finally, we show that epithelium renewal is an essential component of Drosophila defense against oral bacterial infection. Altogether, these results indicate that gut homeostasis is achieved by a complex interregulation of the immune response, gut microbiota, and stem cell activity. PMID:19797770

  3. Controlling anoxic tolerance in adult Drosophila via the cGMP–PKG pathway

    PubMed Central

    Dawson-Scully, K.; Bukvic, D.; Chakaborty-Chatterjee, M.; Ferreira, R.; Milton, S. L.; Sokolowski, M. B.

    2010-01-01

    In this study we identify a cGMP-dependent protein kinase (PKG) cascade as a biochemical pathway critical for controlling low-oxygen tolerance in the adult fruit fly, Drosophila melanogaster. Even though adult Drosophila can survive in 0% oxygen (anoxia) environments for hours, air with less than 2% oxygen rapidly induces locomotory failure resulting in an anoxic coma. We use natural genetic variation and an induced mutation in the foraging (for) gene, which encodes a Drosophila PKG, to demonstrate that the onset of anoxic coma is correlated with PKG activity. Flies that have lower PKG activity demonstrate a significant increase in time to the onset of anoxic coma. Further, in vivo pharmacological manipulations reveal that reducing either PKG or protein phosphatase 2A (PP2A) activity increases tolerance of behavior to acute hypoxic conditions. Alternatively, PKG activation and phosphodiesterase (PDE5/6) inhibition significantly reduce the time to the onset of anoxic coma. By manipulating these targets in paired combinations, we characterized a specific PKG cascade, with upstream and downstream components. Further, using genetic variants of PKG expression/activity subjected to chronic anoxia over 6 h, ~50% of animals with higher PKG activity survive, while only ~25% of those with lower PKG activity survive after a 24 h recovery. Therefore, in this report we describe the PKG pathway and the differential protection of function vs survival in a critically low oxygen environment. PMID:20581270

  4. Cooperative control of Drosophila immune responses by the JNK and NF-κB signaling pathways

    PubMed Central

    Delaney, Joseph R; Stöven, Svenja; Uvell, Hanna; Anderson, Kathryn V; Engström, Ylva; Mlodzik, Marek

    2006-01-01

    Jun N-terminal kinase (JNK) signaling is a highly conserved pathway that controls both cytoskeletal remodeling and transcriptional regulation in response to a wide variety of signals. Despite the importance of JNK in the mammalian immune response, and various suggestions of its importance in Drosophila immunity, the actual contribution of JNK signaling in the Drosophila immune response has been unclear. Drosophila TAK1 has been implicated in the NF-κB/Relish-mediated activation of antimicrobial peptide genes. However, we demonstrate that Relish activation is intact in dTAK1 mutant animals, and that the immune response in these mutant animals was rescued by overexpression of a downstream JNKK. The expression of a JNK inhibitor and induction of JNK loss-of-function clones in immune responsive tissue revealed a general requirement for JNK signaling in the expression of antimicrobial peptides. Our data indicate that dTAK1 is not required for Relish activation, but instead is required in JNK signaling for antimicrobial peptide gene expression. PMID:16763552

  5. Drosophila glia use a conserved cotransporter mechanism to regulate extracellular volume

    PubMed Central

    Leiserson, William M.; Forbush, Biff; Keshishian, Haig

    2010-01-01

    The nervous system is protected by blood barriers that use multiple systems to control extracellular solute composition, osmotic pressure, and fluid volume. In the human nervous system, misregulation of the extracellular volume poses serious health threats. Here we show that the glial cells that form the Drosophila blood-nerve barrier have a conserved molecular mechanism that regulates extracellular volume: the Serine/Threonine kinase Fray, which we previously showed is an ortholog of mammalian PASK/SPAK; and the Na-K-Cl cotransporter NCC69, which we show is an ortholog of human NKCC1. In mammals, PASK/SPAK binds to NKCC1 and regulates its activity. In Drosophila, larvae mutant for NCC69 develop a peripheral neuropathy, where fluid accumulates between glia and axons. The accumulation of fluid has no detectable impact on action potential conduction, suggesting that the role of NCC69 is to maintain volume or osmotic homeostasis. Drosophila NCC69 has kinetics similar to human NKCC1, and NKCC1 can rescue NCC69, suggesting that they function in a conserved physiological mechanism. We show that fray and NCC69 are coexpressed in nerve glia, interact in a yeast-two-hybrid assay, and have an essentially identical bulging nerve phenotype. We propose that normally functioning nerves generate extracellular solutes that are removed by NCC69 under the control of Fray. This mechanism may perform a similar role in humans, given that NKCC1 is expressed at the blood-brain barrier. PMID:21125654

  6. Aging and CaMKII alter intracellular Ca2+ transients and heart rhythm in Drosophila melanogaster.

    PubMed

    Santalla, Manuela; Valverde, Carlos A; Harnichar, Ezequiel; Lacunza, Ezequiel; Aguilar-Fuentes, Javier; Mattiazzi, Alicia; Ferrero, Paola

    2014-01-01

    Aging is associated to disrupted contractility and rhythmicity, among other cardiovascular alterations. Drosophila melanogaster shows a pattern of aging similar to human beings and recapitulates the arrhythmogenic conditions found in the human heart. Moreover, the kinase CaMKII has been characterized as an important regulator of heart function and an arrhythmogenic molecule that participate in Ca2+ handling. Using a genetically engineered expressed Ca2+ indicator, we report changes in cardiac Ca2+ handling at two different ages. Aging prolonged relaxation, reduced spontaneous heart rate (HR) and increased the occurrence of arrhythmias, ectopic beats and asystoles. Alignment between Drosophila melanogaster and human CaMKII showed a high degree of conservation and indicates that relevant phosphorylation sites in humans are also present in the fruit fly. Inhibition of CaMKII by KN-93 (CaMKII-specific inhibitor), reduced HR without significant changes in other parameters. By contrast, overexpression of CaMKII increased HR and reduced arrhythmias. Moreover, it increased fluorescence amplitude, maximal rate of rise of fluorescence and reduced time to peak fluorescence. These results suggest that CaMKII in Drosophila melanogaster acts directly on heart function and that increasing CaMKII expression levels could be beneficial to improve contractility. PMID:25003749

  7. Aging and CaMKII Alter Intracellular Ca2+ Transients and Heart Rhythm in Drosophila melanogaster

    PubMed Central

    Santalla, Manuela; Valverde, Carlos A.; Harnichar, Ezequiel; Lacunza, Ezequiel; Aguilar-Fuentes, Javier; Mattiazzi, Alicia; Ferrero, Paola

    2014-01-01

    Aging is associated to disrupted contractility and rhythmicity, among other cardiovascular alterations. Drosophila melanogaster shows a pattern of aging similar to human beings and recapitulates the arrhythmogenic conditions found in the human heart. Moreover, the kinase CaMKII has been characterized as an important regulator of heart function and an arrhythmogenic molecule that participate in Ca2+ handling. Using a genetically engineered expressed Ca2+ indicator, we report changes in cardiac Ca2+ handling at two different ages. Aging prolonged relaxation, reduced spontaneous heart rate (HR) and increased the occurrence of arrhythmias, ectopic beats and asystoles. Alignment between Drosophila melanogaster and human CaMKII showed a high degree of conservation and indicates that relevant phosphorylation sites in humans are also present in the fruit fly. Inhibition of CaMKII by KN-93 (CaMKII-specific inhibitor), reduced HR without significant changes in other parameters. By contrast, overexpression of CaMKII increased HR and reduced arrhythmias. Moreover, it increased fluorescence amplitude, maximal rate of rise of fluorescence and reduced time to peak fluorescence. These results suggest that CaMKII in Drosophila melanogaster acts directly on heart function and that increasing CaMKII expression levels could be beneficial to improve contractility. PMID:25003749

  8. Differential Selection within the Drosophila Retinal Determination Network and Evidence for Functional Divergence between Paralog Pairs

    PubMed Central

    Datta, Rhea R.; Cruickshank, Tami; Kumar, Justin P.

    2011-01-01

    The retinal determination (RD) network in Drosophila comprises fourteen known nuclear proteins that include DNA binding proteins, transcriptional co-activators, kinases and phosphatases. The composition of the network varies considerably throughout the animal kingdom, with the network in several basal insects having fewer members and with vertebrates having potentially significantly higher numbers of retinal determination genes. One important contributing factor for the variation in gene number within the network is gene duplication. For example, ten members of the RD network in Drosophila are derived from duplication events. Here we present an analysis of the coding regions of the five pairs of duplicate genes from within the retinal determination network of several different Drosophila species. We demonstrate that there is differential selection across the coding regions of all RD genes. Additionally, some of the most significant differences in ratios of non-silent to silent site substitutions (dN/dS) between paralog pairs are found within regions that have no ascribed function. Previous structure/function analyses of several duplicate genes have identified areas within one gene that contain novel activities when compared to its paralog. The evolutionary analysis presented here identifies these same areas in the paralogs as being under high levels of relaxed selection. We suggest that sequence divergence between paralogs and selection signatures can be used as a reasonable predictor of functional changes in rapidly evolving motifs. PMID:21210943

  9. Drosophila melanogaster as a model organism for Alzheimer’s disease

    PubMed Central

    2013-01-01

    Drosophila melanogaster provides an important resource for in vivo modifier screens of neurodegenerative diseases. To study the underlying pathogenesis of Alzheimer’s disease, fly models that address Tau or amyloid toxicity have been developed. Overexpression of human wild-type or mutant Tau causes age-dependent neurodegeneration, axonal transport defects and early death. Large-scale screens utilizing a neurodegenerative phenotype induced by eye-specific overexpression of human Tau have identified several kinases and phosphatases, apoptotic regulators and cytoskeleton proteins as determinants of Tau toxicity in vivo. The APP ortholog of Drosophila (dAPPl) shares the characteristic domains with vertebrate APP family members, but does not contain the human Aβ42 domain. To circumvent this drawback, researches have developed strategies by either direct secretion of human Aβ42 or triple transgenic flies expressing human APP, β-secretase and Drosophila γ-secretase presenilin (dPsn). Here, we provide a brief overview of how fly models of AD have contributed to our knowledge of the pathomechanisms of disease. PMID:24267573

  10. Functional characterisation of human synaptic genes expressed in the Drosophila brain.

    PubMed

    Zografos, Lysimachos; Tang, Joanne; Hesse, Franziska; Wanker, Erich E; Li, Ka Wan; Smit, August B; Davies, R Wayne; Armstrong, J Douglas

    2016-01-01

    Drosophila melanogaster is an established and versatile model organism. Here we describe and make available a collection of transgenic Drosophila strains expressing human synaptic genes. The collection can be used to study and characterise human synaptic genes and their interactions and as controls for mutant studies. It was generated in a way that allows the easy addition of new strains, as well as their combination. In order to highlight the potential value of the collection for the characterisation of human synaptic genes we also use two assays, investigating any gain-of-function motor and/or cognitive phenotypes in the strains in this collection. Using these assays we show that among the strains made there are both types of gain-of-function phenotypes investigated. As an example, we focus on the three strains expressing human tyrosine protein kinase Fyn, the small GTPase Rap1a and human Arc, respectively. Of the three, the first shows a cognitive gain-of-function phenotype while the second a motor gain-of-function phenotype. By contrast, Arc, which has no Drosophila ortholog, shows no gain-of-function phenotype. PMID:27069252

  11. Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway.

    PubMed

    Kapahi, Pankaj; Zid, Brian M; Harper, Tony; Koslover, Daniel; Sapin, Viveca; Benzer, Seymour

    2004-05-25

    In many species, reducing nutrient intake without causing malnutrition extends lifespan. Like DR (dietary restriction), modulation of genes in the insulin-signaling pathway, known to alter nutrient sensing, has been shown to extend lifespan in various species. In Drosophila, the target of rapamycin (TOR) and the insulin pathways have emerged as major regulators of growth and size. Hence we examined the role of TOR pathway genes in regulating lifespan by using Drosophila. We show that inhibition of TOR signaling pathway by alteration of the expression of genes in this nutrient-sensing pathway, which is conserved from yeast to human, extends lifespan in a manner that may overlap with known effects of dietary restriction on longevity. In Drosophila, TSC1 and TSC2 (tuberous sclerosis complex genes 1 and 2) act together to inhibit TOR (target of rapamycin), which mediates a signaling pathway that couples amino acid availability to S6 kinase, translation initiation, and growth. We find that overexpression of dTsc1, dTsc2, or dominant-negative forms of dTOR or dS6K all cause lifespan extension. Modulation of expression in the fat is sufficient for the lifespan-extension effects. The lifespan extensions are dependent on nutritional condition, suggesting a possible link between the TOR pathway and dietary restriction. PMID:15186745

  12. Regulation of Lifespan in Drosophila by Modulation of Genes in the TOR Signaling Pathway

    PubMed Central

    Kapahi, Pankaj; Zid, Brian M.; Harper, Tony; Koslover, Daniel; Sapin, Viveca; Benzer, Seymour

    2009-01-01

    Summary In many species, reducing nutrient intake without causing malnutrition extends lifespan [1-3]. Like DR (dietary restriction), modulation of genes in the insulin-signaling pathway, known to alter nutrient sensing, has been shown to extend lifespan in various species [1-4]. In Drosophila, the target of rapamycin (TOR) and the insulin pathways have emerged as major regulators of growth and size. Hence we examined the role of TOR pathway genes in regulating lifespan by using Drosophila. We show that inhibition of TOR signaling pathway by alteration of the expression of genes in this nutrient-sensing pathway, which is conserved from yeast to human, extends lifespan in a manner that may overlap with known effects of dietary restriction on longevity. In Drosophila , TSC1 and TSC2 (tuberous sclerosis complex genes 1 and 2) act together to inhibit TOR (target of rapamycin), which mediates a signaling pathway that couples amino acid availability to S6 kinase, translation initiation, and growth [5]. We find that overexpression of dTsc1, dTsc2, or dominant-negative forms of dTOR or dS6K all cause lifespan extension. Modulation of expression in the fat is sufficient for the lifespan-extension effects. The lifespan extensions are dependent on nutritional condition, suggesting a possible link between the TOR pathway and dietary restriction. PMID:15186745

  13. Functional characterisation of human synaptic genes expressed in the Drosophila brain

    PubMed Central

    Zografos, Lysimachos; Tang, Joanne; Hesse, Franziska; Wanker, Erich E.; Li, Ka Wan; Smit, August B.; Davies, R. Wayne; Armstrong, J. Douglas

    2016-01-01

    ABSTRACT Drosophila melanogaster is an established and versatile model organism. Here we describe and make available a collection of transgenic Drosophila strains expressing human synaptic genes. The collection can be used to study and characterise human synaptic genes and their interactions and as controls for mutant studies. It was generated in a way that allows the easy addition of new strains, as well as their combination. In order to highlight the potential value of the collection for the characterisation of human synaptic genes we also use two assays, investigating any gain-of-function motor and/or cognitive phenotypes in the strains in this collection. Using these assays we show that among the strains made there are both types of gain-of-function phenotypes investigated. As an example, we focus on the three strains expressing human tyrosine protein kinase Fyn, the small GTPase Rap1a and human Arc, respectively. Of the three, the first shows a cognitive gain-of-function phenotype while the second a motor gain-of-function phenotype. By contrast, Arc, which has no Drosophila ortholog, shows no gain-of-function phenotype. PMID:27069252

  14. Intercellular cooperation and competition in brain cancers: lessons from Drosophila and human studies.

    PubMed

    Waghmare, Indrayani; Roebke, Austin; Minata, Mutsuko; Kango-Singh, Madhuri; Nakano, Ichiro

    2014-11-01

    Glioblastoma (GBM) is a primary brain cancer with an extremely poor prognosis. GBM tumors contain heterogeneous cellular components, including a small subpopulation of tumor cells termed glioma stem cells (GSCs). GSCs are characterized as chemotherapy- and radiotherapy-resistant cells with prominent tumorigenic ability. Studies in Drosophila cancer models demonstrated that interclonal cooperation and signaling from apoptotic clones provokes aggressive growth of neighboring tumorigenic clones, via compensatory proliferation or apoptosis induced proliferation. Mechanistically, these aggressive tumors depend on activation of Jun-N-terminal kinase (upstream of c-JUN), and Drosophila Wnt (Wg) in the apoptotic clones. Consistent with these nonmammalian studies, data from several mammalian studies have shown that c-JUN and Wnt are hyperactivated in aggressive tumors (including GBM). However, it remains elusive whether compensatory proliferation is an evolutionarily conserved mechanism in cancers. In the present report, we summarize recent studies in Drosophila models and mammalian models (e.g., xenografts of human cancer cells into small animals) to elucidate the intercellular interactions between the apoptosis-prone cancer cells (e.g., non-GSCs) and the hyperproliferative cancer cells (e.g., GSCs). These evolving investigations will yield insights about molecular signaling interactions in the context of post-therapeutic phenotypic changes in human cancers. Furthermore, these studies are likely to revise our understanding of the genetic changes and post-therapeutic cell-cell interactions, which is a vital area of cancer biology with wide applications to many cancer types in humans. PMID:25232184

  15. Activity-based kinase profiling of approved tyrosine kinase inhibitors.

    PubMed

    Kitagawa, Daisuke; Yokota, Koichi; Gouda, Masaki; Narumi, Yugo; Ohmoto, Hiroshi; Nishiwaki, Eiji; Akita, Kensaku; Kirii, Yasuyuki

    2013-02-01

    The specificities of nine approved tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, lapatinib, sorafenib, sunitinib, and pazopanib) were determined by activity-based kinase profiling using a large panel of human recombinant active kinases. This panel consisted of 79 tyrosine kinases, 199 serine/threonine kinases, three lipid kinases, and 29 disease-relevant mutant kinases. Many potential targets of each inhibitor were identified by kinase profiling at the K(m) for ATP. In addition, profiling at a physiological ATP concentration (1 mm) was carried out, and the IC(50) values of the inhibitors against each kinase were compared with the estimated plasma-free concentration (calculated from published pharmacokinetic parameters of plasma C(trough) and C(max) values). This analysis revealed that the approved kinase inhibitors were well optimized for their target kinases. This profiling also implicates activity at particular off-target kinases in drug side effects. Thus, large-scale kinase profiling at both K(m) and physiological ATP concentrations could be useful in characterizing the targets and off-targets of kinase inhibitors. PMID:23279183

  16. Two retrotransposons maintain telomeres in Drosophila

    PubMed Central

    Pardue, M.-L.; Rashkova, S.; Casacuberta, E.; DeBaryshe, P.G.; George, J. A.; Traverse, K.L.

    2005-01-01

    Telomeres across the genus Drosophila are maintained, not by telomerase, but by two non-LTR retrotransposons, HeT-A and TART, that transpose specifically to chromosome ends. Successive transpositions result in long head-to-tail arrays of these elements. Thus Drosophila telomeres, like those produced by telomerase, consist of repeated sequences reverse transcribed from RNA templates. The Drosophila repeats, complete and 5′-truncated copies of HeT-A and TART, are more complex than telomerase repeats; nevertheless these evolutionary variants have functional similarities to the more common telomeres. Like other telomeres, the Drosophila arrays are dynamic, fluctuating around an average length that can be changed by changes in the genetic background. Several proteins that interact with telomeres in other species have been found to have homologues that interact with Drosophila telomeres. Although they have hallmarks of non-LTR retrotransposons, HeT-A and TART appear to have a special relationship to Drosophila. Their Gag proteins are efficiently transported into diploid nuclei where HeT-A Gag recruits TART Gag to chromosome ends. Gags of other non-LTR elements remain predominantly in the cytoplasm. These studies provide intriguing evolutionary links between telomeres and retrotransposable elements. PMID:16132810

  17. A Drosophila Model for Screening Antiobesity Agents

    PubMed Central

    Men, Tran Thanh; Thanh, Duong Ngoc Van; Yamaguchi, Masamitsu; Suzuki, Takayoshi; Hattori, Gen; Arii, Masayuki; Huy, Nguyen Tien; Kamei, Kaeko

    2016-01-01

    Although triacylglycerol, the major component for lipid storage, is essential for normal physiology, its excessive accumulation causes obesity in adipose tissue and is associated with organ dysfunction in nonadipose tissue. Here, we focused on the Drosophila model to develop therapeutics for preventing obesity. The brummer (bmm) gene in Drosophila melanogaster is known to be homologous with human adipocyte triglyceride lipase, which is related to the regulation of lipid storage. We established a Drosophila model for monitoring bmm expression by introducing the green fluorescent protein (GFP) gene as a downstream reporter of the bmm promoter. The third-instar larvae of Drosophila showed the GFP signal in all tissues observed and specifically in the salivary gland nucleus. To confirm the relationship between bmm expression and obesity, the effect of oral administration of glucose diets on bmm promoter activity was analyzed. The Drosophila flies given high-glucose diets showed higher lipid contents, indicating the obesity phenotype; this was suggested by a weaker intensity of the GFP signal as well as reduced bmm mRNA expression. These results demonstrated that the transgenic Drosophila model established in this study is useful for screening antiobesity agents. We also report the effects of oral administration of histone deacetylase inhibitors and some vegetables on the bmm promoter activity. PMID:27247940

  18. Postharvest treatment of strawberries with methyl bromide to control spotted wing drosophila, Drosophila suzukii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Spotted wing drosophila (SWD), Drosophila suzukii, is a pest of concern to countries that import strawberries from California USA. The purpose of this investigation was to verify elimination of SWD in California-grown fresh strawberries under conditions consistent with export to Australia; a 3-h fum...

  19. Current Recommendations for Managing Spotted Wing Drosophila (SWD), Drosophila suzukii, in PNW Strawberries

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The spotted wing Drosophila (SWD), Drosophila suzukii, was reported in the Pacific Northwest (Oregon, Washington, British Columbia) in 2009. The fly is able to oviposit directly into intact ripe and ripening fruit, so it is of great economic concern to the small fruit industries in region. Fruit i...

  20. Current Recommendations for Managing Spotted Wing Drosophila (SWD), Drosophila suzukii, in PNW Blueberries

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The spotted wing Drosophila (SWD), Drosophila suzukii, was reported in the Pacific Northwest (Oregon, Washington, British Columbia) in 2009. The fly is able to oviposit directly into intact ripe and ripening fruit, so it is of great economic concern to the small fruit industries in region. Fruit i...

  1. Current Recommendations for Managing Spotted Wing Drosophila (SWD), Drosophila suzukii, in PNW Caneberries

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The spotted wing Drosophila (SWD), Drosophila suzukii, was reported in the Pacific Northwest (Oregon, Washington, British Columbia) in 2009. The fly is able to oviposit directly into intact ripe and ripening fruit, so it is of great economic concern to the small fruit industries in region. Fruit i...

  2. The susceptibility of small fruits and cherries to Spotted Wing Drosophila, Drosophila suzukii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: The Spotted Wing Drosophila (SWD), Drosophila suzukii Matsumura, is native to Asia and has been detected in the North American mainland and Europe in 2008-10. SWD is a serious economic pest because it lays eggs within ripening fruit before harvest which can lead to crop loss. The aim ...

  3. Behavioral and antennal responses of spotted wing drosophila, drosophila suzukii, to volatiles from fruit extracts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Native to Southeast Asia, the spotted wing drosophila, Drosophila suzukii, has become a serious pest of soft-skinned fruit crops since its introduction into North America and Europe in 2008. Current monitoring strategies use baits based on fermentation products; however, to date, no fruit-based vola...

  4. Invasion biology of Spotted Wing Drosophila (Drosophila suzukii): a global perspective and future priorities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Asian vinegar fly species Drosophila suzukii (spotted-wing Drosophila or SWD) has emerged as an important invasive insect pest of small and stone fruits in both the Americas and Europe since the late 2000’s. While research efforts have rapidly progressed in Asia, North America, and Europe over ...

  5. Spotted wing drosophila, Drosophila suzukii (Matsumura)(Diptera: drosophilidae), trapped with combinations of wines and vinegars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Field trapping experiments evaluated wine and vinegar baits for spotted wing drosophila flies, Drosophila suzukii (Matsumura), and assessed variance in biat attractiveness with wit type, vinegar type, and bait age. A mixture of apple cider vinegar and a Merlot wine attracted more flies than a mixtur...

  6. Homotypic Regulatory Clusters in Drosophila

    PubMed Central

    Lifanov, Alexander P.; Makeev, Vsevolod J.; Nazina, Anna G.; Papatsenko, Dmitri A.

    2003-01-01

    Cis-regulatory modules (CRMs) are transcription regulatory DNA segments (∼1 Kb range) that control the expression of developmental genes in higher eukaryotes. We analyzed clustering of known binding motifs for transcription factors (TFs) in over 60 known CRMs from 20 Drosophila developmental genes, and we present evidence that each type of recognition motif forms significant clusters within the regulatory regions regulated by the corresponding TF. We demonstrate how a search with a single binding motif can be applied to explore gene regulatory networks and to discover coregulated genes in the genome. We also discuss the potential of the clustering method in interpreting the differential response of genes to various levels of transcriptional regulators. PMID:12670999

  7. Investigating Spermatogenesis in Drosophila melanogaster

    PubMed Central

    Demarco, Rafael S.; Eikenes, Åsmund H.; Haglund, Kaisa; Jones, D. Leanne

    2014-01-01

    The process of spermatogenesis in Drosophila melanogaster provides a powerful model system to probe a variety of developmental and cell biological questions, such as the characterization of mechanisms that regulate stem cell behavior, cytokinesis, meiosis, and mitochondrial dynamics. Classical genetic approaches, together with binary expression systems, FRT-mediated recombination, and novel imaging systems to capture single cell behavior, are rapidly expanding our knowledge of the molecular mechanisms regulating all aspects of spermatogenesis. This methods chapter provides a detailed description of the system, a review of key questions chapter that have been addressed or remain unanswered thus far, and an introduction to tools and techniques available to probe each stage of spermatogenesis. PMID:24798812

  8. Repositioning of a cyclin-dependent kinase inhibitor GW8510 as a ribonucleotide reductase M2 inhibitor to treat human colorectal cancer.

    PubMed

    Hsieh, Y-Y; Chou, C-J; Lo, H-L; Yang, P-M

    2016-01-01

    Colorectal cancer (CRC) is the second leading cause of cancer-related death in males and females in the world. It is of immediate importance to develop novel therapeutics. Human ribonucleotide reductase (RRM1/RRM2) has an essential role in converting ribonucleoside diphosphate to 2'-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools. RRM2 is a prognostic biomarker and predicts poor survival of CRC. In addition, increased RRM2 activity is associated with malignant transformation and tumor cell growth. Bioinformatics analyses show that RRM2 was overexpressed in CRC and might be an attractive target for treating CRC. Therefore, we attempted to search novel RRM2 inhibitors by using a gene expression signature-based approach, connectivity MAP (CMAP). The result predicted GW8510, a cyclin-dependent kinase inhibitor, as a potential RRM2 inhibitor. Western blot analysis indicated that GW8510 inhibited RRM2 expression through promoting its proteasomal degradation. In addition, GW8510 induced autophagic cell death. In addition, the sensitivities of CRC cells to GW8510 were associated with the levels of RRM2 and endogenous autophagic flux. Taken together, our study indicates that GW8510 could be a potential anti-CRC agent through targeting RRM2. PMID:27551518

  9. Repositioning of a cyclin-dependent kinase inhibitor GW8510 as a ribonucleotide reductase M2 inhibitor to treat human colorectal cancer

    PubMed Central

    Hsieh, Y-Y; Chou, C-J; Lo, H-L; Yang, P-M

    2016-01-01

    Colorectal cancer (CRC) is the second leading cause of cancer-related death in males and females in the world. It is of immediate importance to develop novel therapeutics. Human ribonucleotide reductase (RRM1/RRM2) has an essential role in converting ribonucleoside diphosphate to 2′-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools. RRM2 is a prognostic biomarker and predicts poor survival of CRC. In addition, increased RRM2 activity is associated with malignant transformation and tumor cell growth. Bioinformatics analyses show that RRM2 was overexpressed in CRC and might be an attractive target for treating CRC. Therefore, we attempted to search novel RRM2 inhibitors by using a gene expression signature-based approach, connectivity MAP (CMAP). The result predicted GW8510, a cyclin-dependent kinase inhibitor, as a potential RRM2 inhibitor. Western blot analysis indicated that GW8510 inhibited RRM2 expression through promoting its proteasomal degradation. In addition, GW8510 induced autophagic cell death. In addition, the sensitivities of CRC cells to GW8510 were associated with the levels of RRM2 and endogenous autophagic flux. Taken together, our study indicates that GW8510 could be a potential anti-CRC agent through targeting RRM2. PMID:27551518

  10. Phenotypic analysis of separation-of-function alleles of MEI-41, Drosophila ATM/ATR.

    PubMed Central

    Laurençon, Anne; Purdy, Amanda; Sekelsky, Jeff; Hawley, R Scott; Su, Tin Tin

    2003-01-01

    ATM/ATR kinases act as signal transducers in eukaryotic DNA damage and replication checkpoints. Mutations in ATM/ATR homologs have pleiotropic effects that range from sterility to increased killing by genotoxins in humans, mice, and Drosophila. Here we report the generation of a null allele of mei-41, Drosophila ATM/ATR homolog, and the use of it to document a semidominant effect on a larval mitotic checkpoint and methyl methanesulfonate (MMS) sensitivity. We also tested the role of mei-41 in a recently characterized checkpoint that delays metaphase/anaphase transition after DNA damage in cellular embryos. We then compare five existing mei-41 alleles to the null with respect to known phenotypes (female sterility, cell cycle checkpoints, and MMS resistance). We find that not all phenotypes are affected equally by each allele, i.e., the functions of MEI-41 in ensuring fertility, cell cycle regulation, and resistance to genotoxins are genetically separable. We propose that MEI-41 acts not in a single rigid signal transduction pathway, but in multiple molecular contexts to carry out its many functions. Sequence analysis identified mutations, which, for most alleles, fall in the poorly characterized region outside the kinase domain; this allowed us to tentatively identify additional functional domains of MEI-41 that could be subjected to future structure-function studies of this key molecule. PMID:12807779

  11. A Maternal Screen for Genes Regulating Drosophila Oocyte Polarity Uncovers New Steps in Meiotic Progression

    PubMed Central

    Barbosa, Vitor; Kimm, Naomi; Lehmann, Ruth

    2007-01-01

    Meiotic checkpoints monitor chromosome status to ensure correct homologous recombination, genomic integrity, and chromosome segregation. In Drosophila, the persistent presence of double-strand DNA breaks (DSB) activates the ATR/Mei-41 checkpoint, delays progression through meiosis, and causes defects in DNA condensation of the oocyte nucleus, the karyosome. Checkpoint activation has also been linked to decreased levels of the TGFα-like molecule Gurken, which controls normal eggshell patterning. We used this easy-to-score eggshell phenotype in a germ-line mosaic screen in Drosophila to identify new genes affecting meiotic progression, DNA condensation, and Gurken signaling. One hundred eighteen new ventralizing mutants on the second chromosome fell into 17 complementation groups. Here we describe the analysis of 8 complementation groups, including Kinesin heavy chain, the SR protein kinase cuaba, the cohesin-related gene dPds5/cohiba, and the Tudor-domain gene montecristo. Our findings challenge the hypothesis that checkpoint activation upon persistent DSBs is exclusively mediated by ATR/Mei-41 kinase and instead reveal a more complex network of interactions that link DSB formation, checkpoint activation, meiotic delay, DNA condensation, and Gurken protein synthesis. PMID:17507684

  12. Resources for Biological Annotation of the Drosophila Genome

    SciTech Connect

    Gerald M. Rubin

    2005-08-08

    This project supported seed money for the development of cDNA and genetic resources to support studies of the Drosophila melanogaster genome. Key publications supported by this work that provide additional detail: (1) ''The Drosophila gene collection: identification of putative full-length cDNAs for 70% of D. melanogaster genes''; and (2) ''The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes''.

  13. Two Drosophila suppressors of cytokine signaling (SOCS) differentially regulate JAK and EGFR pathway activities

    PubMed Central

    Rawlings, Jason S; Rennebeck, Gabriela; Harrison, Susan MW; Xi, Rongwen; Harrison, Douglas A

    2004-01-01

    Background The Janus kinase (JAK) cascade is an essential and well-conserved pathway required to transduce signals for a variety of ligands in both vertebrates and invertebrates. While activation of the pathway is essential to many processes, mutations from mammals and Drosophila demonstrate that regulation is also critical. The SOCS (Suppressor Of Cytokine Signaling) proteins in mammals are regulators of the JAK pathway that participate in a negative feedback loop, as they are transcriptionally activated by JAK signaling. Examination of one Drosophila SOCS homologue, Socs36E, demonstrated that its expression is responsive to JAK pathway activity and it is capable of downregulating JAK signaling, similar to the well characterized mammalian SOCS. Results Based on sequence analysis of the Drosophila genome, there are three identifiable SOCS homologues in flies. All three are most similar to mammalian SOCS that have not been extensively characterized: Socs36E is most similar to mammalian SOCS5, while Socs44A and Socs16D are most similar to mammalian SOCS6 and 7. Although Socs44A is capable of repressing JAK activity in some tissues, its expression is not regulated by the pathway. Furthermore, Socs44A can enhance the activity of the EGFR/MAPK signaling cascade, in contrast to Socs36E. Conclusions Two Drosophila SOCS proteins have some overlapping and some distinct capabilities. While Socs36E behaves similarly to the canonical vertebrate SOCS, Socs44A is not part of a JAK pathway negative feedback loop. Nonetheless, both SOCS regulate JAK and EGFR signaling pathways, albeit differently. The non-canonical properties of Socs44A may be representative of the class of less characterized vertebrate SOCS with which it shares greatest similarity. PMID:15488148

  14. Broad RNA interference-mediated antiviral immunity and virus-specific inducible responses in Drosophila.

    PubMed

    Kemp, Cordula; Mueller, Stefanie; Goto, Akira; Barbier, Vincent; Paro, Simona; Bonnay, François; Dostert, Catherine; Troxler, Laurent; Hetru, Charles; Meignin, Carine; Pfeffer, Sébastien; Hoffmann, Jules A; Imler, Jean-Luc

    2013-01-15

    The fruit fly Drosophila melanogaster is a good model to unravel the molecular mechanisms of innate immunity and has led to some important discoveries about the sensing and signaling of microbial infections. The response of Drosophila to virus infections remains poorly characterized and appears to involve two facets. On the one hand, RNA interference involves the recognition and processing of dsRNA into small interfering RNAs by the host RNase Dicer-2 (Dcr-2), whereas, on the other hand, an inducible response controlled by the evolutionarily conserved JAK-STAT pathway contributes to the antiviral host defense. To clarify the contribution of the small interfering RNA and JAK-STAT pathways to the control of viral infections, we have compared the resistance of flies wild-type and mutant for Dcr-2 or the JAK kinase Hopscotch to infections by seven RNA or DNA viruses belonging to different families. Our results reveal a unique susceptibility of hop mutant flies to infection by Drosophila C virus and cricket paralysis virus, two members of the Dicistroviridae family, which contrasts with the susceptibility of Dcr-2 mutant flies to many viruses, including the DNA virus invertebrate iridescent virus 6. Genome-wide microarray analysis confirmed that different sets of genes were induced following infection by Drosophila C virus or by two unrelated RNA viruses, Flock House virus and Sindbis virus. Overall, our data reveal that RNA interference is an efficient antiviral mechanism, operating against a large range of viruses, including a DNA virus. By contrast, the antiviral contribution of the JAK-STAT pathway appears to be virus specific. PMID:23255357

  15. Enhancing Undergraduate Teaching and Research with a "Drosophila" Virginizing System

    ERIC Educational Resources Information Center

    Venema, Dennis R.

    2006-01-01

    Laboratory exercises using "Drosophila" crosses are an effective pedagogical method to complement traditional lecture and textbook presentations of genetics. Undergraduate thesis research is another common setting for using "Drosophila." A significant barrier to using "Drosophila" for undergraduate teaching or research is the time and skill…

  16. Interplay of Notch and FGF signaling restricts cell fate and MAPK activation in the Drosophila trachea.

    PubMed

    Ikeya, T; Hayashi, S

    1999-10-01

    The patterned branching in the Drosophila tracheal system is triggered by the FGF-like ligand Branchless that activates a receptor tyrosine kinase Breathless and the MAP kinase pathway. A single fusion cell at the tip of each fusion branch expresses the zinc-finger gene escargot, leads branch migration in a stereotypical pattern and contacts with another fusion cell to mediate fusion of the branches. A high level of MAP kinase activation is also limited to the tip of the branches. Restriction of such cell specialization events to the tip is essential for tracheal tubulogenesis. Here we show that Notch signaling plays crucial roles in the singling out process of the fusion cell. We found that Notch is activated in tracheal cells by Branchless signaling through stimulation of &Dgr; expression at the tip of tracheal branches and that activated Notch represses the fate of the fusion cell. In addition, Notch is required to restrict activation of MAP kinase to the tip of the branches, in part through the negative regulation of Branchless expression. Notch-mediated lateral inhibition in sending and receiving cells is thus essential to restrict the inductive influence of Branchless on the tracheal tubulogenesis. PMID:10498681

  17. Phosphatidylinositol kinase from rabbit reticulocytes

    SciTech Connect

    Tuazon, P.T.; Heng, A.B.W.; Traugh, J.A.

    1986-05-01

    Phosphatidylinositol (PI) kinase was isolated from the postribosomal supernatant of rabbit reticulocytes. This activity was identified by the formation of a product that comigrated with phosphatidylinositol-4-phosphate (PIP) when purified PI was phosphorylated in the presence of (/sup 32/P)ATP and Mg/sup 2 +/. Three major peaks of PI kinase activity were resolved by chromatography on DEAE-cellulose. The first peak eluted at 50-100 mM NaCl together with several serine protein kinases, casein kinase (CK) I and protease activated kinase (PAK) I and II. The PI kinase was subsequently separated from the protein kinases by chromatography on phosphocellulose. The second peak eluted at 125-160 mM NaCl and contained another lipid kinase activity that produced a product which comigrated with phosphatidic acid on thin layer chromatography. The third peak, which eluted at 165-200 mM NaCl, partly comigrated with casein kinase (CK) II and an active protein kinase(s) which phosphorylated mixed histone and histone I. CK II and the histone kinase activities were also separated by chromatography on phosphocelluslose. The different forms of PI kinase were characterized and compared with respect to substrate and salt requirements.

  18. Environmental ethanol as an ecological constraint on dietary breadth of Spotted-Wing Drosophila, Drosophila suzukii Mat. (Diptera: Drosophilidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Spotted-wing Drosophila (SWD), Drosophila suzukii, is a recent fruit pest of the Americas whose destructiveness stems from its subcutaneous insertion of eggs into cultivated berries via a female’s prominent double bladed and serrated ovipositor. Atypical of most other Drosophila, D. suzukii adults a...

  19. 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.

  20. Development of dendrite polarity in Drosophila neurons

    PubMed Central

    2012-01-01

    Background Drosophila neurons have dendrites that contain minus-end-out microtubules. This microtubule arrangement is different from that of cultured mammalian neurons, which have mixed polarity microtubules in dendrites. Results To determine whether Drosophila and mammalian dendrites have a common microtubule organization during development, we analyzed microtubule polarity in Drosophila dendritic arborization neuron dendrites at different stages of outgrowth from the cell body in vivo. As dendrites initially extended, they contained mixed polarity microtubules, like mammalian neurons developing in culture. Over a period of several days this mixed microtubule array gradually matured to a minus-end-out array. To determine whether features characteristic of dendrites were localized before uniform polarity was attained, we analyzed dendritic markers as dendrites developed. In all cases the markers took on their characteristic distribution while dendrites had mixed polarity. An axonal marker was also quite well excluded from dendrites throughout development, although this was perhaps more efficient in mature neurons. To confirm that dendrite character could be acquired in Drosophila while microtubules were mixed, we genetically disrupted uniform dendritic microtubule organization. Dendritic markers also localized correctly in this case. Conclusions We conclude that developing Drosophila dendrites initially have mixed microtubule polarity. Over time they mature to uniform microtubule polarity. Dendrite identity is established before the mature microtubule arrangement is attained, during the period of mixed microtubule polarity. PMID:23111238

  1. Gut-associated microbes of Drosophila melanogaster

    PubMed Central

    Broderick, Nichole; Lemaitre, Bruno

    2012-01-01

    There is growing interest in using Drosophila melanogaster to elucidate mechanisms that underlie the complex relationships between a host and its microbiota. In addition to the many genetic resources and tools Drosophila provides, its associated microbiota is relatively simple (1–30 taxa), in contrast to the complex diversity associated with vertebrates (> 500 taxa). These attributes highlight the potential of this system to dissect the complex cellular and molecular interactions that occur between a host and its microbiota. In this review, we summarize what is known regarding the composition of gut-associated microbes of Drosophila and their impact on host physiology. We also discuss these interactions in the context of their natural history and ecology and describe some recent insights into mechanisms by which Drosophila and its gut microbiota interact. “Workers with Drosophila have been considered fortunate in that they deal with the first multicellular invertebrate to be cultured monoxenically (Delcourt and Guyenot, 1910); the first to be handled axenically on a semisynthetic diet (Guyenot, 1917); and the first to be grown on a defined diet (Schultz et al., 1946). This list of advantages is somewhat embarrassing, since it implies an interest in nutrition that, in reality, was only secondary. The very first studies were concerned with the reduction of variability in genetic experiments (Delcourt and Guyenot, 1910) and standardization of the nutritional environment.” -James Sang, 1959 Ann NY Acad 1 PMID:22572876

  2. Expression patterns of protein kinase D 3 during mouse development

    PubMed Central

    Ellwanger, Kornelia; Pfizenmaier, Klaus; Lutz, Sylke; Hausser, Angelika

    2008-01-01

    Background The PKD family of serine/threonine kinases comprises a single member in Drosophila (dPKD), two isoforms in C. elegans (DKF-1 and 2) and three members, PKD1, PKD2 and PKD3 in mammals. PKD1 and PKD2 have been the focus of most studies up to date, which implicate these enzymes in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, immune responses, apoptosis and cell proliferation. Concerning PKD3, a role in the formation of vesicular transport carriers at the trans-Golgi network (TGN) and in basal glucose transport has been inferred from in vitro studies. So far, however, the physiological functions of the kinase during development remain unknown. Results We have examined the expression pattern of PKD3 during the development of mouse embryos by immunohistochemistry. Using a PKD3 specific antibody we demonstrate that the kinase is differentially expressed during organogenesis. In the developing heart a strong PKD3 expression is constantly detected from E10 to E16.5. From E12.5 on PKD3 is increasingly expressed in neuronal as well as in the supporting connective tissue and in skeletal muscles. Conclusion The data presented support an important role for PKD3 during development of these tissues. PMID:18439271

  3. Sphingosine kinases and their metabolites modulate endolysosomal trafficking in photoreceptors

    PubMed Central

    Yonamine, Ikuko; Bamba, Takeshi; Nirala, Niraj K.; Jesmin, Nahid; Kosakowska-Cholody, Teresa; Nagashima, Kunio; Fukusaki, Eiichiro

    2011-01-01

    Internalized membrane proteins are either transported to late endosomes and lysosomes for degradation or recycled to the plasma membrane. Although proteins involved in trafficking and sorting have been well studied, far less is known about the lipid molecules that regulate the intracellular trafficking of membrane proteins. We studied the function of sphingosine kinases and their metabolites in endosomal trafficking using Drosophila melanogaster photoreceptors as a model system. Gain- and loss-of-function analyses show that sphingosine kinases affect trafficking of the G protein–coupled receptor Rhodopsin and the light-sensitive transient receptor potential (TRP) channel by modulating the levels of dihydrosphingosine 1 phosphate (DHS1P) and sphingosine 1 phosphate (S1P). An increase in DHS1P levels relative to S1P leads to the enhanced lysosomal degradation of Rhodopsin and TRP and retinal degeneration in wild-type photoreceptors. Our results suggest that sphingosine kinases and their metabolites modulate photoreceptor homeostasis by influencing endolysosomal trafficking of Rhodopsin and TRP. PMID:21321100

  4. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Linn, Anning

    1996-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK.

  5. In vivo tracking of phosphoinositides in Drosophila photoreceptors

    PubMed Central

    Hardie, Roger C.; Liu, Che-Hsiung; Randall, Alexander S.; Sengupta, Sukanya

    2015-01-01

    ABSTRACT In order to monitor phosphoinositide turnover during phospholipase C (PLC)-mediated Drosophila phototransduction, fluorescently tagged lipid probes were expressed in photoreceptors and imaged both in dissociated cells, and in eyes of intact living flies. Of six probes tested, TbR332H (a mutant of the Tubby protein pleckstrin homology domain) was judged the best reporter for phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], and the P4M domain from Legionella SidM for phosphatidylinositol 4-phosphate (PtdIns4P). Using accurately calibrated illumination, we found that only ∼50% of PtdIns(4,5)P2 and very little PtdIns4P were depleted by full daylight intensities in wild-type flies, but both were severely depleted by ∼100-fold dimmer intensities in mutants lacking Ca2+-permeable transient receptor potential (TRP) channels or protein kinase C (PKC). Resynthesis of PtdIns4P (t½ ∼12 s) was faster than PtdIns(4,5)P2 (t½ ∼40 s), but both were greatly slowed in mutants of DAG kinase (rdgA) or PtdIns transfer protein (rdgB). The results indicate that Ca2+- and PKC-dependent inhibition of PLC is required for enabling photoreceptors to maintain phosphoinositide levels despite high rates of hydrolysis by PLC, and suggest that phosphorylation of PtdIns4P to PtdIns(4,5)P2 is the rate-limiting step of the cycle. PMID:26483384

  6. The putative HORMA domain protein Atg101 dimerizes and is required for starvation-induced and selective autophagy in Drosophila.

    PubMed

    Hegedűs, Krisztina; Nagy, Péter; Gáspári, Zoltán; Juhász, Gábor

    2014-01-01

    The large-scale turnover of intracellular material including organelles is achieved by autophagy-mediated degradation in lysosomes. Initiation of autophagy is controlled by a protein kinase complex consisting of an Atg1-family kinase, Atg13, FIP200/Atg17, and the metazoan-specific subunit Atg101. Here we show that loss of Atg101 impairs both starvation-induced and basal autophagy in Drosophila. This leads to accumulation of protein aggregates containing the selective autophagy cargo ref(2)P/p62. Mapping experiments suggest that Atg101 binds to the N-terminal HORMA domain of Atg13 and may also interact with two unstructured regions of Atg1. Another HORMA domain-containing protein, Mad2, forms a conformational homodimer. We show that Drosophila Atg101 also dimerizes, and it is predicted to fold into a HORMA domain. Atg101 interacts with ref(2)P as well, similar to Atg13, Atg8a, Atg16, Atg18, Keap1, and RagC, a known regulator of Tor kinase which coordinates cell growth and autophagy. These results raise the possibility that the interactions and dimerization of the putative HORMA domain protein Atg101 play critical roles in starvation-induced autophagy and proteostasis, by promoting the formation of protein aggregate-containing autophagosomes. PMID:24895579

  7. [18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity.

    PubMed

    Kim, Woosuk; Le, Thuc M; Wei, Liu; Poddar, Soumya; Bazzy, Jimmy; Wang, Xuemeng; Uong, Nhu T; Abt, Evan R; Capri, Joseph R; Austin, Wayne R; Van Valkenburgh, Juno S; Steele, Dalton; Gipson, Raymond M; Slavik, Roger; Cabebe, Anthony E; Taechariyakul, Thotsophon; Yaghoubi, Shahriar S; Lee, Jason T; Sadeghi, Saman; Lavie, Arnon; Faull, Kym F; Witte, Owen N; Donahue, Timothy R; Phelps, Michael E; Herschman, Harvey R; Herrmann, Ken; Czernin, Johannes; Radu, Caius G

    2016-04-12

    Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring. PMID:27035974

  8. Apoptosis in Drosophila: which role for mitochondria?

    PubMed

    Clavier, Amandine; Rincheval-Arnold, Aurore; Colin, Jessie; Mignotte, Bernard; Guénal, Isabelle

    2016-03-01

    It is now well established that the mitochondrion is a central regulator of mammalian cell apoptosis. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, mainly because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and cell death in Drosophila occurs at the mitochondrial level. Numerous proteins, including RHG proteins and proteins of the Bcl-2 family that are key regulators of Drosophila apoptosis, constitutively or transiently localize in mitochondria. These proteins participate in the cell death process at different levels such as degradation of Diap1, a Drosophila IAP, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. Here, we review these mitochondrial events that might have their counterpart in human. PMID:26679112

  9. The digestive tract of Drosophila melanogaster.

    PubMed

    Lemaitre, Bruno; Miguel-Aliaga, Irene

    2013-01-01

    The digestive tract plays a central role in the digestion and absorption of nutrients. Far from being a passive tube, it provides the first line of defense against pathogens and maintains energy homeostasis by exchanging neuronal and endocrine signals with other organs. Historically neglected, the gut of the fruit fly Drosophila melanogaster has recently come to the forefront of Drosophila research. Areas as diverse as stem cell biology, neurobiology, metabolism, and immunity are benefitting from the ability to study the genetics of development, growth regulation, and physiology in the same organ. In this review, we summarize our knowledge of the Drosophila digestive tract, with an emphasis on the adult midgut and its functional underpinnings. PMID:24016187

  10. Viruses and Antiviral Immunity in Drosophila

    PubMed Central

    Xu, Jie; Cherry, Sara

    2013-01-01

    Viral pathogens present many challenges to organisms, driving the evolution of a myriad of antiviral strategies to combat infections. A wide variety of viruses infect invertebrates, including both natural pathogens that are insect-restricted, and viruses that are transmitted to vertebrates. Studies using the powerful tools available in the model organism Drosophila have expanded our understanding of antiviral defenses against diverse viruses. In this review, we will cover three major areas. First, we will describe the tools used to study viruses in Drosophila. Second, we will survey the major viruses that have been studied in Drosophila. And lastly, we will discuss the well-characterized mechanisms that are active against these diverse pathogens, focusing on non-RNAi mediated antiviral mechanisms. Antiviral RNAi is discussed in another paper in this issue. PMID:23680639

  11. Developmental Toxicity Assays Using the Drosophila Model

    PubMed Central

    Rand, Matthew D.; Montgomery, Sara L.; Prince, Lisa; Vorojeikina, Daria

    2014-01-01

    The fruit fly (Drosophila melanogaster) has long been a premier model for developmental biologists and geneticists. The utility of Drosophila for toxicology studies has only recently gained broader recognition as a tool to elaborate molecular genetic mechanisms of toxic substances. In this article two practical applications of Drosophila for developmental toxicity assays are described. The first assay takes advantage of newly developed methods to render the fly embryo accessible to small molecules, toxicants and drugs. The second assay engages straightforward exposures to developing larvae and easy to score outcomes of adult development. With the extensive collections of flies that are publicly available and the ease with which to create transgenic flies, these two assays have a unique power for identifying and characterizing molecular mechanisms and cellular pathways specific to the mode of action of a number of toxicants and drugs. PMID:24789363

  12. Xotch, the Xenopus homolog of Drosophila notch.

    PubMed

    Coffman, C; Harris, W; Kintner, C

    1990-09-21

    During the development of a vertebrate embryo, cell fate is determined by inductive signals passing between neighboring tissues. Such determinative interactions have been difficult to characterize fully without knowledge of the molecular mechanisms involved. Mutations of Drosophila and the nematode Caenorhabditis elegans have been isolated that define a family of related gene products involved in similar types of cellular inductions. One of these genes, the Notch gene from Drosophila, is involved with cell fate choices in the neurogenic region of the blastoderm, in the developing nervous system, and in the eye-antennal imaginal disc. Complementary DNA clones were isolated from Xenopus embryos with Notch DNA in order to investigate whether cell-cell interactions in vertebrate embryos also depend on Notch-like molecules. This approach identified a Xenopus molecule, Xotch, which is remarkably similar to Drosophila Notch in both structure and developmental expression. PMID:2402639

  13. Live cell imaging in Drosophila melanogaster.

    PubMed

    Parton, Richard M; Vallés, Ana Maria; Dobbie, Ian M; Davis, Ilan

    2010-04-01

    Although many of the techniques of live cell imaging in Drosophila melanogaster are also used by the greater community of cell biologists working on other model systems, studying living fly tissues presents unique difficulties with regard to keeping the cells alive, introducing fluorescent probes, and imaging through thick, hazy cytoplasm. This article outlines the major tissue types amenable to study by time-lapse cinematography and different methods for keeping the cells alive. It describes various imaging and associated techniques best suited to following changes in the distribution of fluorescently labeled molecules in real time in these tissues. Imaging, in general, is a rapidly developing discipline, and recent advances in imaging technology are able to greatly extend what can be achieved with live cell imaging of Drosophila tissues. As far as possible, this article includes the latest technical developments and discusses likely future developments in imaging methods that could have an impact on research using Drosophila. PMID:20360379

  14. Volatile pheromone signalling in Drosophila.

    PubMed

    Smith, Dean P

    2012-03-01

    Once captured by the antenna, 11-cis vaccenyl acetate (cVA) binds to an extracellular binding protein called LUSH that undergoes a conformational shift upon cVA binding. The stable LUSH-cVA complex is the activating ligand for pheromone receptors present on the dendrites of the aT1 neurones, comprising the only neurones that detect cVA pheromone. This mechanism explains the single molecule sensitivity of insect pheromone detection systems. The receptor that recognizes activated LUSH consists of a complex of several proteins, including Or67d, a member of the tuning odourant receptor family, Orco, a co-receptor ion channel, and SNMP, a CD36 homologue that may be an inhibitory subunit. In addition, genetic screens and reconstitution experiments reveal additional factors that are important for pheromone detection. Identification and functional dissection of these factors in Drosophila melanogaster Meigen should permit the identification of homologous factors in pathogenic insects and agricultural pests, which, in turn, may be viable candidates for novel classes of compounds to control populations of target insect species without impacting beneficial species. PMID:24347807

  15. Trithorax regulates systemic signaling during Drosophila imaginal disc regeneration.

    PubMed

    Skinner, Andrea; Khan, Sumbul Jawed; Smith-Bolton, Rachel K

    2015-10-15

    Although tissue regeneration has been studied in a variety of organisms, from Hydra to humans, many of the genes that regulate the ability of each animal to regenerate remain unknown. The larval imaginal discs of the genetically tractable model organism Drosophila melanogaster have complex patterning, well-characterized development and a high regenerative capacity, and are thus an excellent model system for studying mechanisms that regulate regeneration. To identify genes that are important for wound healing and tissue repair, we have carried out a genetic screen for mutations that impair regeneration in the wing imaginal disc. Through this screen we identified the chromatin-modification gene trithorax as a key regeneration gene. Here we show that animals heterozygous for trithorax are unable to maintain activation of a developmental checkpoint that allows regeneration to occur. This defect is likely to be caused by abnormally high expression of puckered, a negative regulator of Jun N-terminal kinase (JNK) signaling, at the wound site. Insufficient JNK signaling leads to insufficient expression of an insulin-like peptide, dILP8, which is required for the developmental checkpoint. Thus, trithorax regulates regeneration signaling and capacity. PMID:26487779

  16. Lifespan Extension by Preserving Proliferative Homeostasis in Drosophila

    PubMed Central

    Supoyo, Stephen; DeGennaro, Matthew; Lehmann, Ruth; Jasper, Heinrich

    2010-01-01

    Regenerative processes are critical to maintain tissue homeostasis in high-turnover tissues. At the same time, proliferation of stem and progenitor cells has to be carefully controlled to prevent hyper-proliferative diseases. Mechanisms that ensure this balance, thus promoting proliferative homeostasis, are expected to be critical for longevity in metazoans. The intestinal epithelium of Drosophila provides an accessible model in which to test this prediction. In aging flies, the intestinal epithelium degenerates due to over-proliferation of intestinal stem cells (ISCs) and mis-differentiation of ISC daughter cells, resulting in intestinal dysplasia. Here we show that conditions that impair tissue renewal lead to lifespan shortening, whereas genetic manipulations that improve proliferative homeostasis extend lifespan. These include reduced Insulin/IGF or Jun-N-terminal Kinase (JNK) signaling activities, as well as over-expression of stress-protective genes in somatic stem cell lineages. Interestingly, proliferative activity in aging intestinal epithelia correlates with longevity over a range of genotypes, with maximal lifespan when intestinal proliferation is reduced but not completely inhibited. Our results highlight the importance of the balance between regenerative processes and strategies to prevent hyperproliferative disorders and demonstrate that promoting proliferative homeostasis in aging metazoans is a viable strategy to extend lifespan. PMID:20976250

  17. GLAD: an Online Database of Gene List Annotation for Drosophila.

    PubMed

    Hu, Yanhui; Comjean, Aram; Perkins, Lizabeth A; Perrimon, Norbert; Mohr, Stephanie E

    2015-01-01

    We present a resource of high quality lists of functionally related Drosophila genes, e.g. based on protein domains (kinases, transcription factors, etc.) or cellular function (e.g. autophagy, signal transduction). To establish these lists, we relied on different inputs, including curation from databases or the literature and mapping from other species. Moreover, as an added curation and quality control step, we asked experts in relevant fields to review many of the lists. The resource is available online for scientists to search and view, and is editable based on community input. Annotation of gene groups is an ongoing effort and scientific need will typically drive decisions regarding which gene lists to pursue. We anticipate that the number of lists will increase over time; that the composition of some lists will grow and/or change over time as new information becomes available; and that the lists will benefit the scientific community, e.g. at experimental design and data analysis stages. Based on this, we present an easily updatable online database, available at www.flyrnai.org/glad, at which gene group lists can be viewed, searched and downloaded. PMID:26157507

  18. Genetic analysis of the Drosophila Gs(alpha) gene.

    PubMed Central

    Wolfgang, W J; Hoskote, A; Roberts, I J; Jackson, S; Forte, M

    2001-01-01

    One of the best understood signal transduction pathways activated by receptors containing seven transmembrane domains involves activation of heterotrimeric G-protein complexes containing Gs(alpha), the subsequent stimulation of adenylyl cyclase, production of cAMP, activation of protein kinase A (PKA), and the phosphorylation of substrates that control a wide variety of cellular responses. Here, we report the identification of "loss-of-function" mutations in the Drosophila Gs(alpha) gene (dgs). Seven mutants have been identified that are either complemented by transgenes representing the wild-type dgs gene or contain nucleotide sequence changes resulting in the production of altered Gs(alpha) protein. Examination of mutant alleles representing loss-of-Gs(alpha) function indicates that the phenotypes generated do not mimic those created by mutational elimination of PKA. These results are consistent with the conclusion reached in previous studies that activation of PKA, at least in these developmental contexts, does not depend on receptor-mediated increases in intracellular cAMP, in contrast to the predictions of models developed primarily on the basis of studies in cultured cells. PMID:11454767

  19. Drosophila embryos lacking N-myristoyltransferase have multiple developmental defects.

    PubMed

    Ntwasa, M; Aapies, S; Schiffmann, D A; Gay, N J

    2001-01-15

    Lipid modification of proteins by the addition of myristic acid to the N-terminal is important in a number of critical cellular processes, for example, signal transduction and the modulation of membrane association by myristoyl switches. Myristic acid is added to proteins by the enzyme N-myristoyltransferase (NMT) and in this paper we detail the effects on embryonic development of a null mutation in the Drosophila NMT gene. Mutant embryos display a range of phenotypes, including failures of head involution, dorsal closure, and germ-band retraction, morphogenetic processes that require cellular movements. Embryos with milder phenotypes have more specific defects in the central nervous system, including thinning of the ventral nerve chord and, in some embryos, specific scission at parasegment 10. Staining of mutant embryos with phalloidin shows that the mutant embryos have a disrupted actin cytoskeleton and abnormal cell morphology. These phenotypes are strikingly similar to those caused by genes involved in dynamic rearrangement of the actin cytoskeleton. For example the myristoylated nonreceptor tyrosine kinases Dsrc42A and Dsrc64B were shown recently to be key regulators of dorsal closure. In addition, analysis of cell death reveals widespread ectopic apoptosis. Our findings are consistent with the hypothesis that the myristoyl switches and signaling pathways characterized at the biochemical level have important functions in fundamental morphogenetic processes. PMID:11139338

  20. Fasciclin II controls proneural gene expression in Drosophila.

    PubMed Central

    García-Alonso, L; VanBerkum, M F; Grenningloh, G; Schuster, C; Goodman, C S

    1995-01-01

    Fasciclin II (Fas II), an NCAM-like cell adhesion molecule in Drosophila, is expressed on a subset of embryonic axons and controls selective axon fasiculation. Fas II is also expressed in imaginal discs. Here we use genetic analysis to show that Fas II is required for the control of proneural gene expression. Clusters of cells in the eye-antennal imaginal disc express the achaete proneural gene and give rise to mechanosensory neurons; other clusters of cells express the atonal gene and give rise to ocellar photoreceptor neurons. In fasII loss-of-function mutants, the expression of both proneural genes is absent in certain locations, and, as a result, the corresponding sensory precursors fail to develop. In fasII gain-of-function conditions, extra sensory structures arise from this same region of the imaginal disc. Mutations in the Abelson tyrosine kinase gene show dominant interactions with fasII mutations, suggesting that Abl and Fas II function in a signaling pathway that controls proneural gene expression. Images Fig. 1 Fig. 2 PMID:7479828

  1. Evaluation of Traditional Medicines for Neurodegenerative Diseases Using Drosophila Models

    PubMed Central

    Lee, Soojin; Bang, Se Min; Lee, Joon Woo; Cho, Kyoung Sang

    2014-01-01

    Drosophila is one of the oldest and most powerful genetic models and has led to novel insights into a variety of biological processes. Recently, Drosophila has emerged as a model system to study human diseases, including several important neurodegenerative diseases. Because of the genomic similarity between Drosophila and humans, Drosophila neurodegenerative disease models exhibit a variety of human-disease-like phenotypes, facilitating fast and cost-effective in vivo genetic modifier screening and drug evaluation. Using these models, many disease-associated genetic factors have been identified, leading to the identification of compelling drug candidates. Recently, the safety and efficacy of traditional medicines for human diseases have been evaluated in various animal disease models. Despite the advantages of the Drosophila model, its usage in the evaluation of traditional medicines is only nascent. Here, we introduce the Drosophila model for neurodegenerative diseases and some examples demonstrating the successful application of Drosophila models in the evaluation of traditional medicines. PMID:24790636

  2. Rosa damascena decreased mortality in adult Drosophila.

    PubMed

    Jafari, Mahtab; Zarban, Asghar; Pham, Steven; Wang, Thomas

    2008-03-01

    The effects of a rose-flower extract, Rosa damascena, on the mortality rate of Drosophila melanogaster was evaluated in this study. R. damascena is a potent antioxidant that has many therapeutic uses in addition to its perfuming effects. Supplementing Drosophila with this rose extract resulted in a statistically significant decrease in mortality rate in male and female flies. Moreover, the observed anti-aging effects were not associated with common confounds of anti-aging properties, such as a decrease in fecundity or metabolic rate. PMID:18361732

  3. Neural control of aggression in Drosophila.

    PubMed

    Hoopfer, Eric D

    2016-06-01

    Like most animal species, fruit flies fight to obtain and defend resources essential to survival and reproduction. Aggressive behavior in Drosophila is genetically specified and also strongly influenced by the fly's social context, past experiences and internal states, making it an excellent framework for investigating the neural mechanisms that regulate complex social behaviors. Here, I summarize our current knowledge of the neural control of aggression in Drosophila and discuss recent advances in understanding the sensory pathways that influence the decision to fight or court, the neuromodulatory control of aggression, the neural basis by which internal states can influence both fighting and courtship, and how social experience modifies aggressive behavior. PMID:27179788

  4. The secret lives of Drosophila flies.

    PubMed

    Markow, Therese Ann

    2015-01-01

    Flies of the genus Drosophila, and particularly those of the species Drosophila melanogaster, are best known as laboratory organisms. As with all model organisms, they were domesticated for empirical studies, but they also continue to exist as wild populations. Decades of research on these flies in the laboratory have produced astounding and important insights into basic biological processes, but we have only scratched the surface of what they have to offer as research organisms. An outstanding challenge now is to build on this knowledge and explore how natural history has shaped D. melanogaster in order to advance our understanding of biology more generally. PMID:26041333

  5. The PNT domain from Drosophila pointed-P2 contains a dynamic N-terminal helix preceded by a disordered phosphoacceptor sequence.

    PubMed

    Lau, Desmond K W; Okon, Mark; McIntosh, Lawrence P

    2012-11-01

    Pointed-P2, the Drosophila ortholog of human ETS1 and ETS2, is a transcription factor involved in Ras/MAP kinase-regulated gene expression. In addition to a DNA-binding ETS domain, Pointed-P2 contains a PNT (or SAM) domain that serves as a docking module to enhance phosphorylation of an adjacent phosphoacceptor threonine by the ERK2 MAP kinase Rolled. Using NMR chemical shift, ¹⁵N relaxation, and amide hydrogen exchange measurements, we demonstrate that the Pointed-P2 PNT domain contains a dynamic N-terminal helix H0 appended to a core conserved five-helix bundle diagnostic of the SAM domain fold. Neither the secondary structure nor dynamics of the PNT domain is perturbed significantly upon in vitro ERK2 phosphorylation of three threonine residues in a disordered sequence immediately preceding this domain. These data thus confirm that the Drosophila Pointed-P2 PNT domain and phosphoacceptors are highly similar to those of the well-characterized human ETS1 transcription factor. NMR-monitored titrations also revealed that the phosphoacceptors and helix H0, as well as region of the core helical bundle identified previously by mutational analyses as a kinase docking site, are selectively perturbed upon ERK2 binding by Pointed-P2. Based on a homology model derived from the ETS1 PNT domain, helix H0 is predicted to partially occlude the docking interface. Therefore, this dynamic helix must be displaced to allow both docking of the kinase, as well as binding of Mae, a Drosophila protein that negatively regulates Pointed-P2 by competing with the kinase for its docking site. PMID:22936607

  6. Targeting cancer with kinase inhibitors

    PubMed Central

    Gross, Stefan; Rahal, Rami; Stransky, Nicolas; Lengauer, Christoph; Hoeflich, Klaus P.

    2015-01-01

    Kinase inhibitors have played an increasingly prominent role in the treatment of cancer and other diseases. Currently, more than 25 oncology drugs that target kinases have been approved, and numerous additional therapeutics are in various stages of clinical evaluation. In this Review, we provide an in-depth analysis of activation mechanisms for kinases in cancer, highlight recent successes in drug discovery, and demonstrate the clinical impact of selective kinase inhibitors. We also describe the substantial progress that has been made in designing next-generation inhibitors to circumvent on-target resistance mechanisms, as well as ongoing strategies for combining kinase inhibitors in the clinic. Last, there are numerous prospects for the discovery of novel kinase targets, and we explore cancer immunotherapy as a new and promising research area for studying kinase biology. PMID:25932675

  7. Phosphorylation of the RNA-binding protein Dazl by MAPKAP kinase 2 regulates spermatogenesis.

    PubMed

    Williams, Patrick A; Krug, Michael S; McMillan, Emily A; Peake, Jasmine D; Davis, Tara L; Cocklin, Simon; Strochlic, Todd I

    2016-08-01

    Developing male germ cells are exquisitely sensitive to environmental insults such as heat and oxidative stress. An additional characteristic of these cells is their unique dependence on RNA-binding proteins for regulating posttranscriptional gene expression and translational control. Here we provide a mechanistic link unifying these two features. We show that the germ cell-specific RNA-binding protein deleted in azoospermia-like (Dazl) is phosphorylated by MAPKAP kinase 2 (MK2), a stress-induced protein kinase activated downstream of p38 MAPK. We demonstrate that phosphorylation of Dazl by MK2 on an evolutionarily conserved serine residue inhibits its interaction with poly(A)-binding protein, resulting in reduced translation of Dazl-regulated target RNAs. We further show that transgenic expression of wild-type human Dazl but not a phosphomimetic form in the Drosophila male germline can restore fertility to flies deficient in boule, the Drosophila orthologue of human Dazl. These results illuminate a novel role for MK2 in spermatogenesis, expand the repertoire of RNA-binding proteins phosphorylated by this kinase, and suggest that signaling by the p38-MK2 pathway is a negative regulator of spermatogenesis via phosphorylation of Dazl. PMID:27280388

  8. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2005-03-08

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  9. Oncoprotein protein kinase

    DOEpatents

    Davis, Roger; Derijard, Benoit; Karin, Michael; Hibi, Masahiko; Lin, Anning

    2005-01-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  10. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1999-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  11. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  12. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1998-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  13. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2003-02-04

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  14. Oncoprotein protein kinase

    DOEpatents

    Karin, M.; Hibi, M.; Lin, A.

    1997-02-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE is disclosed. The polypeptide has serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences. The method of detection of JNK is also provided. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites. 44 figs.

  15. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  16. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Lin, Anning

    1999-11-30

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  17. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2004-03-16

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  18. Cyclin-dependent kinases

    PubMed Central

    2014-01-01

    Summary Cyclin-dependent kinases (CDKs) are protein kinases characterized by needing a separate subunit - a cyclin - that provides domains essential for enzymatic activity. CDKs play important roles in the control of cell division and modulate transcription in response to several extra- and intracellular cues. The evolutionary expansion of the CDK family in mammals led to the division of CDKs into three cell-cycle-related subfamilies (Cdk1, Cdk4 and Cdk5) and five transcriptional subfamilies (Cdk7, Cdk8, Cdk9, Cdk11 and Cdk20). Unlike the prototypical Cdc28 kinase of budding yeast, most of these CDKs bind one or a few cyclins, consistent with functional specialization during evolution. This review summarizes how, although CDKs are traditionally separated into cell-cycle or transcriptional CDKs, these activities are frequently combined in many family members. Not surprisingly, deregulation of this family of proteins is a hallmark of several diseases, including cancer, and drug-targeted inhibition of specific members has generated very encouraging results in clinical trials. PMID:25180339

  19. Drosophila Bitter Taste(s)

    PubMed Central

    French, Alice; Ali Agha, Moutaz; Mitra, Aniruddha; Yanagawa, Aya; Sellier, Marie-Jeanne; Marion-Poll, Frédéric

    2015-01-01

    Most animals possess taste receptors neurons detecting potentially noxious compounds. In humans, the ligands which activate these neurons define a sensory space called “bitter”. By extension, this term has been used in animals and insects to define molecules which induce aversive responses. In this review, based on our observations carried out in Drosophila, we examine how bitter compounds are detected and if bitter-sensitive neurons respond only to molecules bitter to humans. Like most animals, flies detect bitter chemicals through a specific population of taste neurons, distinct from those responding to sugars or to other modalities. Activating bitter-sensitive taste neurons induces aversive reactions and inhibits feeding. Bitter molecules also contribute to the suppression of sugar-neuron responses and can lead to a complete inhibition of the responses to sugar at the periphery. Since some bitter molecules activate bitter-sensitive neurons and some inhibit sugar detection, bitter molecules are represented by two sensory spaces which are only partially congruent. In addition to molecules which impact feeding, we recently discovered that the activation of bitter-sensitive neurons also induces grooming. Bitter-sensitive neurons of the wings and of the legs can sense chemicals from the gram negative bacteria, Escherichia coli, thus adding another biological function to these receptors. Bitter-sensitive neurons of the proboscis also respond to the inhibitory pheromone, 7-tricosene. Activating these neurons by bitter molecules in the context of sexual encounter inhibits courting and sexual reproduction, while activating these neurons with 7-tricosene in a feeding context will inhibit feeding. The picture that emerges from these observations is that the taste system is composed of detectors which monitor different “categories” of ligands, which facilitate or inhibit behaviors depending on the context (feeding, sexual reproduction, hygienic behavior), thus

  20. Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

    PubMed

    Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

    2016-07-29

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. PMID:27226575

  1. A High-Throughput Radiometric Kinase Assay.

    PubMed

    Duong-Ly, Krisna C; Peterson, Jeffrey R

    2016-01-01

    Aberrant kinase signaling has been implicated in a number of diseases. While kinases have become attractive drug targets, only a small fraction of human protein kinases have validated inhibitors. Screening of libraries of compounds against a kinase or kinases of interest is routinely performed during kinase inhibitor development to identify promising scaffolds for a particular target and to identify kinase targets for compounds of interest. Screening of more focused compound libraries may also be conducted in the later stages of inhibitor development to improve potency and optimize selectivity. The dot blot kinase assay is a robust, high-throughput kinase assay that can be used to screen a number of small-molecule compounds against one kinase of interest or several kinases. Here, a protocol for a dot blot kinase assay used for measuring insulin receptor kinase activity is presented. This protocol can be readily adapted for use with other protein kinases. PMID:26501904

  2. Drosophila Melanogaster as an Experimental Organism.

    ERIC Educational Resources Information Center

    Rubin, Gerald M.

    1988-01-01

    Discusses the role of the fruit fly in genetics research requiring a multidisciplinary approach. Describes embryological and genetic methods used in the experimental analysis of this organism. Outlines the use of Drosophila in the study of the development and function of the nervous system. (RT)

  3. Functional Neuroanatomy of "Drosophila" Olfactory Memory Formation

    ERIC Educational Resources Information Center

    Guven-Ozkan, Tugba; Davis, Ronald L.

    2014-01-01

    New approaches, techniques and tools invented over the last decade and a half have revolutionized the functional dissection of neural circuitry underlying "Drosophila" learning. The new methodologies have been used aggressively by researchers attempting to answer three critical questions about olfactory memories formed with appetitive…

  4. Second-Order Conditioning in "Drosophila"

    ERIC Educational Resources Information Center

    Tabone, Christopher J.; de Belle, J. Steven

    2011-01-01

    Associative conditioning in "Drosophila melanogaster" has been well documented for several decades. However, most studies report only simple associations of conditioned stimuli (CS, e.g., odor) with unconditioned stimuli (US, e.g., electric shock) to measure learning or establish memory. Here we describe a straightforward second-order conditioning…

  5. Organization of descending neurons in Drosophila melanogaster.

    PubMed

    Hsu, Cynthia T; Bhandawat, Vikas

    2016-01-01

    Neural processing in the brain controls behavior through descending neurons (DNs) - neurons which carry signals from the brain to the spinal cord (or thoracic ganglia in insects). Because DNs arise from multiple circuits in the brain, the numerical simplicity and availability of genetic tools make Drosophila a tractable model for understanding descending motor control. As a first step towards a comprehensive study of descending motor control, here we estimate the number and distribution of DNs in the Drosophila brain. We labeled DNs by backfilling them with dextran dye applied to the neck connective and estimated that there are ~1100 DNs distributed in 6 clusters in Drosophila. To assess the distribution of DNs by neurotransmitters, we labeled DNs in flies in which neurons expressing the major neurotransmitters were also labeled. We found DNs belonging to every neurotransmitter class we tested: acetylcholine, GABA, glutamate, serotonin, dopamine and octopamine. Both the major excitatory neurotransmitter (acetylcholine) and the major inhibitory neurotransmitter (GABA) are employed equally; this stands in contrast to vertebrate DNs which are predominantly excitatory. By comparing the distribution of DNs in Drosophila to those reported previously in other insects, we conclude that the organization of DNs in insects is highly conserved. PMID:26837716

  6. Organization of descending neurons in Drosophila melanogaster

    PubMed Central

    Hsu, Cynthia T.; Bhandawat, Vikas

    2016-01-01

    Neural processing in the brain controls behavior through descending neurons (DNs) - neurons which carry signals from the brain to the spinal cord (or thoracic ganglia in insects). Because DNs arise from multiple circuits in the brain, the numerical simplicity and availability of genetic tools make Drosophila a tractable model for understanding descending motor control. As a first step towards a comprehensive study of descending motor control, here we estimate the number and distribution of DNs in the Drosophila brain. We labeled DNs by backfilling them with dextran dye applied to the neck connective and estimated that there are ~1100 DNs distributed in 6 clusters in Drosophila. To assess the distribution of DNs by neurotransmitters, we labeled DNs in flies in which neurons expressing the major neurotransmitters were also labeled. We found DNs belonging to every neurotransmitter class we tested: acetylcholine, GABA, glutamate, serotonin, dopamine and octopamine. Both the major excitatory neurotransmitter (acetylcholine) and the major inhibitory neurotransmitter (GABA) are employed equally; this stands in contrast to vertebrate DNs which are predominantly excitatory. By comparing the distribution of DNs in Drosophila to those reported previously in other insects, we conclude that the organization of DNs in insects is highly conserved. PMID:26837716

  7. A Drosophila Model of Epidermolysis Bullosa Simplex.

    PubMed

    Bohnekamp, Jens; Cryderman, Diane E; Paululat, Achim; Baccam, Gabriel C; Wallrath, Lori L; Magin, Thomas M

    2015-08-01

    The blistering skin disorder epidermolysis bullosa simplex (EBS) results from dominant mutations in keratin 5 (K5) or keratin 14 (K14) genes, encoding the intermediate filament (IF) network of basal epidermal keratinocytes. The mechanisms governing keratin network formation and collapse due to EBS mutations remain incompletely understood. Drosophila lacks cytoplasmic IFs, providing a 'null' environment to examine the formation of keratin networks and determine mechanisms by which mutant keratins cause pathology. Here, we report that ubiquitous co-expression of transgenes encoding wild-type human K14 and K5 resulted in the formation of extensive keratin networks in Drosophila epithelial and non-epithelial tissues, causing no overt phenotype. Similar to mammalian cells, treatment of transgenic fly tissues with phosphatase inhibitors caused keratin network collapse, validating Drosophila as a genetic model system to investigate keratin dynamics. Co-expression of K5 and a K14(R125C) mutant that causes the most severe form of EBS resulted in widespread formation of EBS-like cytoplasmic keratin aggregates in epithelial and non-epithelial fly tissues. Expression of K14(R125C)/K5 caused semi-lethality; adult survivors developed wing blisters and were flightless due to a lack of intercellular adhesion during wing heart development. This Drosophila model of EBS is valuable for the identification of pathways altered by mutant keratins and for the development of EBS therapies. PMID:25830653

  8. The 5S genes of Drosophila melanogaster.

    PubMed

    Artavanis-Tsakonas, S; Schedl, P; Tschudi, C; Pirrotta, V; Steward, R; Gehring, W J

    1977-12-01

    We have cloned embryonic Drosophila DNA using the poly (dA-DT) connector method (Lobban and Kaiser, 1973) and the ampicillin-resistant plasmid pSF2124 (So, Gill and Falkow, 1975) as a cloning vehicle. Two clones, containing hybrid plasmids with sequences complementary to a 5S RNA probe isolated from Drosophila tissue culture cells, were identified by the Grunstein and Hogness (1975) colony hybridization procedure. One hybrid plasmid has a Drosophila insert which is comprised solely of tandem repeats of the 5S gene plus spacer sequences. The other plasmid contains an insert which has about 20 tandem 5S repeat units plus an additional 4 kilobases of adjacent sequences. The size of the 5S repeat unit was determined by gel electrophoresis and was found to be approximately 375 base pairs. We present a restriction map of both plasmids, and a detailed map of of the5S repeat unit. The 5S repat unit shows slight length and sequence heterogeneity. We present evidence suggesting that the 5S genes in Drosophila melanogaster may be arranged in a single continuous cluster. PMID:413625

  9. [Recombination in Drosophila in space flight].

    PubMed

    Filatova, L P; Vaulina, E N; Lapteva, N Sh; Grozdova, T Ia

    1988-04-01

    An experiment with Drosophila melanogaster males was performed aboard the Artificial Satellite "Kosmos-1667". Mutagenic effects of a 7-day space flight on intergene recombination in chromosome 2 were studied. The space flight factors decreased the frequency of recombination. A model experiment on a laboratory centrifuge demonstrated insignificant increase in recombination frequency caused by acceleration. PMID:3135244

  10. Open-Ended Laboratory Investigations with Drosophila.

    ERIC Educational Resources Information Center

    Mertens, Thomas R.

    1983-01-01

    Background information, laboratory procedures (including matings performed), and results are presented for an open-ended investigation using the fruitfly Drosophila melanogaster. Once data are collected, students develop hypotheses to explain results as well as devise additional experiments to test their hypotheses. Calculation of chi-square for…

  11. Measurement of Cytoplasmic Streaming in Drosophila Melanogaster

    NASA Astrophysics Data System (ADS)

    Ganguly, Sujoy; Williams, Lucy; Palacios, Isabel; Goldstein, Raymond

    2010-11-01

    During stage 9 of Drosophila melanogastor oogenesis flow of the oocyte cytoplasm, driven by kinesin 1 motor protein is observed. This cytoplasmic streaming is analyzed by PIV in both wild type and kinesin light chain mutants, revealing striking statistical differences. Further measurements of the rheology of the oocyte allow for estimations of the mechanical energy needed to generate the observed flows.

  12. Analysis of Phagocytosis in the Drosophila Ovary.

    PubMed

    Meehan, Tracy L; Serizier, Sandy B; Kleinsorge, Sarah E; McCall, Kimberly

    2016-01-01

    Programmed cell death (PCD) is essential for health and development. Generally, the last step of PCD is clearance, or engulfment, by phagocytes. Engulfment can be broken down into five basic steps: attraction of the phagocyte, recognition of the dying cell, internalization, phagosome maturation, and acidification of the engulfed material. The Drosophila melanogaster ovary serves as an excellent model to study diverse types of PCD and engulfment by epithelial cells. Here, we describe several methods to detect and analyze multiple steps of engulfment in the Drosophila ovary: recognition, vesicle uptake, phagosome maturation, and acidification. Annexin V detects phosphatidylserine, which is flipped to the outer leaflet of the plasma membrane of apoptotic cells, serving as an "eat me" signal. Several germline markers including tral-GFP, Orb, and cleaved Dcp-1 can all be used to label the germline and visualize its uptake into engulfing follicle cells. Drosophila strains expressing GFP and mCherry protein fusions can enable a detailed analysis of phagosome maturation. LysoTracker labels highly acidified compartments, marking phagolysosomes. Together these labels can be used to mark the progression of engulfment in Drosophila follicle cells. PMID:27557574

  13. Characterization of novel microsatellites from Drosophila transversa.

    PubMed

    Räisänen, L; Roininen, E; Liimatainen, J O

    2009-03-01

    We investigated a partial genomic library of Drosophila transversa for microsatellites and developed 12 markers for genetic analyses. This is the first time that microsatellite primers from the quinaria species group have been described. Four loci were cross-amplified in D. phalerata. Nine out of the 12 microsatellite markers developed are likely to be on the X chromosome. PMID:21564716

  14. Redox Regulation of Protein Kinases

    PubMed Central

    Truong, Thu H.; Carroll, Kate S.

    2015-01-01

    Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous H2O2 by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiologic and pathological H2O2 responses. PMID:23639002

  15. Myt1 inhibition of Cyclin A/Cdk1 is essential for fusome integrity and premeiotic centriole engagement in Drosophila spermatocytes.

    PubMed

    Varadarajan, Ramya; Ayeni, Joseph; Jin, Zhigang; Homola, Ellen; Campbell, Shelagh D

    2016-07-01

    Regulation of cell cycle arrest in premeiotic G2 phase coordinates germ cell maturation and meiotic cell division with hormonal and developmental signals by mechanisms that control Cyclin B synthesis and inhibitory phosphorylation of the M-phase kinase, Cdk1. In this study, we investigated how inhibitory phosphorylation of Cdk1 by Myt1 kinase regulates premeiotic G2 phase of Drosophila male meiosis. Immature spermatocytes lacking Myt1 activity exhibit two distinct defects: disrupted intercellular bridges (fusomes) and premature centriole disengagement. As a result, the myt1 mutant spermatocytes enter meiosis with multipolar spindles. These myt1 defects can be suppressed by depletion of Cyclin A activity or ectopic expression of Wee1 (a partially redundant Cdk1 inhibitory kinase) and phenocopied by expression of a Cdk1F mutant defective for inhibitory phosphorylation. We therefore conclude that Myt1 inhibition of Cyclin A/Cdk1 is essential for normal fusome behavior and centriole engagement during premeiotic G2 arrest of Drosophila male meiosis. The novel meiotic functions we discovered for Myt1 kinase are spatially and temporally distinct from previously described functions of Myt1 as an inhibitor of Cyclin B/Cdk1 to regulate G2/MI timing. PMID:27170181

  16. Genomics of Ecological Adaptation in Cactophilic Drosophila

    PubMed Central

    Guillén, Yolanda; Rius, Núria; Delprat, Alejandra; Williford, Anna; Muyas, Francesc; Puig, Marta; Casillas, Sònia; Ràmia, Miquel; Egea, Raquel; Negre, Barbara; Mir, Gisela; Camps, Jordi; Moncunill, Valentí; Ruiz-Ruano, Francisco J.; Cabrero, Josefa; de Lima, Leonardo G.; Dias, Guilherme B.; Ruiz, Jeronimo C.; Kapusta, Aurélie; Garcia-Mas, Jordi; Gut, Marta; Gut, Ivo G.; Torrents, David; Camacho, Juan P.; Kuhn, Gustavo C.S.; Feschotte, Cédric; Clark, Andrew G.; Betrán, Esther; Barbadilla, Antonio; Ruiz, Alfredo

    2015-01-01

    Cactophilic Drosophila species provide a valuable model to study gene–environment interactions and ecological adaptation. Drosophila buzzatii and Drosophila mojavensis are two cactophilic species that belong to the repleta group, but have very different geographical distributions and primary host plants. To investigate the genomic basis of ecological adaptation, we sequenced the genome and developmental transcriptome of D. buzzatii and compared its gene content with that of D. mojavensis and two other noncactophilic Drosophila species in the same subgenus. The newly sequenced D. buzzatii genome (161.5 Mb) comprises 826 scaffolds (>3 kb) and contains 13,657 annotated protein-coding genes. Using RNA sequencing data of five life-stages we found expression of 15,026 genes, 80% protein-coding genes, and 20% noncoding RNA genes. In total, we detected 1,294 genes putatively under positive selection. Interestingly, among genes under positive selection in the D. mojavensis lineage, there is an excess of genes involved in metabolism of heterocyclic compounds that are abundant in Stenocereus cacti and toxic to nonresident Drosophila species. We found 117 orphan genes in the shared D. buzzatii–D. mojavensis lineage. In addition, gene duplication analysis identified lineage-specific expanded families with functional annotations associated with proteolysis, zinc ion binding, chitin binding, sensory perception, ethanol tolerance, immunity, physiology, and reproduction. In summary, we identified genetic signatures of adaptation in the shared D. buzzatii–D. mojavensis lineage, and in the two separate D. buzzatii and D. mojavensis lineages. Many of the novel lineage-specific genomic features are promising candidates for explaining the adaptation of these species to their distinct ecological niches. PMID:25552534

  17. Optogenetic pacing in Drosophila melanogaster (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Alex, Aneesh; Li, Airong; Men, Jing; Jerwick, Jason; Tanzi, Rudolph E.; Zhou, Chao

    2016-03-01

    A non-invasive, contact-less cardiac pacing technology can be a powerful tool in basic cardiac research and in clinics. Currently, electrical pacing is the gold standard for cardiac pacing. Although highly effective in controlling the cardiac function, the invasive nature, non-specificity to cardiac tissues and possible tissue damage limits its capabilities. Optical pacing of heart is a promising alternative, which is non-invasive and more specific, has high spatial and temporal precision, and avoids shortcomings in electrical stimulation. Optical coherence tomography has been proved to be an effective technique in non-invasive imaging in vivo with ultrahigh resolution and imaging speed. In the last several years, non-invasive specific optical pacing in animal hearts has been reported in quail, zebrafish, and rabbit models. However, Drosophila Melanogaster, which is a significant model with orthologs of 75% of human disease genes, has rarely been studied concerning their optical pacing in heart. Here, we combined optogenetic control of Drosophila heartbeat with optical coherence microscopy (OCM) technique for the first time. The light-gated cation channel, channelrhodopsin-2 (ChR2) was specifically expressed by transgene as a pacemaker in drosophila heart. By stimulating the pacemaker with 472 nm pulsed laser light at different frequencies, we achieved non-invasive and more specific optical control of the Drosophila heart rhythm, which demonstrates the wide potential of optical pacing for studying cardiac dynamics and development. Imaging capability of our customized OCM system was also involved to observe the pacing effect visually. No tissue damage was found after long exposure to laser pulses, which proved the safety of optogenetic control of Drosophila heart.

  18. Rab8, POSH, and TAK1 regulate synaptic growth in a Drosophila model of frontotemporal dementia

    PubMed Central

    West, Ryan J.H.; Lu, Yubing; Marie, Bruno; Gao, Fen-Biao

    2015-01-01

    Mutations in genes essential for protein homeostasis have been identified in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) patients. Why mature neurons should be particularly sensitive to such perturbations is unclear. We identified mutations in Rab8 in a genetic screen for enhancement of an FTD phenotype associated with ESCRT-III dysfunction. Examination of Rab8 mutants or motor neurons expressing a mutant ESCRT-III subunit, CHMP2BIntron5, at the Drosophila melanogaster neuromuscular junction synapse revealed synaptic overgrowth and endosomal dysfunction. Expression of Rab8 rescued overgrowth phenotypes generated by CHMP2BIntron5. In Rab8 mutant synapses, c-Jun N-terminal kinase (JNK)/activator protein-1 and TGF-β signaling were overactivated and acted synergistically to potentiate synaptic growth. We identify novel roles for endosomal JNK-scaffold POSH (Plenty-of-SH3s) and a JNK kinase kinase, TAK1, in regulating growth activation in Rab8 mutants. Our data uncover Rab8, POSH, and TAK1 as regulators of synaptic growth responses and point to recycling endosome as a key compartment for synaptic growth regulation during neurodegenerative processes. PMID:25800055

  19. PDK1 regulates growth through Akt and S6K in Drosophila

    PubMed Central

    Rintelen, Felix; Stocker, Hugo; Thomas, George; Hafen, Ernst

    2001-01-01

    The insulin/insulin-like growth factor-1 signaling pathway promotes growth in invertebrates and vertebrates by increasing the levels of phosphatidylinositol 3,4,5-triphosphate through the activation of p110 phosphatidylinositol 3-kinase. Two key effectors of this pathway are the phosphoinositide-dependent protein kinase 1 (PDK1) and Akt/PKB. Although genetic analysis in Caenorhabditis elegans has implicated Akt as the only relevant PDK1 substrate, cell culture studies have suggested that PDK1 has additional targets. Here we show that, in Drosophila, dPDK1 controls cellular and organism growth by activating dAkt and S6 kinase, dS6K. Furthermore, dPDK1 genetically interacts with dRSK but not with dPKN, encoding two substrates of PDK1 in vitro. Thus, the results suggest that dPDK1 is required for dRSK but not dPKN activation and that it regulates insulin-mediated growth through two main effector branches, dAkt and dS6K. PMID:11752451

  20. Drosophila ATM and ATR have distinct activities in the regulation of meiotic DNA damage and repair.

    PubMed

    Joyce, Eric F; Pedersen, Michael; Tiong, Stanley; White-Brown, Sanese K; Paul, Anshu; Campbell, Shelagh D; McKim, Kim S

    2011-10-31

    Ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-related (ATR) kinases are conserved regulators of cellular responses to double strand breaks (DSBs). During meiosis, however, the functions of these kinases in DSB repair and the deoxyribonucleic acid (DNA) damage checkpoint are unclear. In this paper, we show that ATM and ATR have unique roles in the repair of meiotic DSBs in Drosophila melanogaster. ATR mutant analysis indicated that it is required for checkpoint activity, whereas ATM may not be. Both kinases phosphorylate H2AV (γ-H2AV), and, using this as a reporter for ATM/ATR activity, we found that the DSB repair response is surprisingly dynamic at the site of DNA damage. γ-H2AV is continuously exchanged, requiring new phosphorylation at the break site until repair is completed. However, most surprising is that the number of γ-H2AV foci is dramatically increased in the absence of ATM, but not ATR, suggesting that the number of DSBs is increased. Thus, we conclude that ATM is primarily required for the meiotic DSB repair response, which includes functions in DNA damage repair and negative feedback control over the level of programmed DSBs during meiosis. PMID:22024169

  1. Genetic and evolutionary analysis of the Drosophila larval neuromuscular junction

    NASA Astrophysics Data System (ADS)

    Campbell, Megan

    Although evolution of brains and behaviors is of fundamental biological importance, we lack comprehensive understanding of the general principles governing these processes or the specific mechanisms and molecules through which the evolutionary changes are effected. Because synapses are the basic structural and functional units of nervous systems, one way to address these problems is to dissect the genetic and molecular pathways responsible for morphological evolution of a defined synapse. I have undertaken such an analysis by examining morphology of the larval neuromuscular junction (NMJ) in wild caught D. melanogaster as well as in over 20 other species of Drosophila. Whereas variation in NMJ morphology within a species is limited, I discovered a surprisingly extensive variation among different species. Compared with evolution of other morphological traits, NMJ morphology appears to be evolving very rapidly. Moreover, my data indicate that natural selection rather than genetic drift is primarily responsible for evolution of NMJ morphology. To dissect underlying molecular mechanisms that may govern NMJ growth and evolutionary divergence, I focused on a naturally occurring variant in D. melanogaster that causes NMJ overgrowth. I discovered that the variant mapped to Mob2, a gene encoding a kinase adapter protein originally described in yeast as a member of the Mitotic Exit Network (MEN). I have subsequently examined mutations in the Drosophila orthologs of all the core components of the yeast MEN and found that all of them function as part of a common pathway that acts presynaptically to negatively regulate NMJ growth. As in the regulation of yeast cytokinesis, these components of the MEN appear to act ultimately by regulating actin dynamics during the process of bouton growth and division. These studies have thus led to the discovery of an entirely new role for the MEN---regulation of synaptic growth---that is separate from its function in cell division. This work

  2. MAP kinase dynamics in yeast.

    PubMed

    van Drogen, F; Peter, M

    2001-09-01

    MAP kinase pathways play key roles in cellular responses towards extracellular signals. In several cases, the three core kinases interact with a scaffold molecule, but the function of these scaffolds is poorly understood. They have been proposed to contribute to signal specificity, signal amplification, or subcellular localization of MAP kinases. Several MAP kinases translocate to the nucleus in response to their activation, suggesting that nuclear transport may provide a regulatory mechanism. Here we describe new applications for Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP), to study dynamic translocations of MAPKs between different subcellular compartments. We have used these methods to measure the nuclear/cytoplasmic dynamics of several yeast MAP kinases, and in particular to address the role of scaffold proteins for MAP-kinase signaling. PMID:11730324

  3. A protein kinase that phosphorylates the C-terminal repeat domain of the largest subunit of RNA polymerase II.

    PubMed Central

    Lee, J M; Greenleaf, A L

    1989-01-01

    The unique C-terminal repeat domain (CTD) of the largest subunit (IIa) of eukaryotic RNA polymerase II consists of multiple repeats of the heptapeptide consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The number of repeats ranges from 26 in yeast to 42 in Drosophila to 52 in mouse. The CTD is essential in vivo, but its structure and function are not yet understood. The CTD can be phosphorylated at multiple serine and threonine residues, generating a form of the largest subunit (II0) with markedly reduced mobility in NaDodSO4/polyacrylamide gels. To investigate this extensive phosphorylation, which presumably modulates functional properties of RNA polymerase II, we began efforts to purify a specific CTD kinase. Using CTD-containing fusion proteins as substrates, we have purified a CTD kinase from the yeast Saccharomyces cerevisiae. The enzyme extensively phosphorylates the CTD portion of both the fusion proteins and intact subunit IIa, producing products with reduced electrophoretic mobilities. The properties of the CTD kinase suggest that it is distinct from previously described protein kinases. Analogous activities were also detected in Drosophila and HeLa cell extracts. Images PMID:2657724

  4. Organization and Evolution of Drosophila Terminin: Similarities and Differences between Drosophila and Human Telomeres.

    PubMed

    Raffa, Grazia D; Cenci, Giovanni; Ciapponi, Laura; Gatti, Maurizio

    2013-01-01

    Drosophila lacks telomerase and fly telomeres are elongated by occasional transposition of three specialized retroelements. Drosophila telomeres do not terminate with GC-rich repeats and are assembled independently of the sequence of chromosome ends. Recent work has shown that Drosophila telomeres are capped by the terminin complex, which includes the fast-evolving proteins HOAP, HipHop, Moi, and Ver. These proteins, which are not conserved outside Drosophilidae and closely related Diptera, localize and function exclusively at telomeres, protecting them from fusion events. Other proteins required to prevent end-to-end fusion in flies include HP1, Eff/UbcD1, ATM, the components of the Mre11-Rad50-Nbs (MRN) complex, and the Woc transcription factor. These proteins do not share the terminin properties; they are evolutionarily conserved non-fast-evolving proteins that do not accumulate only at telomeres and do not serve telomere-specific functions. We propose that following telomerase loss, Drosophila rapidly evolved terminin to bind chromosome ends in a sequence-independent manner. This hypothesis suggests that terminin is the functional analog of the shelterin complex that protects human telomeres. The non-terminin proteins are instead likely to correspond to ancestral telomere-associated proteins that did not evolve as rapidly as terminin because of the functional constraints imposed by their involvement in diverse cellular processes. Thus, it appears that the main difference between Drosophila and human telomeres is in the protective complexes that specifically associate with the DNA termini. We believe that Drosophila telomeres offer excellent opportunities for investigations on human telomere biology. The identification of additional Drosophila genes encoding non-terminin proteins involved in telomere protection might lead to the discovery of novel components of human telomeres. PMID:23675571

  5. Organization and Evolution of Drosophila Terminin: Similarities and Differences between Drosophila and Human Telomeres

    PubMed Central

    Raffa, Grazia D.; Cenci, Giovanni; Ciapponi, Laura; Gatti, Maurizio

    2013-01-01

    Drosophila lacks telomerase and fly telomeres are elongated by occasional transposition of three specialized retroelements. Drosophila telomeres do not terminate with GC-rich repeats and are assembled independently of the sequence of chromosome ends. Recent work has shown that Drosophila telomeres are capped by the terminin complex, which includes the fast-evolving proteins HOAP, HipHop, Moi, and Ver. These proteins, which are not conserved outside Drosophilidae and closely related Diptera, localize and function exclusively at telomeres, protecting them from fusion events. Other proteins required to prevent end-to-end fusion in flies include HP1, Eff/UbcD1, ATM, the components of the Mre11-Rad50-Nbs (MRN) complex, and the Woc transcription factor. These proteins do not share the terminin properties; they are evolutionarily conserved non-fast-evolving proteins that do not accumulate only at telomeres and do not serve telomere-specific functions. We propose that following telomerase loss, Drosophila rapidly evolved terminin to bind chromosome ends in a sequence-independent manner. This hypothesis suggests that terminin is the functional analog of the shelterin complex that protects human telomeres. The non-terminin proteins are instead likely to correspond to ancestral telomere-associated proteins that did not evolve as rapidly as terminin because of the functional constraints imposed by their involvement in diverse cellular processes. Thus, it appears that the main difference between Drosophila and human telomeres is in the protective complexes that specifically associate with the DNA termini. We believe that Drosophila telomeres offer excellent opportunities for investigations on human telomere biology. The identification of additional Drosophila genes encoding non-terminin proteins involved in telomere protection might lead to the discovery of novel components of human telomeres. PMID:23675571

  6. Phosphatidylinositol 3-kinase in myogenesis.

    PubMed

    Kaliman, P; Zorzano, A

    1997-08-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been cloned and characterized in a wide range of organisms. PI 3-kinases are activated by a diversity of extracellular stimuli and are involved in multiple cell processes such as cell proliferation, protein trafficking, cell motility, differentiation, regulation of cytoskeletal structure, and apoptosis. It has recently been shown that PI 3-kinase is a crucial second messenger in the signaling of myogenesis. Two structurally unrelated highly specific inhibitors of PI 3-kinase-wortmannin and LY294002-block the morphological and biochemical differentiation program of different skeletal-muscle cell models. Moreover, L6E9 myoblasts overexpressing a dominant-negative mutant of PI 3-kinase p85 regulatory subunit (Δp85) are unable to differentiate. Furthermore, PI 3-kinase is specifically involved in the insulinlike growth factor (IGF)-dependent myogenic pathway. Indeed, the ability of IGF-I, des-1,3-IGF-I, and IGF-II to promote cell fusion and muscle-specific protein expression is impaired after treatment with PI 3-kinase inhibitors or in cells overexpressing Δp85. The identification of additional key downstream elements of the IGF/PI 3-kinase myogenic cascade is crucial to a detailed understanding of the process of muscle differentiation and may generate new tools for skeletal and cardiac muscle regeneration therapies. (Trends Cardiovasc Med 1997;7:198-202). © 1997, Elsevier Science Inc. PMID:21235885

  7. [Kinase inhibitors and their resistance].

    PubMed

    Togashi, Yosuke; Nishio, Kazuto

    2015-08-01

    Kinase cascades are involved in all stages of tumorigenesis through modulation of transformation and differentiation, cell-cycle progression, and motility. Advances in molecular targeted drug development allow the design and synthesis of inhibitors targeting cancer-associated signal transduction pathways. Potent selective inhibitors with low toxicity can benefit patients especially with several malignancies harboring an oncogenic driver addictive signal. This article evaluates information on solid tumor-related kinase signals and inhibitors, including receptor tyrosine kinase or serine/threonine kinase signals that lead to successful application in clinical settings. In addition, the resistant mechanisms to the inhibitors is summarized. PMID:26281685

  8. A Drosophila XPD model links cell cycle coordination with neuro-development and suggests links to cancer

    PubMed Central

    Stettler, Karin; Li, Xiaoming; Sandrock, Björn; Braga-Lagache, Sophie; Heller, Manfred; Dümbgen, Lutz; Suter, Beat

    2015-01-01

    XPD functions in transcription, DNA repair and in cell cycle control. Mutations in human XPD (also known as ERCC2) mainly cause three clinical phenotypes: xeroderma pigmentosum (XP), Cockayne syndrome (XP/CS) and trichothiodystrophy (TTD), and only XP patients have a high predisposition to developing cancer. Hence, we developed a fly model to obtain novel insights into the defects caused by individual hypomorphic alleles identified in human XP-D patients. This model revealed that the mutations that displayed the greatest in vivo UV sensitivity in Drosophila did not correlate with those that led to tumor formation in humans. Immunoprecipitations followed by targeted quantitative MS/MS analysis showed how different xpd mutations affected the formation or stability of different transcription factor IIH (TFIIH) subcomplexes. The XP mutants most clearly linked to high cancer risk, Xpd R683W and R601L, showed a reduced interaction with the core TFIIH and also an abnormal interaction with the Cdk-activating kinase (CAK) complex. Interestingly, these two XP alleles additionally displayed high levels of chromatin loss and free centrosomes during the rapid nuclear division phase of the Drosophila embryo. Finally, the xpd mutations showing defects in the coordination of cell cycle timing during the Drosophila embryonic divisions correlated with those human mutations that cause the neurodevelopmental abnormalities and developmental growth defects observed in XP/CS and TTD patients. PMID:25431422

  9. Identification of Light-Sensitive Phosphorylation Sites on PERIOD That Regulate the Pace of Circadian Rhythms in Drosophila.

    PubMed

    Yildirim, Evrim; Chiu, Joanna C; Edery, Isaac

    2016-03-01

    The main components regulating the pace of circadian (≅24 h) clocks in animals are PERIOD (PER) proteins, transcriptional regulators that undergo daily changes in levels and nuclear accumulation by means of complex multisite phosphorylation programs. In the present study, we investigated the function of two phosphorylation sites, at Ser826 and Ser828, located in a putative nuclear localization signal (NLS) on the Drosophila melanogaster PER protein. These sites are phosphorylated by DOUBLETIME (DBT; Drosophila homolog of CK1δ/ε), the key circadian kinase regulating the daily changes in PER stability and phosphorylation. Mutant flies in which phosphorylation at Ser826/Ser828 is blocked manifest behavioral rhythms with periods slightly longer than 1 h and with altered temperature compensation properties. Intriguingly, although phosphorylation at these sites does not influence PER stability, timing of nuclear entry, or transcriptional autoinhibition, the phospho-occupancy at Ser826/Ser828 is rapidly stimulated by light and blocked by TIMELESS (TIM), the major photosensitive clock component in Drosophila and a crucial binding partner of PER. Our findings identify the first phosphorylation sites on core clock proteins that are acutely regulated by photic cues and suggest that some phosphosites on PER proteins can modulate the pace of downstream behavioral rhythms without altering central aspects of the clock mechanism. PMID:26711257

  10. Partial venom gland transcriptome of a Drosophila parasitoid wasp, Leptopilina heterotoma, reveals novel and shared bioactive profiles with stinging Hymenoptera.

    PubMed

    Heavner, Mary E; Gueguen, Gwenaelle; Rajwani, Roma; Pagan, Pedro E; Small, Chiyedza; Govind, Shubha

    2013-09-10

    Analysis of natural host-parasite relationships reveals the evolutionary forces that shape the delicate and unique specificity characteristic of such interactions. The accessory long gland-reservoir complex of the wasp Leptopilina heterotoma (Figitidae) produces venom with virus-like particles. Upon delivery, venom components delay host larval development and completely block host immune responses. The host range of this Drosophila endoparasitoid notably includes the highly-studied model organism, Drosophila melanogaster. Categorization of 827 unigenes, using similarity as an indicator of putative homology, reveals that approximately 25% are novel or classified as hypothetical proteins. Most of the remaining unigenes are related to processes involved in signaling, cell cycle, and cell physiology including detoxification, protein biogenesis, and hormone production. Analysis of L. heterotoma's predicted venom gland proteins demonstrates conservation among endo- and ectoparasitoids within the Apocrita (e.g., this wasp and the jewel wasp Nasonia vitripennis) and stinging aculeates (e.g., the honey bee and ants). Enzyme and KEGG pathway profiling predicts that kinases, esterases, and hydrolases may contribute to venom activity in this unique wasp. To our knowledge, this investigation is among the first functional genomic studies for a natural parasitic wasp of Drosophila. Our findings will help explain how L. heterotoma shuts down its hosts' immunity and shed light on the molecular basis of a natural arms race between these insects. PMID:23688557

  11. Partial venom gland transcriptome of a Drosophila parasitoid wasp, Leptopilina heterotoma, reveals novel and shared bioactive profiles with stinging Hymenoptera

    PubMed Central

    Heavner, Mary E.; Gueguen, Gwenaelle; Rajwani, Roma; Pagan, Pedro E.; Small, Chiyedza; Govind, Shubha

    2013-01-01

    Analysis of natural host-parasite relationships reveals the evolutionary forces that shape the delicate and unique specificity characteristic of such interactions. The accessory long gland-reservoir complex of the wasp Leptopilina heterotoma (Figitidae) produces venom with virus-like particles. Upon delivery, venom components delay host larval development and completely block host immune responses. The host range of this Drosophila endoparasitoid notably includes the highly-studied model organism, Drosophila melanogaster. Categorization of 827 unigenes, using similarity as an indicator of putative homology, reveals that approximately 25% are novel or classified as hypothetical proteins. Most of the remaining unigenes are related to processes involved in signaling, cell cycle, and cell physiology including detoxification, protein biogenesis, and hormone production. Analysis of L. heterotoma’s predicted venom gland proteins demonstrates conservation among endo- and ectoparasitoids within the Apocrita (e.g., this wasp and the jewel wasp Nasonia vitripennis) and stinging aculeates (e.g., the honey bee and ants). Enzyme and KEGG pathway profiling predicts that kinases, esterases, and hydrolases may contribute to venom activity in this unique wasp. To our knowledge, this investigation marks the first functional genomic study for a natural parasitic wasp of Drosophila. Our findings will help explain how L. heterotoma shuts down its hosts’ immunity and shed light on the molecular basis of a natural arms race between these insects. PMID:23688557

  12. Two roles for the Drosophila IKK complex in the activation of Relish and the induction of antimicrobial peptide genes

    PubMed Central

    Ertürk-Hasdemir, Deniz; Broemer, Meike; Leulier, François; Lane, William S.; Paquette, Nicholas; Hwang, Daye; Kim, Chan-Hee; Stöven, Svenja; Meier, Pascal; Silverman, Neal

    2009-01-01

    The Drosophila NF-κB transcription factor Relish is an essential regulator of antimicrobial peptide gene induction after Gram-negative bacterial infection. Relish is a bipartite NF-κB precursor protein, with an N-terminal Rel homology domain and a C-terminal IκB-like domain, similar to mammalian p100 and p105. Unlike these mammalian homologs, Relish is endoproteolytically cleaved after infection, allowing the N-terminal NF-κB module to translocate to the nucleus. Signal-dependent activation of Relish, including cleavage, requires both the Drosophila IκB kinase (IKK) and death-related ced-3/Nedd2-like protein (DREDD), the Drosophila caspase-8 like protease. In this report, we show that the IKK complex controls Relish by direct phosphorylation on serines 528 and 529. Surprisingly, these phosphorylation sites are not required for Relish cleavage, nuclear translocation, or DNA binding. Instead they are critical for recruitment of RNA polymerase II and antimicrobial peptide gene induction, whereas IKK functions noncatalytically to support Dredd-mediated cleavage of Relish. PMID:19497884

  13. The first complete Mag family retrotransposons discovered in Drosophila.

    PubMed

    Glukhov, I A; Kotnova, A P; Stefanov, Y E; Ilyin, Y V

    2016-01-01

    A retrotransposon of the Mag family was found in the Drosophila simulans genome for the first time. We also identified novel transposable elements representing the Mag family in seven Drosophila species. The high similarity between the 3' and 5' long terminal repeats in the found copies of transposable elements indicates that their retrotransposition has occurred relatively recently. Thus, the Mag family of retrotransposons is quite common for the genus Drosophila. PMID:27025475

  14. High-dose irradiation induces cell cycle arrest, apoptosis, and developmental defects during Drosophila oogenesis.

    PubMed

    Shim, Hee Jin; Lee, Eun-Mi; Nguyen, Long Duy; Shim, Jaekyung; Song, Young-Han

    2014-01-01

    Ionizing radiation (IR) treatment induces a DNA damage response, including cell cycle arrest, DNA repair, and apoptosis in metazoan somatic cells. Because little has been reported in germline cells, we performed a temporal analysis of the DNA damage response utilizing Drosophila oogenesis as a model system. Oogenesis in the adult Drosophila female begins with the generation of 16-cell cyst by four mitotic divisions of a cystoblast derived from the germline stem cells. We found that high-dose irradiation induced S and G2 arrests in these mitotically dividing germline cells in a grp/Chk1- and mnk/Chk2-dependent manner. However, the upstream kinase mei-41, Drosophila ATR ortholog, was required for the S-phase checkpoint but not for the G2 arrest. As in somatic cells, mnk/Chk2 and dp53 were required for the major cell death observed in early oogenesis when oocyte selection and meiotic recombination occurs. Similar to the unscheduled DNA double-strand breaks (DSBs) generated from defective repair during meiotic recombination, IR-induced DSBs produced developmental defects affecting the spherical morphology of meiotic chromosomes and dorsal-ventral patterning. Moreover, various morphological abnormalities in the ovary were detected after irradiation. Most of the IR-induced defects observed in oogenesis were reversible and were restored between 24 and 96 h after irradiation. These defects in oogenesis severely reduced daily egg production and the hatch rate of the embryos of irradiated female. In summary, irradiated germline cells induced DSBs, cell cycle arrest, apoptosis, and developmental defects resulting in reduction of egg production and defective embryogenesis. PMID:24551207

  15. Genome-wide RNAi screening implicates the E3 ubiquitin ligase Sherpa in mediating innate immune signaling by Toll in Drosophila adults.

    PubMed

    Kanoh, Hirotaka; Tong, Li-Li; Kuraishi, Takayuki; Suda, Yamato; Momiuchi, Yoshiki; Shishido, Fumi; Kurata, Shoichiro

    2015-10-27

    The Drosophila Toll pathway plays important roles in innate immune responses against Gram-positive bacteria and fungi. To identify previously uncharacterized components of this pathway, we performed comparative, ex vivo, genome-wide RNA interference screening. In four screens, we overexpressed the Toll adaptor protein dMyd88, the downstream kinase Pelle, or the nuclear factor κB (NF-κB) homolog Dif, or we knocked down Cactus, the Drosophila homolog of mammalian inhibitor of NF-κB. On the basis of these screens, we identified the E3 ubiquitin ligase Sherpa as being necessary for the activation of Toll signaling. A loss-of-function sherpa mutant fly exhibited compromised production of antimicrobial peptides and enhanced susceptibility to infection by Gram-positive bacteria. In cultured cells, Sherpa mediated ubiquitylation of dMyd88 and Sherpa itself, and Sherpa and Drosophila SUMO (small ubiquitin-like modifier) were required for the proper membrane localization of an adaptor complex containing dMyd88. These findings highlight a role for Sherpa in Drosophila host defense and suggest the SUMOylation-mediated regulation of dMyd88 functions in Toll innate immune signaling. PMID:26508789

  16. The Drosophila insulin-degrading enzyme restricts growth by modulating the PI3K pathway in a cell-autonomous manner

    PubMed Central

    Galagovsky, Diego; Katz, Maximiliano J.; Acevedo, Julieta M.; Sorianello, Eleonora; Glavic, Alvaro; Wappner, Pablo

    2014-01-01

    Mammalian insulin-degrading enzyme (IDE) cleaves insulin, among other peptidic substrates, but its function in insulin signaling is elusive. We use the Drosophila system to define the function of IDE in the regulation of growth and metabolism. We find that either loss or gain of function of Drosophila IDE (dIDE) can restrict growth in a cell-autonomous manner by affecting both cell size and cell number. dIDE can modulate Drosophila insulin-like peptide 2 levels, thereby restricting activation of the phosphatidylinositol-3-phosphate kinase pathway and promoting activation of Drosophila forkhead box, subgroup O transcription factor. Larvae reared in high sucrose exhibit delayed developmental timing due to insulin resistance. We find that dIDE loss of function exacerbates this phenotype and that mutants display increased levels of circulating sugar, along with augmented expression of a lipid biosynthesis marker. We propose that dIDE is a modulator of insulin signaling and that its loss of function favors insulin resistance, a hallmark of diabetes mellitus type II. PMID:24430872

  17. MUMMY, A UDP-N-ACETYLGLUCOSAMINE PYROPHOSPHORYLASE, MODULATES DPP SIGNALING IN THE EMBRYONIC EPIDERMIS OF DROSOPHILA

    PubMed Central

    HUMPHREYS, GREGORY B.; JUD, MOLLY C.; MONROE, KATHRYN M.; KIMBALL, SUZANNE S.; HIGLEY, MATTHEW; SHIPLEY, DANIELLE; VRABLIK, MARIE CLOUGHERTY; BATES, KATHERINE L.; LETSOU, ANTHEA

    2013-01-01

    The evolutionarily conserved JNK/AP-1 (Jun N-terminal kinase/activator protein 1) and BMP (Bone Morphogenetic Protein) signaling cascades are deployed hierarchically to regulate dorsal closure in the fruit fly Drosophila melanogaster. In this developmental context, the JNK/AP-1 signaling cascade transcriptionally activates BMP signaling in leading edge epidermal cells. Here we show that the mummy (mmy) gene product, which is required for dorsal closure, functions as a BMP signaling antagonist. Genetic and biochemical tests of Mmy’s role as a BMP-antagonist indicate that its function is independent of AP-1, the transcriptional trigger of BMP signal transduction in leading edge cells. pMAD (phosphorylated Mothers Against Dpp) activity data show the mmy gene product to be a new type of epidermal BMP regulator – one which transforms a BMP ligand from a long- to a short-range signal. mmy codes for the single UDP-N-acetylglucosamine pyrophosphorylase in Drosophila, and its requirement for attenuating epidermal BMP signaling during dorsal closure points to a new role for glycosylation in defining a highly restricted BMP activity field in the fly. These findings add a new dimension to our understanding of mechanisms modulating the BMP signaling gradient. PMID:23796903

  18. Regulation of the activity of the tumor suppressor PTEN by thioredoxin in Drosophila melanogaster

    SciTech Connect

    Song, Zuohe; Saghafi, Negin; Gokhale, Vijay; Brabant, Marc; Meuillet, Emmanuelle J. . E-mail: emeuillet@azcc.arizona.edu

    2007-04-01

    Human Thioredoxin-1 (hTrx-1) is a small redox protein with a molecular weight of 12 kDa that contains two cysteine residues found in its catalytic site. HTrx-1 plays an important role in cell growth, apoptosis, and cancer patient prognosis. Recently, we have demonstrated that hTrx-1 binds to the C2 domain of the human tumor suppressor, PTEN, in a redox dependent manner. This binding leads to the inhibition of PTEN lipid phosphatase activity in mammalian tissue culture systems. In this study, we show that over-expression of hTrx-1 in Drosophila melanogaster promotes cell growth and proliferation during eye development as measured by eye size and ommatidia size. Furthermore, hTrx-1 rescues the small eye phenotype induced by the over-expression of PTEN. We demonstrate that this rescue of the PTEN-induced eye size phenotype requires cysteine-218 in the C2 domain of PTEN. We also show that hTrx-1 over-expression results in increased Akt phosphorylation in fly head extracts supporting our observations that the hTrx-1-induced eye size increase results from the inhibition of PTEN activity. Our study confirms the redox regulation of PTEN through disulfide bond formation with the hTrx-1 in Drosophila and suggests conserved mechanisms for thioredoxins and their interactions with the phosphatidylinositol-3-kinase signaling pathway in humans and fruit flies.

  19. A dual function for Deep orange in programmed autophagy in the Drosophila melanogaster fat body

    SciTech Connect

    Lindmo, Karine; Simonsen, Anne; Brech, Andreas; Finley, Kim; Rusten, Tor Erik; Stenmark, Harald . E-mail: stenmark@ulrik.uio.no

    2006-07-01

    Lysosomal degradation of cytoplasm by way of autophagy is essential for cellular amino acid homeostasis and for tissue remodeling. In insects such as Drosophila, autophagy is developmentally upregulated in the larval fat body prior to metamorphosis. Here, autophagy is induced by the hormone ecdysone through down-regulation of the autophagy-suppressive phosphoinositide 3-kinase (PI3K) signaling pathway. In yeast, Vps18 and other members of the HOPS complex have been found essential for autophagic degradation. In Drosophila, the Vps18 homologue Deep orange (Dor) has previously been shown to mediate fusion of multivesicular endosomes with lysosomes. A requirement of Dor for ecdysone-mediated chromosome puffing has also been reported. In the present report, we have tested the hypothesis that Dor may control programmed autophagy at the level of ecdysone signaling as well as by mediating autophagosome-to-lysosome fusion. We show that dor mutants are defective in programmed autophagy and provide evidence that autophagy is blocked at two levels. First, PI3K activity was not down-regulated correctly in dor larvae, which correlated with a decrease in ecdysone reporter activity. The down-regulation of PI3K activity was restored by feeding ecdysone to the mutant larvae. Second, neither exogenous ecdysone nor overexpression of PTEN, a silencer of PI3K signaling, restored fusion of autophagosomes with lysosomes in the fat body of dor mutants. These results indicate that Dor controls autophagy indirectly, via ecdysone signaling, as well as directly, via autolysosomal fusion.

  20. Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts

    PubMed Central

    Wang, Hongyan; Somers, Gregory W.; Bashirullah, Arash; Heberlein, Ulrike; Yu, Fengwei; Chia, William

    2006-01-01

    The choice of self-renewal versus differentiation is a fundamental issue in stem cell and cancer biology. Neural progenitors of the Drosophila post-embryonic brain, larval neuroblasts (NBs), divide asymmetrically in a stem cell-like fashion to generate a self-renewing NB and a Ganglion Mother Cell (GMC), which divides terminally to produce two differentiating neuronal/glial daughters. Here we show that Aurora-A (AurA) acts as a tumor suppressor by suppressing NB self-renewal and promoting neuronal differentiation. In aurA loss-of-function mutants, supernumerary NBs are produced at the expense of neurons. AurA suppresses tumor formation by asymmetrically localizing atypical protein kinase C (aPKC), an NB proliferation factor. Numb, which also acts as a tumor suppressor in larval brains, is a major downstream target of AurA and aPKC. Notch activity is up-regulated in aurA and numb larval brains, and Notch signaling is necessary and sufficient to promote NB self-renewal and suppress differentiation in larval brains. Our data suggest that AurA, aPKC, Numb, and Notch function in a pathway that involved a series of negative genetic interactions. We have identified a novel mechanism for controlling the balance between self-renewal and neuronal differentiation during the asymmetric division of Drosophila larval NBs. PMID:17182870

  1. PKC in motorneurons underlies self-learning, a form of motor learning in Drosophila

    PubMed Central

    Colomb, Julien

    2016-01-01

    Tethering a fly for stationary flight allows for exquisite control of its sensory input, such as visual or olfactory stimuli or a punishing infrared laser beam. A torque meter measures the turning attempts of the tethered fly around its vertical body axis. By punishing, say, left turning attempts (in a homogeneous environment), one can train a fly to restrict its behaviour to right turning attempts. It was recently discovered that this form of operant conditioning (called operant self-learning), may constitute a form of motor learning in Drosophila. Previous work had shown that Protein Kinase C (PKC) and the transcription factor dFoxP were specifically involved in self-learning, but not in other forms of learning. These molecules are specifically involved in various forms of motor learning in other animals, such as compulsive biting in Aplysia, song-learning in birds, procedural learning in mice or language acquisition in humans. Here we describe our efforts to decipher which PKC gene is involved in self-learning in Drosophila. We also provide evidence that motorneurons may be one part of the neuronal network modified during self-learning experiments. The collected evidence is reminiscent of one of the simplest, clinically relevant forms of motor learning in humans, operant reflex conditioning, which also relies on motorneuron plasticity. PMID:27168980

  2. The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the Drosophila mesoderm

    PubMed Central

    Grigorian, Melina; Mandal, Lolitika; Hakimi, Manuel; Ortiz, Irma; Hartenstein, Volker

    2011-01-01

    Blood progenitors arise from a pool of pluripotential cells (“hemangioblasts”) within the Drosophila embryonic mesoderm. The fact that the cardiogenic mesoderm consists of only a small number of highly stereotypically patterned cells that can be queried individually regarding their gene expression in normal and mutant embryos, is one of the significant advantages that Drosophila offers to dissect the mechanism specifying the fate of these cells. We show in this paper that the expression of the Notch ligand Delta (Dl) reveals segmentally reiterated mesodermal clusters (“cardiogenic clusters”) that constitute the cardiogenic mesoderm. These clusters give rise to cardioblasts, blood progenitors and nephrocytes. Cardioblasts emerging from the cardiogenic clusters accumulate high levels of Dl, which is required to prevent more cells from adopting the cardioblast fate. In embryos lacking Dl function, all cells of the cardiogenic clusters become cardioblasts, and blood progenitors are lacking. Concomitant activation of the Mitogen Activated Protein Kinase (MAPK) pathway by Epidermal Growth Factor Receptor (EGFR) and Fibroblast Growth Factor Receptor (FGFR) is required for the specification and maintenance of the cardiogenic mesoderm; in addition, the spatially restricted localization of some of the FGFR ligands may be instrumental in controlling the spatial restriction of the Dl ligand to presumptive cardioblasts. PMID:21382367

  3. Neto-Mediated Intracellular Interactions Shape Postsynaptic Composition at the Drosophila Neuromuscular Junction

    PubMed Central

    Ramos, Cathy I.; Igiesuorobo, Oghomwen; Wang, Qi; Serpe, Mihaela

    2015-01-01

    The molecular mechanisms controlling the subunit composition of glutamate receptors are crucial for the formation of neural circuits and for the long-term plasticity underlying learning and memory. Here we use the Drosophila neuromuscular junction (NMJ) to examine how specific receptor subtypes are recruited and stabilized at synaptic locations. In flies, clustering of ionotropic glutamate receptors (iGluRs) requires Neto (Neuropillin and Tolloid-like), a highly conserved auxiliary subunit that is essential for NMJ assembly and development. Drosophila neto encodes two isoforms, Neto-α and Neto-β, with common extracellular parts and distinct cytoplasmic domains. Mutations that specifically eliminate Neto-β or its intracellular domain were generated. When Neto-β is missing or is truncated, the larval NMJs show profound changes in the subtype composition of iGluRs due to reduced synaptic accumulation of the GluRIIA subunit. Furthermore, neto-β mutant NMJs fail to accumulate p21-activated kinase (PAK), a critical postsynaptic component implicated in the synaptic stabilization of GluRIIA. Muscle expression of either Neto-α or Neto-β rescued the synaptic transmission at neto null NMJs, indicating that Neto conserved domains mediate iGluRs clustering. However, only Neto-β restored PAK synaptic accumulation at neto null NMJs. Thus, Neto engages in intracellular interactions that regulate the iGluR subtype composition by preferentially recruiting and/or stabilizing selective receptor subtypes. PMID:25905467

  4. Phagocytosis genes nonautonomously promote developmental cell death in the Drosophila ovary.

    PubMed

    Timmons, Allison K; Mondragon, Albert A; Schenkel, Claire E; Yalonetskaya, Alla; Taylor, Jeffrey D; Moynihan, Katherine E; Etchegaray, Jon Iker; Meehan, Tracy L; McCall, Kimberly

    2016-03-01

    Programmed cell death (PCD) is usually considered a cell-autonomous suicide program, synonymous with apoptosis. Recent research has revealed that PCD is complex, with at least a dozen cell death modalities. Here, we demonstrate that the large-scale nonapoptotic developmental PCD in the Drosophila ovary occurs by an alternative cell death program where the surrounding follicle cells nonautonomously promote death of the germ line. The phagocytic machinery of the follicle cells, including Draper, cell death abnormality (Ced)-12, and c-Jun N-terminal kinase (JNK), is essential for the death and removal of germ-line-derived nurse cells during late oogenesis. Cell death events including acidification, nuclear envelope permeabilization, and DNA fragmentation of the nurse cells are impaired when phagocytosis is inhibited. Moreover, elimination of a small subset of follicle cells prevents nurse cell death and cytoplasmic dumping. Developmental PCD in the Drosophila ovary is an intriguing example of nonapoptotic, nonautonomous PCD, providing insight on the diversity of cell death mechanisms. PMID:26884181

  5. Phagocytosis genes nonautonomously promote developmental cell death in the Drosophila ovary

    PubMed Central

    Timmons, Allison K.; Mondragon, Albert A.; Schenkel, Claire E.; Yalonetskaya, Alla; Taylor, Jeffrey D.; Moynihan, Katherine E.; Etchegaray, Jon Iker; Meehan, Tracy L.; McCall, Kimberly

    2016-01-01

    Programmed cell death (PCD) is usually considered a cell-autonomous suicide program, synonymous with apoptosis. Recent research has revealed that PCD is complex, with at least a dozen cell death modalities. Here, we demonstrate that the large-scale nonapoptotic developmental PCD in the Drosophila ovary occurs by an alternative cell death program where the surrounding follicle cells nonautonomously promote death of the germ line. The phagocytic machinery of the follicle cells, including Draper, cell death abnormality (Ced)-12, and c-Jun N-terminal kinase (JNK), is essential for the death and removal of germ-line–derived nurse cells during late oogenesis. Cell death events including acidification, nuclear envelope permeabilization, and DNA fragmentation of the nurse cells are impaired when phagocytosis is inhibited. Moreover, elimination of a small subset of follicle cells prevents nurse cell death and cytoplasmic dumping. Developmental PCD in the Drosophila ovary is an intriguing example of nonapoptotic, nonautonomous PCD, providing insight on the diversity of cell death mechanisms. PMID:26884181

  6. PKC in motorneurons underlies self-learning, a form of motor learning in Drosophila.

    PubMed

    Colomb, Julien; Brembs, Björn

    2016-01-01

    Tethering a fly for stationary flight allows for exquisite control of its sensory input, such as visual or olfactory stimuli or a punishing infrared laser beam. A torque meter measures the turning attempts of the tethered fly around its vertical body axis. By punishing, say, left turning attempts (in a homogeneous environment), one can train a fly to restrict its behaviour to right turning attempts. It was recently discovered that this form of operant conditioning (called operant self-learning), may constitute a form of motor learning in Drosophila. Previous work had shown that Protein Kinase C (PKC) and the transcription factor dFoxP were specifically involved in self-learning, but not in other forms of learning. These molecules are specifically involved in various forms of motor learning in other animals, such as compulsive biting in Aplysia, song-learning in birds, procedural learning in mice or language acquisition in humans. Here we describe our efforts to decipher which PKC gene is involved in self-learning in Drosophila. We also provide evidence that motorneurons may be one part of the neuronal network modified during self-learning experiments. The collected evidence is reminiscent of one of the simplest, clinically relevant forms of motor learning in humans, operant reflex conditioning, which also relies on motorneuron plasticity. PMID:27168980

  7. 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. PMID:25311449

  8. Ras-activated Dsor1 promotes Wnt signaling in Drosophila development.

    PubMed

    Hall, Eric T; Verheyen, Esther M

    2015-12-15

    Wnt/Wingless (Wg) and Ras-MAPK signaling both play fundamental roles in growth and cell fate determination, and when dysregulated, can lead to tumorigenesis. Several conflicting modes of interaction between Ras-MAPK and Wnt signaling have been identified in specific cellular contexts, causing synergistic or antagonistic effects on target genes. We find novel evidence that the Drosophila homolog of the dual specificity kinases MEK1/2 (also known as MAP2K1/2), Downstream of Raf1 (Dsor1), is required for Wnt signaling. Knockdown of Dsor1 results in loss of Wg target gene expression, as well as reductions in stabilized Armadillo (Arm; Drosophila β-catenin). We identify a close physical interaction between Dsor1 and Arm, and find that catalytically inactive Dsor1 causes a reduction in active Arm. These results suggest that Dsor1 normally counteracts the Axin-mediated destruction of Arm. We find that Ras-Dsor1 activity is independent of upstream activation by EGFR, and instead it appears to be activated by the insulin-like growth factor receptor to promote Wg signaling. Taken together, our results suggest that there is a new crosstalk pathway between insulin and Wg signaling that is mediated by Dsor1. PMID:26542023

  9. The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the Drosophila mesoderm.

    PubMed

    Grigorian, Melina; Mandal, Lolitika; Hakimi, Manuel; Ortiz, Irma; Hartenstein, Volker

    2011-05-01

    Blood progenitors arise from a pool of pluripotential cells ("hemangioblasts") within the Drosophila embryonic mesoderm. The fact that the cardiogenic mesoderm consists of only a small number of highly stereotypically patterned cells that can be queried individually regarding their gene expression in normal and mutant embryos is one of the significant advantages that Drosophila offers to dissect the mechanism specifying the fate of these cells. We show in this paper that the expression of the Notch ligand Delta (Dl) reveals segmentally reiterated mesodermal clusters ("cardiogenic clusters") that constitute the cardiogenic mesoderm. These clusters give rise to cardioblasts, blood progenitors and nephrocytes. Cardioblasts emerging from the cardiogenic clusters accumulate high levels of Dl, which is required to prevent more cells from adopting the cardioblast fate. In embryos lacking Dl function, all cells of the cardiogenic clusters become cardioblasts, and blood progenitors are lacking. Concomitant activation of the Mitogen Activated Protein Kinase (MAPK) pathway by Epidermal Growth Factor Receptor (EGFR) and Fibroblast Growth Factor Receptor (FGFR) is required for the specification and maintenance of the cardiogenic mesoderm; in addition, the spatially restricted localization of some of the FGFR ligands may be instrumental in controlling the spatial restriction of the Dl ligand to presumptive cardioblasts. PMID:21382367

  10. Temperature and functional plasticity of L-type Ca2+ channels in Drosophila.

    PubMed

    Frolov, Roman V; Singh, Satpal

    2013-10-01

    The question of optimization of ion channel function to surrounding temperatures in poikilothermic organisms remains largely uninvestigated. Here, we addressed it by studying the temperature-dependence of L-type Ca2+ channels (LTCCs) in Drosophila larval muscles in the context of their modulation by protein kinase A (PKA). LTCC currents were recorded between 4 and 30°C. Different aspects of LTCC function reached maxima between 15 and 25°C: conductance, tail current amplitude, inactivation rate, and the level of basal up-regulation by PKA (26% at 21°C). Anomalous temperature-dependencies of LTCC conductance and kinetics were similar in control and in the presence of the PKA inhibitor H-89. Analysis of deactivation kinetics revealed excessive tail currents at lower temperatures (up to 15°C), indicative of voltage-dependent facilitation of LTCCs. Tail current magnitude gradually decreased with temperature from a maximum at 15°C until a nearly complete disappearance at 30°C. Elimination of excessive tail currents at higher temperatures coincided with unusual slowing of inactivation, suggesting disruption of the facilitation by rising temperature, possibly through depletion of the pool of contributing channels. Overall, these results suggest the presence of a physiological plasticity optimum of LTCC function in the temperature range of normal Drosophila development. PMID:23993048

  11. Coordinated niche-associated signals promote germline homeostasis in the Drosophila ovary

    PubMed Central

    Liu, Zhong; Zhong, Guohua; Chai, Phing Chian; Luo, Lichao; Liu, Sen; Yang, Ying; Baeg, Gyeong-Hun

    2015-01-01

    Stem cell niches provide localized signaling molecules to promote stem cell fate and to suppress differentiation. The Drosophila melanogaster ovarian niche is established by several types of stromal cells, including terminal filament cells, cap cells, and escort cells (ECs). Here, we show that, in addition to its well-known function as a niche factor expressed in cap cells, the Drosophila transforming growth factor β molecule Decapentaplegic (Dpp) is expressed at a low level in ECs to maintain a pool of partially differentiated germline cells that may dedifferentiate to replenish germline stem cells upon their depletion under normal and stress conditions. Our study further reveals that the Dpp level in ECs is modulated by Hedgehog (Hh) ligands, which originate from both cap cells and ECs. We also demonstrate that Hh signaling exerts its function by suppressing Janus kinase/signal transducer activity, which promotes Dpp expression in ECs. Collectively, our data suggest a complex interplay of niche-associated signals that controls the development of a stem cell lineage. PMID:26504174

  12. JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut

    PubMed Central

    Biteau, Benoît; Hochmuth, Christine E.; Jasper, Heinrich

    2011-01-01

    Metazoans employ cytoprotective and regenerative strategies to maintain tissue homeostasis. Understanding the coordination of these strategies is critical to develop accurate models for aging and associated diseases. Here we show that cytoprotective Jun-N-terminal Kinase (JNK) signaling influences regeneration in the Drosophila gut by directing proliferation of intestinal stem cells (ISCs). Interestingly, this function of JNK contributes to the loss of tissue homeostasis in old and stressed intestines by promoting the accumulation of mis-differentiated ISC daughter cells. Ectopic Delta/Notch signaling in these cells causes their abnormal differentiation, but also limits JNK-induced proliferation. Protective JNK signaling, and control of cell proliferation and differentiation by Delta/Notch signaling thus have to be carefully balanced to ensure tissue homeostasis. Our findings suggest that this balance is lost in old animals, increasing the potential for neoplastic transformation. PMID:18940735

  13. Widespread changes in the posttranscriptional landscape at the Drosophila oocyte-to-embryo transition

    PubMed Central

    Kronja, Iva; Yuan, Bingbing; Eichhorn, Stephen; Dzeyk, Kristina; Krijgsveld, Jeroen; Bartel, David P.

    2014-01-01

    SUMMARY The oocyte-to-embryo transition marks the onset of development. The initial phase of this profound change from the differentiated oocyte to the totipotent embryo occurs in the absence of both transcription and mRNA degradation. Here we combine global polysome profiling, ribosome-footprint profiling, and quantitative mass spectrometry in a comprehensive approach to delineate the translational and proteomic changes at this important transition in Drosophila. Our results show that PNG kinase is a critical regulator of the extensive changes in the translatome, acting uniquely at this developmental window. Analysis of the proteome in png mutants provided insights into the contributions of translation to changes in protein levels, revealing a compensatory dynamic between translation and protein turnover during proteome remodeling at the return to totipotency. The proteome changes additionally suggested new regulators of meiosis and early embryogenesis, including the conserved H3K4 demethylase LID, which we demonstrated is required during this period despite transcriptional inactivity. PMID:24882012

  14. The novel plant homeodomain protein rhinoceros antagonizes Ras signaling in the Drosophila eye.

    PubMed Central

    Voas, Matthew G; Rebay, Ilaria

    2003-01-01

    The sequential specification of cell fates in the Drosophila eye requires repeated activation of the epidermal growth factor receptor (EGFR)/Ras/MAP kinase (MAPK) pathway. Equally important are the multiple layers of inhibitory regulation that prevent excessive or inappropriate signaling. Here we describe the molecular and genetic analysis of a previously uncharacterized gene, rhinoceros (rno), that we propose functions to restrict EGFR signaling in the eye. Loss of rno results in the overproduction of photoreceptors, cone cells, and pigment cells and a corresponding reduction in programmed cell death, all phenotypes characteristic of hyperactivated EGFR signaling. Genetic interactions between rno and multiple EGFR pathway components support this hypothesis. rno encodes a novel but evolutionarily conserved nuclear protein with a PHD zinc-finger domain, a motif commonly found in chromatin-remodeling factors. Future analyses of rno will help to elucidate the regulatory strategies that modulate EGFR signaling in the fly eye. PMID:14704181

  15. Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster.

    PubMed

    Xing, Yalan; Su, Tin Tin; Ruohola-Baker, Hannele

    2015-01-01

    Many types of normal and cancer stem cells are resistant to killing by genotoxins, but the mechanism for this resistance is poorly understood. Here we show that adult stem cells in Drosophila melanogaster germline and midgut are resistant to ionizing radiation (IR) or chemically induced apoptosis and dissect the mechanism for this protection. We find that upon IR the receptor tyrosine kinase Tie/Tie-2 is activated, leading to the upregulation of microRNA bantam that represses FOXO-mediated transcription of pro-apoptotic Smac/DIABLO orthologue, Hid in germline stem cells. Knockdown of the IR-induced putative Tie ligand, Pvf1, a functional homologue of human Angiopoietin, in differentiating daughter cells renders germline stem cells sensitive to IR, suggesting that the dying daughters send a survival signal to protect their stem cells for future repopulation of the tissue. If conserved in cancer stem cells, this mechanism may provide therapeutic options for the eradication of cancer. PMID:25959206

  16. JNK pathway activation is able to synchronize neuronal death and glial phagocytosis in Drosophila

    PubMed Central

    Shklover, J; Mishnaevski, K; Levy-Adam, F; Kurant, E

    2015-01-01

    Glial phagocytosis of superfluous neurons and damaged or aberrant neuronal material is crucial for normal development and maintenance of the CNS. However, the molecular mechanisms underlying the relationship between neuronal death and glial phagocytosis are poorly understood. We describe a novel mechanism that is able to synchronize neuronal cell death and glial phagocytosis of dying neurons in the Drosophila embryonic CNS. This mechanism involves c-Jun N-terminal kinase (JNK) signaling, which is required for developmental apoptosis of specific neurons during embryogenesis. We demonstrate that the dJNK pathway gain-of-function in neurons leads to dJNK signaling in glia, which results in upregulation of glial phagocytosis. Importantly, this promotion of phagocytosis is not mediated by upregulation of the glial phagocytic receptors SIMU and DRPR, but by increasing glial capacity to degrade apoptotic particles inside phagosomes. The proposed mechanism may be important for removal of damaged neurons in the developing and mature CNS. PMID:25695602

  17. Dynamics of cell polarity in tissue morphogenesis: a comparative view from Drosophila and Ciona

    PubMed Central

    Veeman, Michael T.; McDonald, Jocelyn A.

    2016-01-01

    Tissues in developing embryos exhibit complex and dynamic rearrangements that shape forming organs, limbs, and body axes. Directed migration, mediolateral intercalation, lumen formation, and other rearrangements influence the topology and topography of developing tissues. These collective cell behaviors are distinct phenomena but all involve the fine-grained control of cell polarity. Here we review recent findings in the dynamics of polarized cell behavior in both the Drosophila ovarian border cells and the Ciona notochord. These studies reveal the remarkable reorganization of cell polarity during organ formation and underscore conserved mechanisms of developmental cell polarity including the Par/atypical protein kinase C (aPKC) and planar cell polarity pathways. These two very different model systems demonstrate important commonalities but also key differences in how cell polarity is controlled in tissue morphogenesis. Together, these systems raise important, broader questions on how the developmental control of cell polarity contributes to morphogenesis of diverse tissues across the metazoa. PMID:27303647

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

    PubMed

    Jackson, George R; Wiedau-Pazos, Martina; Sang, Tzu-Kang; Wagle, Naveed; Brown, Carlos A; Massachi, Sasan; Geschwind, Daniel H

    2002-05-16

    Pathologic alterations in the microtubule-associated protein tau have been implicated in a number of neurodegenerative disorders, including Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and frontotemporal dementia (FTD). Here, we show that tau overexpression, in combination with phosphorylation by the Drosophila glycogen synthase kinase-3 (GSK-3) homolog and wingless pathway component (Shaggy), exacerbated neurodegeneration induced by tau overexpression alone, leading to neurofibrillary pathology in the fly. Furthermore, manipulation of other wingless signaling molecules downstream from shaggy demonstrated that components of the Wnt signaling pathway modulate neurodegeneration induced by tau pathology in vivo but suggested that tau phosphorylation by GSK-3beta differs from canonical Wnt effects on beta-catenin stability and TCF activity. The genetic system we have established provides a powerful reagent for identification of novel modifiers of tau-induced neurodegeneration that may serve as future therapeutic targets. PMID:12062036

  19. Dscam Expression Levels Determine Presynaptic Arbor Sizes in Drosophila Sensory Neurons

    PubMed Central

    Kim, Jung Hwan; Wang, Xin; Coolon, Rosemary; Ye, Bing

    2013-01-01

    SUMMARY Expression of the Down syndrome cell-adhesion molecule (Dscam) is increased in the brains of patients with several neurological disorders. Although Dscam is critically involved in many aspects of neuronal development, little is known about either the mechanism that regulates its expression or the functional consequences of dysregulated Dscam expression. Here, we show that Dscam expression levels serve as an instructive code for the size control of presynaptic arbor. Two convergent pathways, involving dual leucine zipper kinase (DLK) and fragile X mental retardation protein (FMRP), control Dscam expression through protein translation. Defects in this regulation of Dscam translation lead to exuberant presynaptic arbor growth in Drosophila somatosensory neurons. Our findings demonstrate a previously unknown aspect of Dscam function and provide insights into how dysregulated Dscam may contribute to the pathogenesis of neurological disorders. PMID:23764288

  20. Polo-like kinase, a novel marker for cellular proliferation.

    PubMed Central

    Yuan, J.; Hörlin, A.; Hock, B.; Stutte, H. J.; Rübsamen-Waigmann, H.; Strebhardt, K.

    1997-01-01

    PLK (polo-like kinase) belongs to a family of serine/threonine kinases and represents the human counterpart of polo in Drosophila melanogaster and of CDC5 in Saccharomyces cerevisiae. It is strongly involved in spindle formation and chromosome segregation during mitosis. We have shown previously that PLK mRNA expression correlates with the mitotic activity of cells and the prognosis of lung cancer patients. In this report, the level of PLK protein was analyzed using immunohistochemical techniques. PLK protein was found expressed in the nuclei of tumor cells from lung and breast cancer as well as in several tumor cell lines. Furthermore, in peripheral lymphocytes treated with phytohemagglutinin, elevated proliferative activity of the cells correlated with the up-regulation of PLK protein expression. In contrast, in U937 and HL-60 cells after induction of differentiation with phorbol ester, PLK immunostaining disappeared under conditions of terminal differentiation. Most of the PLK protein was found in the nucleus of proliferating cells with diffuse but distinct staining also in the cytoplasm. Taken together, high levels of PLK protein are associated with cellular proliferation. Combined with other proliferative and oncogene markers, PLK may be useful for improved prediction of the clinical prognosis of cancer patients and for early cancer diagnosis. Due to its activity late in the cell cycle, it may be a target for cancer chemotherapy. Images Figure 1 Figure 2 Figure 3 PMID:9094972

  1. Homeodomain-interacting protein kinase (Hipk) phosphorylates the small SPOC family protein Spenito.

    PubMed

    Dewald, D N; Steinmetz, E L; Walldorf, U

    2014-12-01

    The Drosophila homeodomain-interacting protein kinase (Hipk) is a versatile regulator involved in a variety of pathways, such as Notch and Wingless signalling, thereby acting in processes including the promotion of eye development or control of cell numbers in the nervous system. In vertebrates, extensive studies have related its homologue HIPK2 to important roles in the control of p53-mediated apoptosis and tumour suppression. Spenito (Nito) belongs to the group of small SPOC family proteins and has a role, amongst others, as a regulator of Wingless signalling downstream of Armadillo. In the present study, we show that both proteins have an enzyme-substrate relationship, adding a new interesting component to the broad range of Hipk interactions, and we map several phosphorylation sites of Nito. Furthermore, we were able to define a preliminary consensus motif for Hipk target sites, which will simplify the identification of new substrates of this kinase. PMID:25040100

  2. Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain.

    PubMed

    Huala, E; Oeller, P W; Liscum, E; Han, I S; Larsen, E; Briggs, W R

    1997-12-19

    The NPH1 (nonphototropic hypocotyl 1) gene encodes an essential component acting very early in the signal-transduction chain for phototropism. Arabidopsis NPH1 contains a serine-threonine kinase domain and LOV1 and LOV2 repeats that share similarity (36 to 56 percent) with Halobacterium salinarium Bat, Azotobacter vinelandii NIFL, Neurospora crassa White Collar-1, Escherichia coli Aer, and the Eag family of potassium-channel proteins from Drosophila and mammals. Sequence similarity with a known (NIFL) and a suspected (Aer) flavoprotein suggests that NPH1 LOV1 and LOV2 may be flavin-binding domains that regulate kinase activity in response to blue light-induced redox changes. PMID:9405347

  3. Loss of the starvation-induced gene Rack1 leads to glycogen deficiency and impaired autophagic responses in Drosophila

    PubMed Central

    Érdi, Balázs; Nagy, Péter; Zvara, Ágnes; Varga, Ágnes; Pircs, Karolina; Ménesi, Dalma; Puskás, László G.; Juhász, Gábor

    2012-01-01

    Autophagy delivers cytoplasmic material for lysosomal degradation in eukaryotic cells. Starvation induces high levels of autophagy to promote survival in the lack of nutrients. We compared genome-wide transcriptional profiles of fed and starved control, autophagy-deficient Atg7 and Atg1 null mutant Drosophila larvae to search for novel regulators of autophagy. Genes involved in catabolic processes including autophagy were transcriptionally upregulated in all cases. We also detected repression of genes involved in DNA replication in autophagy mutants compared with control animals. The expression of Rack1 (receptor of activated protein kinase C 1) increased 4.1- to 5.5-fold during nutrient deprivation in all three genotypes. The scaffold protein Rack1 plays a role in a wide range of processes including translation, cell adhesion and migration, cell survival and cancer. Loss of Rack1 led to attenuated autophagic response to starvation, and glycogen stores were decreased 11.8-fold in Rack1 mutant cells. Endogenous Rack1 partially colocalized with GFP-Atg8a and early autophagic structures on the ultrastructural level, suggesting its involvement in autophagosome formation. Endogenous Rack1 also showed a high degree of colocalization with glycogen particles in the larval fat body, and with Shaggy, the Drosophila homolog of glycogen synthase kinase 3B (GSK-3B). Our results, for the first time, demonstrated the fundamental role of Rack1 in autophagy and glycogen synthesis. PMID:22562043

  4. A genetic screen for modifiers of drosophila Src42A identifies mutations in Egfr, rolled and a novel signaling gene.

    PubMed Central

    Zhang, Q; Zheng, Q; Lu, X

    1999-01-01

    Drosophila Src42A, a close relative of the vertebrate c-Src, has been implicated in the Ras-Mapk signaling cascade. An allele of Src42A, Su(Raf)1, dominantly suppresses the lethality of partial loss-of-function Raf mutations. To isolate genes involved in the same pathway where Src42A functions, we carried out genetic screens for dominant suppressor mutations that prevented Su(Raf)1 from suppressing Raf. Thirty-six mutations representing at least five genetic loci were recovered from the second chromosome. These are Drosophila EGF Receptor (Egfr), rolled, Src42A, and two other new loci, one of which was named semang (sag). During embryogenesis, sag affects the development of the head, tail, and tracheal branches, suggesting that it participates in the pathways of Torso and DFGF-R1 receptor tyrosine kinases. sag also disrupts the embryonic peripheral nervous system. During the development of imaginal discs, sag affects two processes known to require Egfr signaling: the recruitment of photoreceptor cells and wing vein formation. Thus sag functions in several receptor tyrosine kinase (RTK)-mediated processes. In addition, sag dominantly enhances the phenotypes associated with loss-of-function Raf and rl, but suppresses those of activated Ras1(V12) mutation. This work provides the first genetic evidence that both Src42A and sag are modulators of RTK signaling. PMID:9927462

  5. Spermatid Cyst Polarization in Drosophila Depends upon apkc and the CPEB Family Translational Regulator orb2

    PubMed Central

    Xu, Shuwa; Tyagi, Sanjay; Schedl, Paul

    2014-01-01

    Mature Drosophila sperm are highly polarized cells—on one side is a nearly 2 mm long flagellar tail that comprises most of the cell, while on the other is the sperm head, which carries the gamete's genetic information. The polarization of the sperm cells commences after meiosis is complete and the 64-cell spermatid cyst begins the process of differentiation. The spermatid nuclei cluster to one side of the cyst, while the flagellar axonemes grows from the other. The elongating spermatid bundles are also polarized with respect to the main axis of the testis; the sperm heads are always oriented basally, while the growing tails extend apically. This orientation within the testes is important for transferring the mature sperm into the seminal vesicles. We show here that orienting cyst polarization with respect to the main axis of the testis depends upon atypical Protein Kinase C (aPKC), a factor implicated in polarity decisions in many different biological contexts. When apkc activity is compromised in the male germline, the direction of cyst polarization within this organ is randomized. Significantly, the mechanisms used to spatially restrict apkc activity to the apical side of the spermatid cyst are different from the canonical cross-regulatory interactions between this kinase and other cell polarity proteins that normally orchestrate polarization. We show that the asymmetric accumulation of aPKC protein in the cyst depends on an mRNA localization pathway that is regulated by the Drosophila CPEB protein Orb2. orb2 is required to properly localize and activate the translation of apkc mRNAs in polarizing spermatid cysts. We also show that orb2 functions not only in orienting cyst polarization with respect to the apical-basal axis of the testis, but also in the process of polarization itself. One of the orb2 targets in this process is its own mRNA. Moreover, the proper execution of this orb2 autoregulatory pathway depends upon apkc. PMID:24830287

  6. Counting calories in Drosophila diet restriction.

    PubMed

    Min, Kyung-Jin; Flatt, Thomas; Kulaots, Indrek; Tatar, Marc

    2007-03-01

    The extension of life span by diet restriction in Drosophila has been argued to occur without limiting calories. Here we directly measure the calories assimilated by flies when maintained on full- and restricted-diets. We find that caloric intake is reduced on all diets that extend life span. Flies on low-yeast diet are long-lived and consume about half the calories of flies on high-yeast diets, regardless of the energetic content of the diet itself. Since caloric intake correlates with yeast concentration and thus with the intake of every metabolite in this dietary component, it is premature to conclude for Drosophila that calories do not explain extension of life span. PMID:17125951

  7. The genome sequence of Drosophila melanogaster.

    SciTech Connect

    2000-03-24

    The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the {approximately}120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes {approximately}13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.

  8. Quantifying and predicting Drosophila larvae crawling phenotypes.

    PubMed

    Günther, Maximilian N; Nettesheim, Guilherme; Shubeita, George T

    2016-01-01

    The fruit fly Drosophila melanogaster is a widely used model for cell biology, development, disease, and neuroscience. The fly's power as a genetic model for disease and neuroscience can be augmented by a quantitative description of its behavior. Here we show that we can accurately account for the complex and unique crawling patterns exhibited by individual Drosophila larvae using a small set of four parameters obtained from the trajectories of a few crawling larvae. The values of these parameters change for larvae from different genetic mutants, as we demonstrate for fly models of Alzheimer's disease and the Fragile X syndrome, allowing applications such as genetic or drug screens. Using the quantitative model of larval crawling developed here we use the mutant-specific parameters to robustly simulate larval crawling, which allows estimating the feasibility of laborious experimental assays and aids in their design. PMID:27323901

  9. [The comeback of mitochondria in Drosophila apoptosis].

    PubMed

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

    2016-05-01

    The role of the mitochondrion in mammalian cell apoptosis has been established since the mid-1990s. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, notably because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and apoptosis in Drosophila cell death occurs at the mitochondrial level. Numerous proteins that appear key for Drosophila apoptosis regulation constitutively or transiently bind to mitochondria. They participate in the cell death process at different levels such as degradation of an IAP caspase inhibitor, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. The aim of this review is to take stock of these events that might have their counterpart in humans. PMID:27225920

  10. Vertebrate eye development as modeled in Drosophila.

    PubMed

    Wawersik, S; Maas, R L

    2000-04-12

    Pax6, a member of the paired-box family of transcription factors, is critical for oculogenesis in both vertebrates and insects. Identification of potential vertebrate Pax6 targets has been guided by studies in Drosophila, where the Pax6 homologs eyeless ( ey ) and twin of eyeless ( toy ) function within a network of genes that synergistically pattern the developing fly eye. These targets, which share homology with the fly genes sine oculis, eyes absent and dachshund, exist in mice and humans as the Six, Eya and Dach gene families. Members of these gene families are present in the developing vertebrate eye, and preliminary studies suggest that they may function in a network analogous to that in the fly. Thus, despite radically different architecture, a similar molecular scaffold underlies both vertebrate and fly eye patterning, suggesting that the considerable power of Drosophila genetics can be harnessed to study mammalian ocular development. PMID:10767315

  11. Remembering Components of Food in Drosophila

    PubMed Central

    Das, Gaurav; Lin, Suewei; Waddell, Scott

    2016-01-01

    Remembering features of past feeding experience can refine foraging and food choice. Insects can learn to associate sensory cues with components of food, such as sugars, amino acids, water, salt, alcohol, toxins and pathogens. In the fruit fly Drosophila some food components activate unique subsets of dopaminergic neurons (DANs) that innervate distinct functional zones on the mushroom bodies (MBs). This architecture suggests that the overall dopaminergic neuron population could provide a potential cellular substrate through which the fly might learn to value a variety of food components. In addition, such an arrangement predicts that individual component memories reside in unique locations. DANs are also critical for food memory consolidation and deprivation-state dependent motivational control of the expression of food-relevant memories. Here, we review our current knowledge of how nutrient-specific memories are formed, consolidated and specifically retrieved in insects, with a particular emphasis on Drosophila. PMID:26924969

  12. Rapamycin preserves gut homeostasis during Drosophila aging.

    PubMed

    Fan, Xiaolan; Liang, Qing; Lian, Ting; Wu, Qi; Gaur, Uma; Li, Diyan; Yang, Deying; Mao, Xueping; Jin, Zhihua; Li, Ying; Yang, Mingyao

    2015-11-01

    Gut homeostasis plays an important role in maintaining the overall body health during aging. Rapamycin, a specific inhibitor of mTOR, exerts prolongevity effects in evolutionarily diverse species. However, its impact on the intestinal homeostasis remains poorly understood. Here, we demonstrate that rapamycin can slow down the proliferation rate of intestinal stem cells (ISCs) in the aging guts and induce autophagy in the intestinal epithelium in Drosophila. Rapamycin can also significantly affect the FOXO associated genes in intestine and up-regulate the negative regulators of IMD/Rel pathway, consequently delaying the microbial expansion in the aging guts. Collectively, these findings reveal that rapamycin can delay the intestinal aging by inhibiting mTOR and thus keeping stem cell proliferation in check. These results will further explain the mechanism of healthspan and lifespan extension by rapamycin in Drosophila. PMID:26431326

  13. Quantifying and predicting Drosophila larvae crawling phenotypes

    PubMed Central

    Günther, Maximilian N.; Nettesheim, Guilherme; Shubeita, George T.

    2016-01-01

    The fruit fly Drosophila melanogaster is a widely used model for cell biology, development, disease, and neuroscience. The fly’s power as a genetic model for disease and neuroscience can be augmented by a quantitative description of its behavior. Here we show that we can accurately account for the complex and unique crawling patterns exhibited by individual Drosophila larvae using a small set of four parameters obtained from the trajectories of a few crawling larvae. The values of these parameters change for larvae from different genetic mutants, as we demonstrate for fly models of Alzheimer’s disease and the Fragile X syndrome, allowing applications such as genetic or drug screens. Using the quantitative model of larval crawling developed here we use the mutant-specific parameters to robustly simulate larval crawling, which allows estimating the feasibility of laborious experimental assays and aids in their design. PMID:27323901

  14. Rapamycin preserves gut homeostasis during Drosophila aging

    PubMed Central

    Lian, Ting; Wu, Qi; Gaur, Uma; Li, Diyan; Yang, Deying; Mao, Xueping; Jin, Zhihua; Li, Ying; Yang, Mingyao

    2015-01-01

    Gut homeostasis plays an important role in maintaining the overall body health during aging. Rapamycin, a specific inhibitor of mTOR, exerts prolongevity effects in evolutionarily diverse species. However, its impact on the intestinal homeostasis remains poorly understood. Here, we demonstrate that rapamycin can slow down the proliferation rate of intestinal stem cells (ISCs) in the aging guts and induce autophagy in the intestinal epithelium in Drosophila. Rapamycin can also significantly affect the FOXO associated genes in intestine and up-regulate the negative regulators of IMD/Rel pathway, consequently delaying the microbial expansion in the aging guts. Collectively, these findings reveal that rapamycin can delay the intestinal aging by inhibiting mTOR and thus keeping stem cell proliferation in check. These results will further explain the mechanism of healthspan and lifespan extension by rapamycin in Drosophila. PMID:26431326

  15. Imaging Calcium in Drosophila at Egg Activation.

    PubMed

    Derrick, Christopher J; York-Andersen, Anna H; Weil, Timothy T

    2016-01-01

    Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca(2+)) often termed a 'Ca(2+) wave'. While the speed and number of oscillations of the Ca(2+) wave varies between species, the change in intracellular Ca(2+) is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton. In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca(2+)-dependent events. Here we present a protocol for imaging the Ca(2+) wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca(2+) wave and the downstream changes associated with it. PMID:27584955

  16. Maintenance of a Drosophila melanogaster Population Cage

    PubMed Central

    Caravaca, Juan Manuel; Lei, Elissa P.

    2016-01-01

    Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle. PMID:27023790

  17. Towards a Molecular Understanding of Drosophila Hearing

    PubMed Central

    Caldwell, Jason C.; Eberl, Daniel F.

    2007-01-01

    The Drosophila auditory system is presented as a powerful new genetic model system for understanding the molecular aspects of development and physiology of hearing organs. The fly’s ear resides in the antenna, with Johnston’s organ serving as the mechanoreceptor. New approaches using electrophysiology and laser vibrometry have provided useful tools to apply to the study of mutations that disrupt hearing. The fundamental developmental processes that generate the peripheral nervous system are fairly well understood, although specific variations of these processes for chordotonal organs (CHO) and especially for Johnston’s organ require more scrutiny. In contrast, even the fundamental physiologic workings of mechanosensitive systems are still poorly understood, but rapid recent progress is beginning to shed light. The identification and analysis of mutations that affect auditory function are summarized here, and prospects for the role of the Drosophila auditory system in understanding both insect and vertebrate hearing are discussed. PMID:12382274

  18. Flightless Flies: Drosophila models of neuromuscular disease

    PubMed Central

    Lloyd, Thomas E.; Taylor, J. Paul

    2010-01-01

    The fruit fly, Drosophila melanogaster, has a long and rich history as an important model organism for biologists. In particular, study of the fruit fly has been essential to much of our fundamental understanding of the development and function of the nervous system. In recent years, studies using fruit flies have provided important insights into the pathogenesis of neurodegenerative and neuromuscular diseases. Fly models of spinal muscular atrophy, spinobulbar muscular atrophy, myotonic dystrophy, dystrophinopathies and other inherited neuromuscular diseases recapitulate many of the key pathologic features of the human disease. The ability to perform genetic screens holds promise for uncovering the molecular mechanisms of disease, and indeed, for identifying novel therapeutic targets. This review will summarize recent progress in developing fly models of neuromuscular diseases and will emphasize the contribution that Drosophila has made to our understanding of these diseases. PMID:20329357

  19. The secret lives of Drosophila flies

    PubMed Central

    Markow, Therese Ann

    2015-01-01

    Abstract Flies of the genus Drosophila, and particularly those of the species Drosophila melanogaster, are best known as laboratory organisms. As with all model organisms, they were domesticated for empirical studies, but they also continue to exist as wild populations. Decades of research on these flies in the laboratory have produced astounding and important insights into basic biological processes, but we have only scratched the surface of what they have to offer as research organisms. An outstanding challenge now is to build on this knowledge and explore how natural history has shaped D. melanogaster in order to advance our understanding of biology more generally. DOI: http://dx.doi.org/10.7554/eLife.06793.001 PMID:26041333

  20. Psychomotor Behavior: A Practical Approach in Drosophila

    PubMed Central

    Iliadi, Konstantin G.; Gluscencova, Oxana B.; Boulianne, Gabrielle L.

    2016-01-01

    Psychomotor behaviors are governed by fine relationships between physical activity and cognitive functions. Disturbances in psychomotor development and performance are a hallmark of many mental illnesses and often appear as observable and measurable behaviors. Here, we describe a new method called an “equilibrist test,” which can be used to quantify psychomotor learning and performance in Drosophila. We also show how this test can be used to quantify motor disturbances at relatively early stages in the development of neurodegenerative diseases.

  1. Detection of Cell Death in Drosophila Tissues

    PubMed Central

    Vasudevan, Deepika; Ryoo, Hyung Don

    2016-01-01

    Drosophila has served as a particularly attractive model to study cell death due to the vast array of tools for genetic manipulation under defined spatial and temporal conditions in vivo as well as in cultured cells. These genetic methods have been well supplemented by enzymatic assays and a panel of antibodies recognizing cell death markers. This chapter discusses reporters, mutants and assays used by various laboratories to study cell death in the context of development and in response to external insults. PMID:27108437

  2. Three-Dimensional Imaging of Drosophila melanogaster

    PubMed Central

    McGurk, Leeanne; Morrison, Harris; Keegan, Liam P.; Sharpe, James; O'Connell, Mary A.

    2007-01-01

    Background The major hindrance to imaging the intact adult Drosophila is that the dark exoskeleton makes it impossible to image through the cuticle. We have overcome this obstacle and describe a method whereby the internal organs of adult Drosophila can be imaged in 3D by bleaching and clearing the adult and then imaging using a technique called optical projection tomography (OPT). The data is displayed as 2D optical sections and also in 3D to provide detail on the shape and structure of the adult anatomy. Methodology We have used OPT to visualize in 2D and 3D the detailed internal anatomy of the intact adult Drosophila. In addition this clearing method used for OPT was tested for imaging with confocal microscopy. Using OPT we have visualized the size and shape of neurodegenerative vacuoles from within the head capsule of flies that suffer from age-related neurodegeneration due to a lack of ADAR mediated RNA-editing. In addition we have visualized tau-lacZ expression in 2D and 3D. This shows that the wholemount adult can be stained without any manipulation and that this stain penetrates well as we have mapped the localization pattern with respect to the internal anatomy. Conclusion We show for the first time that the intact adult Drosophila can be imaged in 3D using OPT, also we show that this method of clearing is also suitable for confocal microscopy to image the brain from within the intact head. The major advantage of this is that organs can be represented in 3D in their natural surroundings. Furthermore optical sections are generated in each of the three planes and are not prone to the technical limitations that are associated with manual sectioning. OPT can be used to dissect mutant phenotypes and to globally map gene expression in both 2D and 3D. PMID:17786206

  3. A Drosophila model to image phagosome maturation.

    PubMed

    Shandala, Tetyana; Lim, Chiaoxin; Sorvina, Alexandra; Brooks, Douglas A

    2013-01-01

    Phagocytosis involves the internalization of extracellular material by invagination of the plasma membrane to form intracellular vesicles called phagosomes, which have functions that include pathogen degradation. The degradative properties of phagosomes are thought to be conferred by sequential fusion with endosomes and lysosomes; however, this maturation process has not been studied in vivo. We employed Drosophila hemocytes, which are similar to mammalian professional macrophages, to establish a model of phagosome maturation. Adult Drosophila females, carrying transgenic Rab7-GFP endosome and Lamp1-GFP lysosome markers, were injected with E. coli DH5α and the hemocytes were collected at 15, 30, 45 and 60 minutes after infection. In wild-type females, E. coli were detected within enlarged Rab7-GFP positive phagosomes at 15 to 45 minutes after infection; and were also observed in enlarged Lamp1-GFP positive phagolysosomes at 45 minutes. Two-photon imaging of hemocytes in vivo confirmed this vesicle morphology, including enlargement of Rab7-GFP and Lamp1-GFP structures that often appeared to protrude from hemocytes. The interaction of endosomes and lysosomes with E. coli phagosomes observed in Drosophila hemocytes was consistent with that previously described for phagosome maturation in human ex vivo macrophages. We also tested our model as a tool for genetic analysis using 14-3-3e mutants, and demonstrated altered phagosome maturation with delayed E. coli internalization, trafficking and/or degradation. These findings demonstrate that Drosophila hemocytes provide an appropriate, genetically amenable, model for analyzing phagosome maturation ex vivo and in vivo. PMID:24709696

  4. Queuine metabolism and cadmium toxicity in Drosophila

    SciTech Connect

    Farkas, W.R.; Siard, T. ); Jacobson, K.B. )

    1991-03-11

    Queuine is a derivative of guanine found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His and Tyr. The transcripts of these tRNAs contain a guanine in this position. This guanine is enzymatically excised and replaced by queuine. The ratio of queuine-containing or (q+) tRNA to its precursor or (q{minus}) tRNA changes throughout the Drosophila life cycle. in the egg 10% of the tRNA is (q+). During the three larval stages this ratio drops to zero. In the one day old adult it is about 10%. It has previously been shown that when flies are selected for the ability to grow in the presence of cadmium, the tolerant flies had 100% (q+) tRNA at the first day after pupation instead of 10%. However, it was not known whether the elevated level of (q+) tRNA was a coincidence or if the elevated levels of (q+) tRNA was protective. The authors explored this problem using germfree Drosophila. The first thing was to determine if Drosophila can synthesize queuine. Sterilized eggs were seeded onto sterile chemically defined medium. The flies were grown to the adult stage. This study showed that Drosophila like mammals cannot synthesize queuine. A second result of this research was the demonstration that the authors could alter the ratio of (q+) to (q{minus}) tRNA by adding exogenous queuine to the medium e.g. at 0.008 mM queuine the (q+) tRNA was 95% instead of {lt} 5% in the last instar stage. Finally, the authors investigated whether or not queuine gave protection against cadmium. The results were that when the flies were grown in the presence of 0.2 mM cadmium queuine at 0.008 mM gave a statistically significant increase in the number of survivors.

  5. Dimethylnitrosamine demethylase activity in Drosophila melanogaster

    SciTech Connect

    Waters, L.C.; Nix, C.E.; Epler, J.L.

    1982-06-15

    A dimethylnitrosamine (DMN) demethylase with levels of activity comparable to that in uninduced rat liver was demonstrated in both larval and adult forms of the Hikone-R strain of Drosophila. A microsomal enzyme, it has many properties of a cytochrome P-450-containing mixed-function oxidase. Kinetic analysis indicates only a single enzyme with an apparent K/sub m/ of 10.5 mM DMN.

  6. The development of the Drosophila larval brain.

    PubMed

    Hartenstein, Volker; Spindler, Shana; Pereanu, Wayne; Fung, Siaumin

    2008-01-01

    In this chapter we will start out by describing in more detail the progenitors of the nervous system, the neuroblasts and ganglion mother cells. Subsequently we will survey the generic cell types that make up the developing Drosophila brain, namely neurons, glial cells and tracheal cells. Finally, we will attempt a synopsis of the neuronal connectivity of the larval brain that can be deduced from the analysis of neural lineages and their relationship to neuropile compartments. PMID:18683635

  7. The Drosophila Forkhead transcription factor FOXO mediates the reduction in cell number associated with reduced insulin signaling

    PubMed Central

    Jünger, Martin A; Rintelen, Felix; Stocker, Hugo; Wasserman, Jonathan D; Végh, Mátyás; Radimerski, Thomas; Greenberg, Michael E; Hafen, Ernst

    2003-01-01

    Background Forkhead transcription factors belonging to the FOXO subfamily are negatively regulated by protein kinase B (PKB) in response to signaling by insulin and insulin-like growth factor in Caenorhabditis elegans and mammals. In Drosophila, the insulin-signaling pathway regulates the size of cells, organs, and the entire body in response to nutrient availability, by controlling both cell size and cell number. In this study, we present a genetic characterization of dFOXO, the only Drosophila FOXO ortholog. Results Ectopic expression of dFOXO and human FOXO3a induced organ-size reduction and cell death in a manner dependent on phosphoinositide (PI) 3-kinase and nutrient levels. Surprisingly, flies homozygous for dFOXO null alleles are viable and of normal size. They are, however, more sensitive to oxidative stress. Furthermore, dFOXO function is required for growth inhibition associated with reduced insulin signaling. Loss of dFOXO suppresses the reduction in cell number but not the cell-size reduction elicited by mutations in the insulin-signaling pathway. By microarray analysis and subsequent genetic validation, we have identified d4E-BP, which encodes a translation inhibitor, as a relevant dFOXO target gene. Conclusion Our results show that dFOXO is a crucial mediator of insulin signaling in Drosophila, mediating the reduction in cell number in insulin-signaling mutants. We propose that in response to cellular stresses, such as nutrient deprivation or increased levels of reactive oxygen species, dFOXO is activated and inhibits growth through the action of target genes such as d4E-BP. PMID:12908874

  8. α-Catenin phosphorylation promotes intercellular adhesion through a dual-kinase mechanism.

    PubMed

    Escobar, David J; Desai, Ridhdhi; Ishiyama, Noboru; Folmsbee, Stephen S; Novak, Megan N; Flozak, Annette S; Daugherty, Rebecca L; Mo, Rigen; Nanavati, Dhaval; Sarpal, Ritu; Leckband, Deborah; Ikura, Mitsu; Tepass, Ulrich; Gottardi, Cara J

    2015-03-15

    The cadherin-catenin adhesion complex is a key contributor to epithelial tissue stability and dynamic cell movements during development and tissue renewal. How this complex is regulated to accomplish these functions is not fully understood. We identified several phosphorylation sites in mammalian αE-catenin (also known as catenin α-1) and Drosophila α-Catenin within a flexible linker located between the middle (M)-region and the carboxy-terminal actin-binding domain. We show that this phospho-linker (P-linker) is the main phosphorylated region of α-catenin in cells and is sequentially modified at casein kinase 2 and 1 consensus sites. In Drosophila, the P-linker is required for normal α-catenin function during development and collective cell migration, although no obvious defects were found in cadherin-catenin complex assembly or adherens junction formation. In mammalian cells, non-phosphorylatable forms of α-catenin showed defects in intercellular adhesion using a mechanical dispersion assay. Epithelial sheets expressing phosphomimetic forms of α-catenin showed faster and more coordinated migrations after scratch wounding. These findings suggest that phosphorylation and dephosphorylation of the α-catenin P-linker are required for normal cadherin-catenin complex function in Drosophila and mammalian cells. PMID:25653389

  9. TDP-43 Phosphorylation by casein kinase Iε promotes oligomerization and enhances toxicity in vivo.

    PubMed

    Choksi, Darshana K; Roy, Bidisha; Chatterjee, Shreyasi; Yusuff, Tanzeen; Bakhoum, Mathieu F; Sengupta, Urmi; Ambegaokar, Suren; Kayed, Rakez; Jackson, George R

    2014-02-15

    Dominant mutations in transactive response DNA-binding protein-43 (TDP-43) cause amyotrophic lateral sclerosis. TDP-43 inclusions occur in neurons, glia and muscle in this disease and in sporadic and inherited forms of frontotemporal lobar degeneration. Cytoplasmic localization, cleavage, aggregation and phosphorylation of TDP-43 at the Ser409/410 epitope have been associated with disease pathogenesis. TDP-43 aggregation is not a common feature of mouse models of TDP-43 proteinopathy, and TDP-43 is generally not thought to acquire an amyloid conformation or form fibrils. A number of putative TDP-43 kinases have been identified, but whether any of these functions to regulate TDP-43 phosphorylation or toxicity in vivo is not known. Here, we demonstrate that human TDP-43(Q331K) undergoes cytoplasmic localization and aggregates when misexpressed in Drosophila when compared with wild-type and M337V forms. Coexpression of Q331K with doubletime (DBT), the fly homolog of casein kinase Iε (CKIε), enhances toxicity. There is at best modest basal phosphorylation of misexpressed human TDP-43 in Drosophila, but coexpression with DBT increases Ser409/410 phosphorylation of all TDP-43 isoforms tested. Phosphorylation of TDP-43 in the fly is specific for DBT, as it is not observed using the validated tau kinases GSK-3β, PAR-1/MARK2 or CDK5. Coexpression of DBT with TDP-43(Q331K) enhances the formation of high-molecular weight oligomeric species coincident with enhanced toxicity, and treatment of recombinant oligomeric TDP-43 with rat CKI strongly enhances its toxicity in mammalian cell culture. These data identify CKIε as a potent TDP-43 kinase in vivo and implicate oligomeric species as the toxic entities in TDP-43 proteinopathies. PMID:24105464

  10. Modulation of Morphogenesis by Egfr during Dorsal Closure in Drosophila

    PubMed Central

    Cormier, Olga; Cheng, David Chung-Pei; Reed, Bruce; Harden, Nicholas

    2013-01-01

    During Drosophila embryogenesis the process of dorsal closure (DC) results in continuity of the embryonic epidermis, and DC is well recognized as a model system for the analysis of epithelial morphogenesis as well as wound healing. During DC the flanking lateral epidermal sheets stretch, align, and fuse along the dorsal midline, thereby sealing a hole in the epidermis occupied by an extra-embryonic tissue known as the amnioserosa (AS). Successful DC requires the regulation of cell shape change via actomyosin contractility in both the epidermis and the AS, and this involves bidirectional communication between these two tissues. We previously demonstrated that transcriptional regulation of myosin from the zipper (zip) locus in both the epidermis and the AS involves the expression of Ack family tyrosine kinases in the AS in conjunction with Dpp secreted from the epidermis. A major function of Ack in other species, however, involves the negative regulation of Egfr. We have, therefore, asked what role Egfr might play in the regulation of DC. Our studies demonstrate that Egfr is required to negatively regulate epidermal expression of dpp during DC. Interestingly, we also find that Egfr signaling in the AS is required to repress zip expression in both the AS and the epidermis, and this may be generally restrictive to the progression of morphogenesis in these tissues. Consistent with this theme of restricting morphogenesis, it has previously been shown that programmed cell death of the AS is essential for proper DC, and we show that Egfr signaling also functions to inhibit or delay AS programmed cell death. Finally, we present evidence that Ack regulates zip expression by promoting the endocytosis of Egfr in the AS. We propose that the general role of Egfr signaling during DC is that of a braking mechanism on the overall progression of DC. PMID:23579691

  11. Regulation of maternal transcript destabilization during egg activation in Drosophila.

    PubMed Central

    Tadros, Wael; Houston, Simon A; Bashirullah, Arash; Cooperstock, Ramona L; Semotok, Jennifer L; Reed, Bruce H; Lipshitz, Howard D

    2003-01-01

    In animals, the transfer of developmental control from maternal RNAs and proteins to zygotically derived products occurs at the midblastula transition. This is accompanied by the destabilization of a subset of maternal transcripts. In Drosophila, maternal transcript destabilization occurs in the absence of fertilization and requires specific cis-acting instability elements. We show here that egg activation is necessary and sufficient to trigger transcript destabilization. We have identified 13 maternal-effect lethal loci that, when mutated, result in failure of maternal transcript degradation. All mutants identified are defective in one or more additional processes associated with egg activation. These include vitelline membrane reorganization, cortical microtubule depolymerization, translation of maternal mRNA, completion of meiosis, and chromosome condensation (the S-to-M transition) after meiosis. The least pleiotropic class of transcript destabilization mutants consists of three genes: pan gu, plutonium, and giant nuclei. These three genes regulate the S-to-M transition at the end of meiosis and are thought to be required for the maintenance of cyclin-dependent kinase (CDK) activity during this cell cycle transition. Consistent with a possible functional connection between this S-to-M transition and transcript destabilization, we show that in vitro-activated eggs, which exhibit aberrant postmeiotic chromosome condensation, fail to initiate transcript degradation. Several genetic tests exclude the possibility that reduction of CDK/cyclin complex activity per se is responsible for the failure to trigger transcript destabilization in these mutants. We propose that the trigger for transcript destabilization occurs coincidently with the S-to-M transition at the end of meiosis and that pan gu, plutonium, and giant nuclei regulate maternal transcript destabilization independent of their role in cell cycle regulation. PMID:12871909

  12. Calcium and egg activation in Drosophila.

    PubMed

    Sartain, Caroline V; Wolfner, Mariana F

    2013-01-01

    In many animals, a rise in intracellular calcium levels is the trigger for egg activation, the process by which an arrested mature oocyte transitions to prepare for embryogenesis. In nearly all animals studied to date, this calcium rise, and thus egg activation, is triggered by the fertilizing sperm. However in the insects that have been examined, fertilization is not necessary to activate their oocytes. Rather, these insects' eggs activate as they transit through the female's reproductive tract, regardless of male contribution. Recent studies in Drosophila have shown that egg activation nevertheless requires calcium and that the downstream events and molecules of egg activation are also conserved, despite the difference in initial trigger. Genetic studies have uncovered essential roles for the calcium-dependent enzyme calcineurin and its regulator calcipressin, and have hinted at roles for calmodulin, in Drosophila egg activation. Physiological and in vitro studies have led to a model in which mechanical forces that impact the Drosophila oocyte as it moves through the reproductive tract triggers the influx of calcium from the external environment, thereby initiating egg activation. Future research will aim to test this model, as well as to determine the spatiotemporal dynamics of cytoplasmic calcium flux and mode of signal propagation in this unique system. PMID:23218670

  13. ``sex Ratio'' Meiotic Drive in Drosophila Testacea

    PubMed Central

    James, A. C.; Jaenike, J.

    1990-01-01

    We document the occurrence of ``sex ratio'' meiotic drive in natural populations of Drosophila testacea. ``Sex ratio'' males sire >95% female offspring. Genetic analysis reveals that this effect is due to a meiotically driven X chromosome, as in other species of Drosophila in which ``sex ratio'' has been found. In contrast to other drosophilids, the ``sex ratio'' and standard chromosomes of D. testacea do not differ in gene arrangement, implying that the effect may be due to a single genetic factor in this species. In all likelihood, the ``sex ratio'' condition has evolved independently in D. testacea and in the Drosophila obscura species group, as the loci responsible for the effect occur on different chromosomal elements. An important ecological consequence of ``sex ratio'' is that natural populations of D. testacea exhibit a strong female bias. Because D. testacea mates, oviposits, and feeds as adults and larvae on mushrooms, this species provides an excellent opportunity to study the selective factors in nature that prevent ``sex ratio'' chromosomes from increasing to fixation and causing the extinction of the species. PMID:2249763

  14. Imprinted control of gene activity in Drosophila.

    PubMed

    Golic, K G; Golic, M M; Pimpinelli, S

    1998-11-19

    Genetic imprinting is defined as a reversible, differential marking of genes or chromosomes that is determined by the sex of the parent from whom the genetic material is inherited [1]. Imprinting was first observed in insects where, in some species, most notably among the coccoids (scale insects and allies), the differential marking of paternally and maternally transmitted chromosome sets leads to inactivation or elimination of paternal chromosomes [2]. Imprinting is also widespread in plants and mammals [3,4], in which paternally and maternally inherited alleles may be differentially expressed. Despite imprinting having been discovered in insects, clear examples of parental imprinting are scarce in the model insect species Drosophila melanogaster. We describe a case of imprint-mediated control of gene expression in Drosophila. The imprinted gene - the white+ eye-color gene - is expressed at a low level when transmitted by males, and at a high level when transmitted by females. Thus, in common with coccoids, Drosophila is capable of generating an imprint, and can respond to that imprint by silencing the paternal allele. PMID:9822579

  15. Visualizing the spindle checkpoint in Drosophila spermatocytes

    PubMed Central

    Rebollo, Elena; González, Cayetano

    2000-01-01

    The spindle assembly checkpoint detects defects in spindle structure or in the alignment of the chromosomes on the metaphase plate and delays the onset of anaphase until defects are corrected. Thus far, the evidence regarding the presence of a spindle checkpoint during meiosis in male Drosophila has been indirect and contradictory. On the one hand, chromosomes without pairing partners do not prevent meiosis progression. On the other hand, some conserved components of the spindle checkpoint machinery are expressed in these cells and behave as their homologue proteins do in systems with an active spindle checkpoint. To establish whether the spindle checkpoint is active in Drosophila spermatocytes we have followed meiosis progression by time-lapse microscopy under conditions where the checkpoint is likely to be activated. We have found that the presence of a relatively high number of misaligned chromosomes or a severe disruption of the meiotic spindle results in a significant delay in the time of entry into anaphase. These observations provide the first direct evidence substantiating the activity of a meiotic spindle checkpoint in male Drosophila. PMID:11256627

  16. ‘Peer pressure’ in larval Drosophila?

    PubMed Central

    Niewalda, Thomas; Jeske, Ines; Michels, Birgit; Gerber, Bertram

    2014-01-01

    ABSTRACT Understanding social behaviour requires a study case that is simple enough to be tractable, yet complex enough to remain interesting. Do larval Drosophila meet these requirements? In a broad sense, this question can refer to effects of the mere presence of other larvae on the behaviour of a target individual. Here we focused in a more strict sense on ‘peer pressure’, that is on the question of whether the behaviour of a target individual larva is affected by what a surrounding group of larvae is doing. We found that innate olfactory preference of a target individual was neither affected (i) by the level of innate olfactory preference in the surrounding group nor (ii) by the expression of learned olfactory preference in the group. Likewise, learned olfactory preference of a target individual was neither affected (iii) by the level of innate olfactory preference of the surrounding group nor (iv) by the learned olfactory preference the group was expressing. We conclude that larval Drosophila thus do not take note of specifically what surrounding larvae are doing. This implies that in a strict sense, and to the extent tested, there is no social interaction between larvae. These results validate widely used en mass approaches to the behaviour of larval Drosophila. PMID:24907371

  17. SPARC–Dependent Cardiomyopathy in Drosophila

    PubMed Central

    Motamedchaboki, Khatereh; Bodmer, Rolf

    2016-01-01

    Background— The Drosophila heart is an important model for studying the genetics underpinning mammalian cardiac function. The system comprises contractile cardiomyocytes, adjacent to which are pairs of highly endocytic pericardial nephrocytes that modulate cardiac function by uncharacterized mechanisms. Identifying these mechanisms and the molecules involved is important because they may be relevant to human cardiac physiology. Methods and Results— This work aimed to identify circulating cardiomodulatory factors of potential relevance to humans using the Drosophila nephrocyte–cardiomyocyte system. A Kruppel-like factor 15 (dKlf15) loss-of-function strategy was used to ablate nephrocytes and then heart function and the hemolymph proteome were analyzed. Ablation of nephrocytes led to a severe cardiomyopathy characterized by a lengthening of diastolic interval. Rendering adult nephrocytes dysfunctional by disrupting their endocytic function or temporally conditional knockdown of dKlf15 led to a similar cardiomyopathy. Proteomics revealed that nephrocytes regulate the circulating levels of many secreted proteins, the most notable of which was the evolutionarily conserved matricellular protein Secreted Protein Acidic and Rich in Cysteine (SPARC), a protein involved in mammalian cardiac function. Finally, reducing SPARC gene dosage ameliorated the cardiomyopathy that developed in the absence of nephrocytes. Conclusions— The data implicate SPARC in the noncell autonomous control of cardiac function in Drosophila and suggest that modulation of SPARC gene expression may ameliorate cardiac dysfunction in humans. PMID:26839388

  18. Flying Drosophila orient to sky polarization.

    PubMed

    Weir, Peter T; Dickinson, Michael H

    2012-01-10

    Insects maintain a constant bearing across a wide range of spatial scales. Monarch butterflies and locusts traverse continents [1, 2], and foraging bees and ants travel hundreds of meters to return to their nests [1, 3, 4], whereas many other insects fly straight for only a few centimeters before changing direction. Despite this variation in spatial scale, the brain region thought to underlie long-distance navigation is remarkably conserved [5, 6], suggesting that the use of a celestial compass is a general and perhaps ancient capability of insects. Laboratory studies of Drosophila have identified a local search mode in which short, straight segments are interspersed with rapid turns [7, 8]. However, this flight mode is inconsistent with measured gene flow between geographically separated populations [9-11], and individual Drosophila can travel 10 km across desert terrain in a single night [9, 12, 13]-a feat that would be impossible without prolonged periods of straight flight. To directly examine orientation behavior under outdoor conditions, we built a portable flight arena in which a fly viewed the natural sky through a liquid crystal device that could experimentally rotate the polarization angle. Our findings indicate that Drosophila actively orient using the sky's natural polarization pattern. PMID:22177905

  19. Mechanisms of systemic wound response in Drosophila.

    PubMed

    Lee, Won-Jae; Miura, Masayuki

    2014-01-01

    In response to cellular and tissue losses caused by physical or chemical injuries, organisms must activate multiple wound repair systems at the cellular, tissue, and organismal levels. The systemic wound response (SWR) that occurs via interorgan communication between local wound sites and remote organs ensures that the host is protected efficiently in response to a local wound. The local wound response around the wound site is fairly well documented, but the molecular mechanisms that allow the host to launch SWR are poorly understood. Recent studies on the Drosophila adult model system have shown that the local wound response is not restricted to the wound site because it plays an essential role in generating signals transmitted to remote organs that subsequently achieve SWR. By exploiting the genetic methods available for investigating Drosophila, we are just beginning to understand the complex interorgan networks that operate during SWRs. This review discusses the basic processes involved with classical integumental wound responses and tissue regeneration, such as epithelial cell movement, hemocyte recruitment, apoptosis, melanization, and generation of reactive oxygen species, as well as the recently described intestinal epithelial cell renewal program that occurs in response to gut cell damages. Furthermore, we discuss how these local wound responses integrate with organ-to-organ communication to launch SWR. Genetic analysis of SWRs using the Drosophila model system will provide a unique opportunity to dissect the molecular mechanisms that control wound-induced organ-to-organ communication. PMID:24512709

  20. dachshund Potentiates Hedgehog Signaling during Drosophila Retinogenesis

    PubMed Central

    Aerts, Stein; Casares, Fernando; Janody, Florence

    2016-01-01

    Proper organ patterning depends on a tight coordination between cell proliferation and differentiation. The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signaling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. Here we show that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, we demonstrate that dac enhances Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis. PMID:27442438

  1. The Ran Pathway in Drosophila melanogaster Mitosis

    PubMed Central

    Chen, Jack W. C.; Barker, Amy R.; Wakefield, James G.

    2015-01-01

    Over the last two decades, the small GTPase Ran has emerged as a central regulator of both mitosis and meiosis, particularly in the generation, maintenance, and regulation of the microtubule (MT)-based bipolar spindle. Ran-regulated pathways in mitosis bear many similarities to the well-characterized functions of Ran in nuclear transport and, as with transport, the majority of these mitotic effects are mediated through affecting the physical interaction between karyopherins and Spindle Assembly Factors (SAFs)—a loose term describing proteins or protein complexes involved in spindle assembly through promoting nucleation, stabilization, and/or depolymerization of MTs, through anchoring MTs to specific structures such as centrosomes, chromatin or kinetochores, or through sliding MTs along each other to generate the force required to achieve bipolarity. As such, the Ran-mediated pathway represents a crucial functional module within the wider spindle assembly landscape. Research into mitosis using the model organism Drosophila melanogaster has contributed substantially to our understanding of centrosome and spindle function. However, in comparison to mammalian systems, very little is known about the contribution of Ran-mediated pathways in Drosophila mitosis. This article sets out to summarize our understanding of the roles of the Ran pathway components in Drosophila mitosis, focusing on the syncytial blastoderm embryo, arguing that it can provide important insights into the conserved functions on Ran during spindle formation. PMID:26636083

  2. Gain-of-function mutations in the Caenorhabditis elegans lin-1 ETS gene identify a C-terminal regulatory domain phosphorylated by ERK MAP kinase.

    PubMed Central

    Jacobs, D; Beitel, G J; Clark, S G; Horvitz, H R; Kornfeld, K

    1998-01-01

    Genetic analysis of lin-1 loss-of-function mutations suggests that lin-1 controls multiple cell-fate decisions during Caenorhabditis elegans development and is negatively regulated by a conserved receptor tyrosine kinase-Ras-ERK mitogen-activated protein (MAP) kinase signal transduction pathway. LIN-1 protein contains an ETS domain and presumably regulates transcription. We identified and characterized six gain-of-function mutations that define a new class of lin-1 allele. These lin-1 alleles appeared to be constitutively active and unresponsive to negative regulation. Each allele has a single-base change that affects the predicted C terminus of LIN-1, suggesting this region is required for negative regulation. The C terminus of LIN-1 was a high-affinity substrate for Erk2 in vitro, suggesting that LIN-1 is directly regulated by ERK MAP kinase. Because mpk-1 ERK MAP kinase controls at least one cell-fate decision that does not require lin-1, our results suggest that MPK-1 contributes to the specificity of this receptor tyrosine kinase-Ras-MAP kinase signal transduction pathway by phosphorylating different proteins in different developmental contexts. These lin-1 mutations all affect a four-amino-acid motif, FQFP, that is conserved in vertebrate and Drosophila ETS proteins that are also phosphorylated by ERK MAP kinase. This sequence may be a substrate recognition motif for the ERK subfamily of MAP kinases. PMID:9691039

  3. Characterization and distribution of NKD, a receptor for Drosophila tachykinin-related peptide 6

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Neuropeptides related to vertebrate tachykinins have been identified in Drosophila and are referred to as drosotachykinins, or DTKs. Two Drosophila G protein-coupled receptors, designated NKD (neurokinin receptor from Drosophila; CG6515) and DTKR (Drosophila tachykinin receptor; CG7887), display seq...

  4. Spotted wing drosophila: a new invasive pest of Mississippi berries

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Spotted Wing Drosophila (SWD) Drosophila suzukii, a native fly of Southeast Asia, is a widely reported and highly invasive pest of fruit crops in North America and Mediterranean Europe. Between 2010 and 2011, SWD was confirmed in most States in eastern North America. During this same period, SWD was...

  5. Cranberries and Spotted Wing Drosophila (SWD) in Wisconsin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Drosophila suzukii, commonly known as spotted wing drosophila (SWD), does not appear to like cranberries very much. Following multiple replicated trials using ripe, under-ripe, and over-ripe organic Wisconsin cranberries, SWD females would not (or could not) insert eggs into under-ripe or ripe cranb...

  6. Porphyromonas gingivalis-host interactions in a Drosophila melanogaster model.

    PubMed

    Igboin, Christina O; Tordoff, Kevin P; Moeschberger, Melvin L; Griffen, Ann L; Leys, Eugene J

    2011-01-01

    Porphyromonas gingivalis is a Gram-negative obligate anaerobe that has been implicated in the etiology of adult periodontitis. We recently introduced a Drosophila melanogaster killing model for examination of P. gingivalis-host interactions. In the current study, the Drosophila killing model was used to characterize the host response to P. gingivalis infection by identifying host components that play a role during infection. Drosophila immune response gene mutants were screened for altered susceptibility to killing by P. gingivalis. The Imd signaling pathway was shown to be important for the survival of Drosophila infected by nonencapsulated P. gingivalis strains but was dispensable for the survival of Drosophila infected by encapsulated P. gingivalis strains. The P. gingivalis capsule was shown to mediate resistance to killing by Drosophila antimicrobial peptides (Imd pathway-regulated cecropinA and drosocin) and human beta-defensin 3. Drosophila thiol-ester protein II (Tep II) and Tep IV and the tumor necrosis factor (TNF) homolog Eiger were also involved in the immune response against P. gingivalis infection, while the scavenger receptors Eater and Croquemort played no roles in the response to P. gingivalis infection. This study demonstrates that the Drosophila killing model is a useful high-throughput model for characterizing the host response to P. gingivalis infection and uncovering novel interactions between the bacterium and the host. PMID:21041486

  7. First foreign exploration for asian parasitoids of Drosophila suzukii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The invasive spotted wing drosophila, Drosophila suzukii Matsumura (Dipt.: Drosophilidae), is a native of East Asia and is now widely established in North America and Europe, where it is a serious pest of small and stone fruit crops. The lack of effective indigenous parasitoids of D. suzukii in the ...

  8. Drosophila suzukii population response to environment and management strategies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Spotted wing drosophila, Drosophila suzukii, quickly emerged as a devastating invasive pest of small and stone fruits in the Americas and Europe. To better understand the population dynamics of D. suzukii, we reviewed recent work on juvenile development, adult reproduction, and seasonal variation in...

  9. Drosophila lacks C20 and C22 polyunsaturated fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Drosophila melanogaster has been considered an ideal model organism to investigate human diseases and genetic pathways. Whether Drosophila is an ideal model for nutrigenomics, especially for fatty acid metabolism, however, remains to be illustrated. This study was to examine the metabolism of C20 an...

  10. Susceptibility of cranberries to Drosophila suzukii (Diptera: Drosophilidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Drosophila suzukii Mastsumura (Diptera: Drosophilidae), commonly referred to as the spotted-wing drosophila, is an exotic species that has proven a troublesome pest of fruit production in the U.S. The fly targets small fruit and thus represents a concern for the U.S. cranberry industry. Two studies ...

  11. The developmental transcriptome of Drosophila melanogaster

    SciTech Connect

    University of Connecticut; Graveley, Brenton R.; Brooks, Angela N.; Carlson, Joseph W.; Duff, Michael O.; Landolin, Jane M.; Yang, Li; Artieri, Carlo G.; van Baren, Marijke J.; Boley, Nathan; Booth, Benjamin W.; Brown, James B.; Cherbas, Lucy; Davis, Carrie A.; Dobin, Alex; Li, Renhua; Lin, Wei; Malone, John H.; Mattiuzzo, Nicolas R.; Miller, David; Sturgill, David; Tuch, Brian B.; Zaleski, Chris; Zhang, Dayu; Blanchette, Marco; Dudoit, Sandrine; Eads, Brian; Green, Richard E.; Hammonds, Ann; Jiang, Lichun; Kapranov, Phil; Langton, Laura; Perrimon, Norbert; Sandler, Jeremy E.; Wan, Kenneth H.; Willingham, Aarron; Zhang, Yu; Zou, Yi; Andrews, Justen; Bicke, Peter J.; Brenner, Steven E.; Brent, Michael R.; Cherbas, Peter; Gingeras, Thomas R.; Hoskins, Roger A.; Kaufman, Thomas C.; Oliver, Brian; Celniker, Susan E.

    2010-12-02

    Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development. Drosophila melanogaster is an important non-mammalian model system that has had a critical role in basic biological discoveries, such as identifying chromosomes as the carriers of genetic information and uncovering the role of genes in development. Because it shares a substantial genic content with humans, Drosophila is increasingly used as a translational model for human development, homeostasis and disease. High-quality maps are needed for all functional genomic elements. Previous studies demonstrated that a rich collection of genes is deployed during the life cycle of the fly. Although expression profiling using microarrays has revealed the expression of, 13,000 annotated genes, it is difficult to map splice junctions and individual base modifications generated by RNA editing using such approaches. Single-base resolution is essential to define precisely the elements that comprise the Drosophila transcriptome. Estimates of the number of transcript isoforms are less accurate than estimates of the number of genes

  12. MAP kinase-interacting kinases--emerging targets against cancer.

    PubMed

    Diab, Sarah; Kumarasiri, Malika; Yu, Mingfeng; Teo, Theodosia; Proud, Christopher; Milne, Robert; Wang, Shudong

    2014-04-24

    Mitogen-activated protein kinase (MAPK)-interacting kinases (Mnks) regulate the initiation of translation through phosphorylation of eukaryotic initiation factor 4E (eIF4E). Mnk-mediated eIF4E activation promotes cancer development and progression. While the phosphorylation of eIF4E is necessary for oncogenic transformation, the kinase activity of Mnks seems dispensable for normal development. For this reason, pharmacological inhibition of Mnks could represent an ideal mechanism-based and nontoxic therapeutic strategy for cancer treatment. In this review, we discuss the current understanding of Mnk biological roles, structures, and functions, as well as clinical implications. Importantly, we propose different strategies for identification of highly selective small molecule inhibitors of Mnks, including exploring a structural feature of their kinase domain, DFD motif, which is unique within the human kinome. We also argue that a combined targeting of Mnks and other pathways should be considered given the complexity of cancer. PMID:24613018

  13. Discovery of Trypanosomatid Parasites in Globally Distributed Drosophila Species

    PubMed Central

    Chandler, James Angus; James, Pamela M.

    2013-01-01

    Microbial parasites of animals include bacteria, viruses, and various unicellular eukaryotes. Because of the difficulty in studying these microorganisms in both humans and disease vectors, laboratory models are commonly used for experimental analysis of host-parasite interactions. Drosophila is one such model that has made significant contributions to our knowledge of bacterial, fungal, and viral infections. Despite this, less is known about other potential parasites associated with natural Drosophila populations. Here, we surveyed sixteen Drosophila populations comprising thirteen species from four continents and Hawaii and found that they are associated with an extensive diversity of trypanosomatids (Euglenozoa, Kinetoplastea). Phylogenetic analysis finds that Drosophila-associated trypanosomatids are closely related to taxa that are responsible for various types of leishmaniases and more distantly related to the taxa responsible for human African trypanosomiasis and Chagas disease. We suggest that Drosophila may provide a powerful system for studying the interactions between trypanosomatids and their hosts. PMID:23658617

  14. An inventory of peroxisomal proteins and pathways in Drosophila melanogaster

    PubMed Central

    Faust, Joseph E.; Verma, Avani; Peng, Chengwei; McNew, James A.

    2012-01-01

    Peroxisomes are ubiquitous organelles housing a variety of essential biochemical pathways. Peroxisome dysfunction causes a spectrum of human diseases known as peroxisome biogenesis disorders (PBD). While much is known regarding the mechanism of peroxisome biogenesis, it is still unclear how peroxisome dysfunction leads to the disease state. Several recent studies have shown that mutations in Drosophila peroxin genes cause phenotypes similar to those seen in humans with PBDs suggesting that Drosophila might be a useful system to model PBDs. We have analyzed the proteome of Drosophila to identify the proteins involved in peroxisomal biogenesis and homeostasis as well as metabolic enzymes that function within the organelle. The subcellular localization of five of these predicted peroxisomal proteins was confirmed. Similar to C. elegans, Drosophila appears to only utilize the peroxisome targeting signal (PTS) type 1 system for matrix protein import. This work will further our understanding of peroxisomes in Drosophila and add to the usefulness of this emerging model system. PMID:22758915

  15. The influence of temperature and photoperiod on the reproductive diapause and cold tolerance of spotted-wing drosophila, Drosophila suzukii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Knowledge regarding the reproductive status of spotted-wing drosophila, Drosophila suzukii (Matsumura)(Diptera: Drosophilidae) is of critical importance in predicting potential infestations of this invasive pest, as eggs are laid in ripe or ripening fruit of several commercially important small frui...

  16. Optimizing postharvest methyl bromide treatments to control spotted wing drosophila, Drosophila suzukii, in sweet cherries from Western USA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methyl bromide (MB) chamber fumigations were evaluated for postharvest control of spotted wing drosophila (SWD), Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), in fresh sweet cherry exports from Western USA. Sweet cherries were infested with SWD, incubated to maximize numbers of the most M...

  17. Regulation of mixed-lineage kinase activation in JNK-dependent morphogenesis

    PubMed Central

    Garlena, Rebecca A.; Gonda, Rebecca L.; Green, Alyssa B.; Pileggi, Rachel M.; Stronach, Beth

    2010-01-01

    Normal cells respond appropriately to various signals, while sustaining proper developmental programs and tissue homeostasis. Inappropriate signal reception, response or attenuation, can upset the normal balance of signaling within cells, leading to dysfunction or tissue malformation. To understand the molecular mechanisms that regulate protein-kinase-based signaling in the context of tissue morphogenesis, we analyzed the domain requirements of Drosophila Slpr, a mixed-lineage kinase (MLK), for Jun N-terminal kinase (JNK) signaling. The N-terminal half of Slpr is involved in regulated signaling whereas the C-terminal half promotes cortical protein localization. The SH3 domain negatively regulates Slpr activity consistent with autoinhibition via a conserved proline motif. Also, like many kinases, conserved residues in the activation segment of the catalytic domain regulate Slpr. Threonine 295, in particular, is essential for function. Slpr activation requires dual input from the MAP4K Misshapen (Msn), through its C-terminal regulatory domain, and the GTPase Rac, which both bind to the LZ–CRIB region of Slpr in vitro. Although Rac is sufficient to activate JNK signaling, our results indicate that there are Slpr-independent functions for Rac in dorsal closure. Finally, expression of various Slpr constructs alone or with upstream activators reveals a wide-ranging response at the cell and tissue level. PMID:20736302

  18. Neuronal migration and protein kinases

    PubMed Central

    Ohshima, Toshio

    2015-01-01

    The formation of the six-layered structure of the mammalian cortex via the inside-out pattern of neuronal migration is fundamental to neocortical functions. Extracellular cues such as Reelin induce intracellular signaling cascades through the protein phosphorylation. Migrating neurons also have intrinsic machineries to regulate cytoskeletal proteins and adhesion properties. Protein phosphorylation regulates these processes. Moreover, the balance between phosphorylation and dephosphorylation is modified by extracellular cues. Multipolar-bipolar transition, radial glia-guided locomotion and terminal translocation are critical steps of radial migration of cortical pyramidal neurons. Protein kinases such as Cyclin-dependent kinase 5 (Cdk5) and c-Jun N-terminal kinases (JNKs) involve these steps. In this review, I shall give an overview the roles of protein kinases in neuronal migration. PMID:25628530

  19. Protein Crystals of Raf Kinase

    NASA Technical Reports Server (NTRS)

    1995-01-01

    This image shows crystals of the protein raf kinase grown on Earth (photo a) and on USML-2 (photo b). The space-grown crystals are an order of magnitude larger. Principal Investigator: Dan Carter of New Century Pharmaceuticals

  20. Isolation of chloroplastic phosphoglycerate kinase

    SciTech Connect

    Macioszek, J.; Anderson, L.E. ); Anderson, J.B. )

    1990-09-01

    We report here a method for the isolation of high specific activity phosphoglycerate kinase (EC 2.7.2.3) from chloroplasts. The enzyme has been purified over 200-fold from pea (Pisum sativum L.) stromal extracts to apparent homogeneity with 23% recovery. Negative cooperativity is observed with the two enzyme phosphoglycerate kinase/glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) couple restored from the purified enzymes when NADPH is the reducing pyridine nucleotide, consistent with earlier results obtained with crude chloroplastic extracts. Michaelis Menten kinetics are observed when 3-phosphoglycerate is held constant and phosphoglycerate kinase is varied, which suggests that phosphoglycerate kinase-bound 1,3-bisphosphoglycerate may be the preferred substrate for glyceraldehyde-3-P dehydrogenase in the chloroplast.

  1. Evolutionary relationships of Drosophila mojavensis geographic host races and their sister species Drosophila arizonae.

    PubMed

    Reed, L K; Nyboer, M; Markow, T A

    2007-03-01

    The cactophilic Drosophila mojavensis species group living in the deserts and dry tropical forests of the southwestern United States and Mexico provides a valuable system for studies in diversification and speciation. Rigorous studies of the relationships between host races of D. mojavensis and the relationships among the members of the species group (D. mojavensis, Drosophila arizona, and Drosophila navojoa) are lacking. We used mitochondrial CO1 sequence data to address the phylogenetics and population genetics of this species group. In this study we have found that the sister species D. mojavensis and D. arizonae share no mitochondrial haplotypes and thus show no evidence for recent introgression. We estimate the divergence time between D. mojavensis and D. arizonae to be between 1.91 and 2.97 million years ago. D. arizonae shows little structure in our population genetic analyses but there is phylogenetic differentiation between southeastern and northern populations of D. arizonae. Drosophila mojavensis shows significant population and phylogenetic structure across the four geographic regions of its distribution. The mitochondrial data support an origin of D. mojavensis on the mainland with early differentiation into the populations now found in the Mojave Desert and the Mainland Sonoran Desert and later colonization of the Baja Peninsula, in contrast to previous models. Also, the sister clade to D. mojavensis/D. arizonae includes D. navojoa and Drosophila huaylasi. By defining the genetic relationships among these populations, we provide a foundation for more sophisticated hypothesis testing regarding the timing of early speciation events and host switches in this species group. PMID:17305857

  2. Tyrosine kinase gene rearrangements in epithelial malignancies

    PubMed Central

    Shaw, Alice T.; Hsu, Peggy P.; Awad, Mark M.; Engelman, Jeffrey A.

    2014-01-01

    Chromosomal rearrangements that lead to oncogenic kinase activation are observed in many epithelial cancers. These cancers express activated fusion kinases that drive the initiation and progression of malignancy, and often have a considerable response to small-molecule kinase inhibitors, which validates these fusion kinases as ‘druggable’ targets. In this Review, we examine the aetiologic, pathogenic and clinical features that are associated with cancers harbouring oncogenic fusion kinases, including anaplastic lymphoma kinase (ALK), ROS1 and RET. We discuss the clinical outcomes with targeted therapies and explore strategies to discover additional kinases that are activated by chromosomal rearrangements in solid tumours. PMID:24132104

  3. Combover/CG10732, a Novel PCP Effector for Drosophila Wing Hair Formation

    PubMed Central

    Fagan, Jeremy K.; Dollar, Gretchen; Lu, Qiuheng; Barnett, Austen; Pechuan Jorge, Joaquin; Schlosser, Andreas; Pfleger, Cathie; Adler, Paul; Jenny, Andreas

    2014-01-01

    The polarization of cells is essential for the proper functioning of most organs. Planar Cell Polarity (PCP), the polarization within the plane of an epithelium, is perpendicular to apical-basal polarity and established by the non-canonical Wnt/Fz-PCP signaling pathway. Within each tissue, downstream PCP effectors link the signal to tissue specific readouts such as stereocilia orientation in the inner ear and hair follicle orientation in vertebrates or the polarization of ommatidia and wing hairs in Drosophila melanogaster. Specific PCP effectors in the wing such as Multiple wing hairs (Mwh) and Rho Kinase (Rok) are required to position the hair at the correct position and to prevent ectopic actin hairs. In a genome-wide screen in vitro, we identified Combover (Cmb)/CG10732 as a novel Rho kinase substrate. Overexpression of Cmb causes the formation of a multiple hair cell phenotype (MHC), similar to loss of rok and mwh. This MHC phenotype is dominantly enhanced by removal of rok or of other members of the PCP effector gene family. Furthermore, we show that Cmb physically interacts with Mwh, and cmb null mutants suppress the MHC phenotype of mwh alleles. Our data indicate that Cmb is a novel PCP effector that promotes to wing hair formation, a function that is antagonized by Mwh. PMID:25207969

  4. Analysis of corkscrew signaling in the Drosophila epidermal growth factor receptor pathway during myogenesis.

    PubMed Central

    Johnson Hamlet, M R; Perkins, L A

    2001-01-01

    The Drosophila nonreceptor protein tyrosine phosphatase, Corkscrew (Csw), functions positively in multiple receptor tyrosine kinase (RTK) pathways, including signaling by the epidermal growth factor receptor (EGFR). Detailed phenotypic analyses of csw mutations have revealed that Csw activity is required in many of the same developmental processes that require EGFR function. However, it is still unclear where in the signaling hierarchy Csw functions relative to other proteins whose activities are also required downstream of the receptor. To address this issue, genetic interaction experiments were performed to place csw gene activity relative to the EGFR, spitz (spi), rhomboid (rho), daughter of sevenless (DOS), kinase-suppressor of ras (ksr), ras1, D-raf, pointed (pnt), and moleskin. We followed the EGFR-dependent formation of VA2 muscle precursor cells as a sensitive assay for these genetic interaction studies. First, we established that Csw has a positive function during mesoderm development. Second, we found that tissue-specific expression of a gain-of-function csw construct rescues loss-of-function mutations in other positive signaling genes upstream of rolled (rl)/MAPK in the EGFR pathway. Third, we were able to infer levels of EGFR signaling in various mutant backgrounds during myogenesis. This work extends previous studies of Csw during Torso and Sevenless RTK signaling to include an in-depth analysis of the role of Csw in the EGFR signaling pathway. PMID:11729154

  5. Activated Drosophila Ras1 is selectively suppressed by isoprenyl transferase inhibitors.

    PubMed Central

    Kauffmann, R C; Qian, Y; Vogt, A; Sebti, S M; Hamilton, A D; Carthew, R W

    1995-01-01

    Ras CAAX (C = cysteine, A = aliphatic amino acid, and X = any amino acid) peptidomimetic inhibitors of farnesyl protein transferase suppress Ras-dependent cell transformation by preventing farnesylation of the Ras oncoprotein. These compounds are potential anticancer agents for tumors associated with Ras mutations. The peptidomimetic FTI-254 was tested for Ras1-inhibiting activity in whole animals by injection of activated Ras1val12 Drosophila larvae. FTI-254 decreased the ability of Ras1val12 to form supernumerary R7 photoreceptor cells in the compound eye of transformed flies. In contrast, it had no effect on the related supernumerary R7 phenotypes of flies transformed with either the activated sevenless receptor tyrosine kinase, Raf kinase, or a chimeric Ras1val12 protein that is membrane associated through myristylation instead of isoprenylation. Therefore, FTI-254 acts as an isoprenylation inhibitor to selectively inhibit Ras1val12 signaling activity in a whole-animal model system. Images Fig. 2 PMID:7479910

  6. Pheromones mediating copulation and attraction in Drosophila.

    PubMed

    Dweck, Hany K M; Ebrahim, Shimaa A M; Thoma, Michael; Mohamed, Ahmed A M; Keesey, Ian W; Trona, Federica; Lavista-Llanos, Sofia; Svatoš, Aleš; Sachse, Silke; Knaden, Markus; Hansson, Bill S

    2015-05-26

    Intraspecific olfactory signals known as pheromones play important roles in insect mating systems. In the model Drosophila melanogaster, a key part of the pheromone-detecting system has remained enigmatic through many years of research in terms of both its behavioral significance and its activating ligands. Here we show that Or47b-and Or88a-expressing olfactory sensory neurons (OSNs) detect the fly-produced odorants methyl laurate (ML), methyl myristate, and methyl palmitate. Fruitless (fru(M))-positive Or47b-expressing OSNs detect ML exclusively, and Or47b- and Or47b-expressing OSNs are required for optimal male copulation behavior. In addition, activation of Or47b-expressing OSNs in the male is sufficient to provide a competitive mating advantage. We further find that the vigorous male courtship displayed toward oenocyte-less flies is attributed to an oenocyte-independent sustained production of the Or47b ligand, ML. In addition, we reveal that Or88a-expressing OSNs respond to all three compounds, and that these neurons are necessary and sufficient for attraction behavior in both males and females. Beyond the OSN level, information regarding the three fly odorants is transferred from the antennal lobe to higher brain centers in two dedicated neural lines. Finally, we find that both Or47b- and Or88a-based systems and their ligands are remarkably conserved over a number of drosophilid species. Taken together, our results close a significant gap in the understanding of the olfactory background to Drosophila mating and attraction behavior; while reproductive isolation barriers between species are created mainly by species-specific signals, the mating enhancing signal in several Drosophila species is conserved. PMID:25964351

  7. Pheromones mediating copulation and attraction in Drosophila

    PubMed Central

    Dweck, Hany K. M.; Ebrahim, Shimaa A. M.; Thoma, Michael; Mohamed, Ahmed A. M.; Keesey, Ian W.; Trona, Federica; Lavista-Llanos, Sofia; Svatoš, Aleš; Sachse, Silke; Knaden, Markus; Hansson, Bill S.

    2015-01-01

    Intraspecific olfactory signals known as pheromones play important roles in insect mating systems. In the model Drosophila melanogaster, a key part of the pheromone-detecting system has remained enigmatic through many years of research in terms of both its behavioral significance and its activating ligands. Here we show that Or47b-and Or88a-expressing olfactory sensory neurons (OSNs) detect the fly-produced odorants methyl laurate (ML), methyl myristate, and methyl palmitate. Fruitless (fruM)-positive Or47b-expressing OSNs detect ML exclusively, and Or47b- and Or47b-expressing OSNs are required for optimal male copulation behavior. In addition, activation of Or47b-expressing OSNs in the male is sufficient to provide a competitive mating advantage. We further find that the vigorous male courtship displayed toward oenocyte-less flies is attributed to an oenocyte-independent sustained production of the Or47b ligand, ML. In addition, we reveal that Or88a-expressing OSNs respond to all three compounds, and that these neurons are necessary and sufficient for attraction behavior in both males and females. Beyond the OSN level, information regarding the three fly odorants is transferred from the antennal lobe to higher brain centers in two dedicated neural lines. Finally, we find that both Or47b- and Or88a-based systems and their ligands are remarkably conserved over a number of drosophilid species. Taken together, our results close a significant gap in the understanding of the olfactory background to Drosophila mating and attraction behavior; while reproductive isolation barriers between species are created mainly by species-specific signals, the mating enhancing signal in several Drosophila species is conserved. PMID:25964351

  8. Spatiotemporal rescue of memory dysfunction in Drosophila.

    PubMed

    McGuire, Sean E; Le, Phuong T; Osborn, Alexander J; Matsumoto, Kunihiro; Davis, Ronald L

    2003-12-01

    We have developed a method for temporal and regional gene expression targeting (TARGET) in Drosophila and show the simultaneous spatial and temporal rescue of a memory defect. The transient expression of the rutabaga-encoded adenylyl cyclase in the mushroom bodies of the adult brain was necessary and sufficient to rescue the rutabaga memory deficit, which rules out a developmental brain defect in the etiology of this deficit and demonstrates an acute role for rutabaga in memory formation in these neurons. The TARGET system offers general utility in simultaneously addressing issues of when and where gene products are required. PMID:14657498

  9. Organ size control: lessons from Drosophila

    PubMed Central

    Hariharan, Iswar K.

    2015-01-01

    Of fundamental interest to biologists is how organs achieve a reproducible size during development. Studies of the developing Drosophila wing have provided many key insights that will help give a conceptual understanding of the process beyond the fly. In the wing, there is evidence for both “top-down” mechanisms, where signals emanating from small subsets of cells direct global proliferation, and “”bottom-up” mechanisms, where the final size is an emergent property of local cell-cell interactions. Mechanical forces also appear to have an important role along with the Hippo pathway, which may integrate multiple types of inputs to regulate the extent of growth. PMID:26267393

  10. Studying tauopathies in Drosophila: A fruitful model.

    PubMed

    Sun, Mingkuan; Chen, Liam

    2015-12-01

    Tauopathies are a group of neurodegenerative disorders that include hereditary frontotemporal dementias (FTDs) such as FTD with parkinsonism linked to chromosome 17 (FTDP-17), as well as sporadic variants of FTDs like progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick's disease. These diverse diseases all have in common the presence of abnormally phosphorylated tau aggregates. In this review, we will summarize key features of transgenic Drosophila models of tauopathies and a number of insights into disease mechanisms as well as therapeutic implications gained from the fruit fly models. PMID:25862286

  11. A connectionist model of the Drosophila blastoderm

    SciTech Connect

    Reinitz, J. . Dept. of Biological Sciences); Mjolsness, E. . Dept. of Computer Science); Sharp, D.H. . Theoretical Div.)

    1990-11-01

    The authors present a phenomenological modeling framework for development, and apply it to the network of segmentation genes operating in the blastoderm of Drosophila. Their purpose is to provide a systematic method for discovering and expressing correlations in experimental data on gene expression and other developmental processes. The modeling framework is based on a connectionist or neural net dynamics for biochemical regulators, coupled to grammatical rules which describe certain features of the birth, growth, and death of cells, synapses and other biological entities. They present preliminary numerical results regarding regulatory interactions between the genes Kruppel and knirps that demonstrate the potential utility of the model. 14 refs., 5 figs.

  12. The intimate genetics of Drosophila fertilization

    PubMed Central

    Loppin, Benjamin; Dubruille, Raphaëlle; Horard, Béatrice

    2015-01-01

    The union of haploid gametes at fertilization initiates the formation of the diploid zygote in sexually reproducing animals. This founding event of embryogenesis includes several fascinating cellular and nuclear processes, such as sperm–egg cellular interactions, sperm chromatin remodelling, centrosome formation or pronuclear migration. In comparison with other aspects of development, the exploration of animal fertilization at the functional level has remained so far relatively limited, even in classical model organisms. Here, we have reviewed our current knowledge of fertilization in Drosophila melanogaster, with a special emphasis on the genes involved in the complex transformation of the fertilizing sperm nucleus into a replicated set of paternal chromosomes. PMID:26246493

  13. Hypergravity-induced altered behavior in Drosophila

    NASA Astrophysics Data System (ADS)

    Hosamani, Ravikumar; Wan, Judy; Marcu, Oana; Bhattacharya, Sharmila

    2012-07-01

    Microgravity and mechanical stress are important factors of the spaceflight environment, and affect astronaut health and behavior. Structural, functional, and behavioral mechanisms of all cells and organisms are adapted to Earth's gravitational force, 1G, while altered gravity can pose challenges to their adaptability to this new environment. On ground, hypergravity paradigms have been used to predict and complement studies on microgravity. Even small changes that take place at a molecular and genetic level during altered gravity may result in changes in phenotypic behavior. Drosophila provides a robust and simple, yet very reliable model system to understand the complexity of hypergravity-induced altered behavior, due to availability of a plethora of genetic tools. Locomotor behavior is a sensitive parameter that reflects the array of molecular adaptive mechanisms recruited during exposure to altered gravity. Thus, understanding the genetic basis of this behavior in a hypergravity environment could potentially extend our understanding of mechanisms of adaptation in microgravity. In our laboratory we are trying to dissect out the cellular and molecular mechanisms underlying hypergravity-induced oxidative stress, and its potential consequences on behavioral alterations by using Drosophila as a model system. In the present study, we employed pan-neuronal and mushroom body specific knock-down adult flies by using Gal4/UAS system to express inverted repeat transgenes (RNAi) to monitor and quantify the hypergravity-induced behavior in Drosophila. We established that acute hypergravity (3G for 60 min) causes a significant and robust decrease in the locomotor behavior in adult Drosophila, and that this change is dependent on genes related to Parkinson's disease, such as DJ-1α , DJ-1β , and parkin. In addition, we also showed that anatomically the control of this behavior is significantly processed in the mushroom body region of the fly brain. This work links a molecular

  14. Evolution of a Distinct Genomic Domain in Drosophila: Comparative Analysis of the Dot Chromosome in Drosophila melanogaster and Drosophila virilis

    PubMed Central

    Leung, Wilson; Shaffer, Christopher D.; Cordonnier, Taylor; Wong, Jeannette; Itano, Michelle S.; Slawson Tempel, Elizabeth E.; Kellmann, Elmer; Desruisseau, David Michael; Cain, Carolyn; Carrasquillo, Robert; Chusak, Tien M.; Falkowska, Katazyna; Grim, Kelli D.; Guan, Rui; Honeybourne, Jacquelyn; Khan, Sana; Lo, Louis; McGaha, Rebecca; Plunkett, Jevon; Richner, Justin M.; Richt, Ryan; Sabin, Leah; Shah, Anita; Sharma, Anushree; Singhal, Sonal; Song, Fine; Swope, Christopher; Wilen, Craig B.; Buhler, Jeremy; Mardis, Elaine R.; Elgin, Sarah C. R.

    2010-01-01

    The distal arm of the fourth (“dot”) chromosome of Drosophila melanogaster is unusual in that it exhibits an amalgamation of heterochromatic properties (e.g., dense packaging, late replication) and euchromatic properties (e.g., gene density similar to euchromatic domains, replication during polytenization). To examine the evolution of this unusual domain, we undertook a comparative study by generating high-quality sequence data and manually curating gene models for the dot chromosome of D. virilis (Tucson strain 15010–1051.88). Our analysis shows that the dot chromosomes of D. melanogaster and D. virilis have higher repeat density, larger gene size, lower codon bias, and a higher rate of gene rearrangement compared to a reference euchromatic domain. Analysis of eight “wanderer” genes (present in a euchromatic chromosome arm in one species and on the dot chromosome in the other) shows that their characteristics are similar to other genes in the same domain, which suggests that these characteristics are features of the domain and are not required for these genes to function. Comparison of this strain of D. virilis with the strain sequenced by the Drosophila 12 Genomes Consortium (Tucson strain 15010–1051.87) indicates that most genes on the dot are under weak purifying selection. Collectively, despite the heterochromatin-like properties of this domain, genes on the dot evolve to maintain function while being responsive to changes in their local environment. PMID:20479145

  15. Drosophila-associated yeast species in vineyard ecosystems.

    PubMed

    Lam, Samuel S T H; Howell, Kate S

    2015-10-01

    Yeast activity during wine fermentation directly contributes to wine quality, but the source and movement of yeasts in vineyards and winery environments have not been resolved. Here, we investigate the yeast species associated with the Drosophila insect vector to help understand yeast dispersal and persistence. Drosophila are commonly found in vineyards and are known to have a mutualistic relationship with yeasts in other ecosystems. Drosophilids were collected from vineyards, grape waste (marc) piles and wineries during grape harvest. Captured flies were identified morphologically, and their associated yeasts were identified. Drosophila melanogaster/D. simulans, D. hydei and Scaptodrosophila lativittata were identified in 296 captured Drosophila flies. These flies were associated with Metschnikowia pulcherrima, Hanseniaspora uvarum, Torulaspora delbrueckii and H. valbyensis yeasts. Yeast and Drosophila species diversity differed between collection locations (vineyard and marc: R = 0.588 for Drosophila and R = 0.644 for yeasts). Surprisingly, the primary wine fermentation yeast, Saccharomyces cerevisiae, was not isolated. Drosophila flies are preferentially associated with different yeast species in the vineyard and winery environments, and this association may help the movement and dispersal of yeast species in the vineyard and winery ecosystem. PMID:26391524

  16. Multi-kinase inhibitors, AURKs and cancer.

    PubMed

    Cicenas, Jonas; Cicenas, Erikas

    2016-05-01

    Inhibitors that impact function of kinases are valuable both for the biological research as well as therapy of kinase-associated diseases, such as different cancers. There are quite a number of inhibitors, which are quite specific for certain kinases and several of them are either already approved for the cancer therapy or are in clinical studies of various phases. However, that does not mean that each single kinase inhibitor is suitable for targeted therapy. Some of them are not effective others might be toxic or fail some other criteria for the use in vivo. On the other hand, even in case of successful therapy, many responders eventually develop resistance to the inhibitors. The limitations of various single kinase inhibitors can be fought using compounds which target multiple kinases. This tactics can increase effectiveness of the inhibitors by the synergistic effect or help to diminish the likelihood of drug resistance. To date, several families of kinases are quite popular targets of the inhibition in cancers, such as tyrosine kinases, cycle-dependent kinases, mitogen-activated protein kinases, phosphoinositide 3-kinases as well as their pathway "players" and aurora kinases. Aurora kinases play an important role in the control of the mitosis and are often altered in diverse human cancers. Here, we will describe the most interesting multi-kinase inhibitors which inhibit aurora kinases among other targets and their use in preclinical and clinical cancer studies. PMID:27038473

  17. S6 Kinase Reflects and Regulates Ethanol-Induced Sedation

    PubMed Central

    Acevedo, Summer F.; Peru y Colón de Portugal, Raniero L.; Gonzalez, Dante A.; Rodan, Aylin R.

    2015-01-01

    Alcohol use disorders (AUDs) affect people at great individual and societal cost. Individuals at risk for AUDs are sensitive to alcohol's rewarding effects and/or resistant to its aversive and sedating effects. The molecular basis for these traits is poorly understood. Here, we show that p70 S6 kinase (S6k), acting downstream of the insulin receptor (InR) and the small GTPase Arf6, is a key mediator of ethanol-induced sedation in Drosophila. S6k signaling in the adult nervous system determines flies' sensitivity to sedation. Furthermore, S6k activity, measured via levels of phosphorylation (P-S6k), is a molecular marker for sedation and overall neuronal activity: P-S6k levels are decreased when neurons are silenced, as well as after acute ethanol sedation. Conversely, P-S6k levels rebound upon recovery from sedation and are increased when neuronal activity is enhanced. Reducing neural activity increases sensitivity to ethanol-induced sedation, whereas neuronal activation decreases ethanol sensitivity. These data suggest that ethanol has acute silencing effects on adult neuronal activity, which suppresses InR/Arf6/S6k signaling and results in behavioral sedation. In addition, we show that activity of InR/Arf6/S6k signaling determines flies' behavioral sensitivity to ethanol-induced sedation, highlighting this pathway in acute responses to ethanol. SIGNIFICANCE STATEMENT Genetic factors play a major role in the development of addiction. Identifying these genes and understanding their molecular mechanisms is a necessary first step in the development of targeted therapeutic intervention. Here, we show that signaling from the insulin receptor in Drosophila neurons determines flies' sensitivity to ethanol-induced sedation. We show that this signaling cascade includes the small GTPase Arf6 and S6 kinase (S6k). In addition, activity of S6k is regulated by acute ethanol exposure and by neuronal activity. S6k activity is therefore both an acute target of ethanol exposure and

  18. Evidence for horizontal transfer of Wolbachia by a Drosophila mite.

    PubMed

    Brown, Amy N; Lloyd, Vett K

    2015-07-01

    Mites are common ectoparasites of Drosophila and have been implicated in bacterial and mobile element invasion of Drosophila stocks. The obligate endobacterium, Wolbachia, has widespread effects on gene expression in their arthropod hosts and alters host reproduction to enhance its survival and propagation, often with deleterious effects in Drosophila hosts. To determine whether Wolbachia could be transferred between Drosophila melanogaster laboratory stocks by the mite Tyrophagus putrescentiae, mites were introduced to Wolbachia-infected Drosophila vials. These vials were kept adjacent to mite-free and Wolbachia-uninfected Drosophila stock vials. The Wolbachia infection statuses of the infected and uninfected flies were checked from generation 1 to 5. Results indicate that Wolbachia DNA could be amplified from mites infesting Wolbachia-infected fly stocks and infection in the previously uninfected stocks arose within generation 1 or 2, concomitant with invasion of mites from the Wolbachia-infected stock. A possible mechanism for the transfer of Wolbachia from flies to mites and vice versa, can be inferred from time-lapse photography of fly and mite interactions. We demonstrated that mites ingest Drosophila corpses, including Wolbachia-infected corpses, and Drosophila larva ingest mites, providing possible sources of Wolbachia infection and transfer. This research demonstrated that T. putrescentiae white mites can facilitate Wolbachia transfer between Drosophila stocks and that this may occur by ingestion of infected corpses. Mite-vectored Wolbachia transfer allows for rapid establishment of Wolbachia infection within a new population. This mode of Wolbachia introduction may be relevant in nature as well as in the laboratory, and could have a variety of biological consequences. PMID:25921489

  19. Genetic effects of plutonium in Drosophila. Final technical report

    SciTech Connect

    1995-07-01

    This three year project, initiated in 1987, involved the genetic effects of alpha radiations on Drosophila. This document represents the final technical report. Plutonium residue was used as the alpha source of radon gas. Spontaneous mutation frequency in the Drosophila stock was very low. In the experiments using alpha radiation from radon gas, radiation doses as low as 20R induced significant numbers of mutations, with higher numbers of mutations at higher doses. If X-ray induced mutation frequencies reported in the literature are used for comparison, it can be concluded that alpha radiation from radon gas induces at least 2 to 3 time more mutations in Drosophila.

  20. A novel, tissue-specific, Drosophila homeobox gene.

    PubMed Central

    Barad, M; Jack, T; Chadwick, R; McGinnis, W

    1988-01-01

    The homeobox gene family of Drosophila appears to control a variety of position-specific patterning decisions during embryonic and imaginal development. Most of these patterning decisions determine groups of cells on the anterior-posterior axis of the Drosophila germ band. We have isolated a novel homeobox gene from Drosophila, designated H2.0. H2.0 has the most diverged homeobox so far characterized in metazoa, and, in contrast to all previously isolated homeobox genes, H2.0 exhibits a tissue-specific pattern of expression. The cells that accumulate transcripts for this novel gene correspond to the visceral musculature and its anlagen. Images PMID:2901348

  1. Drosophila melanogaster as a Model System to Study Mitochondrial Biology

    PubMed Central

    Fernández-Moreno, Miguel Angel; Farr, Carol L.; Kaguni, Laurie S.; Garesse, Rafael

    2016-01-01

    Summary Mitochondria play an essential role in cellular homeostasis. Although in the last few decades our knowledge of mitochondria has increased substantially, the mechanisms involved in the control of mitochondrial biogenesis remain largely unknown. The powerful genetics of Drosophila combined with a wealth of available cell and molecular biology techniques, make this organism an excellent system to study mitochondria. In this chapter we will review briefly the opportunities that Drosophila offers as a model system and describe in detail how to purify mitochondria from Drosophila and to perform the analysis of developmental gene expression using in situ hybridization. PMID:18314716

  2. Jelly Belly Trans-Synaptic Signaling to Anaplastic Lymphoma Kinase Regulates Neurotransmission Strength and Synapse Architecture

    PubMed Central

    Rohrbough, Jeffrey; Kent, Karla S.; Broadie, Kendal; Weiss, Joseph B.

    2012-01-01

    In Drosophila the secreted signaling molecule Jelly Belly (Jeb) activates Anaplastic Lymphoma Kinase (Alk), a receptor tyrosine kinase, in multiple developmental and adult contexts. We have shown previously that Jeb and Alk are highly enriched at Drosophila synapses within the CNS neuropil and neuromuscular junction (NMJ) and postulated a conserved intercellular signaling function. At the embryonic and larval NMJ Jeb is localized in the motor neuron presynaptic terminal whereas Alk is concentrated in the muscle postsynaptic domain surrounding boutons, consistent with anterograde trans-synaptic signaling. Here, we show by functional inhibition of Jeb-Alk signaling that neurotransmission is regulated by Jeb secretion. Jeb is a novel negative regulator of neuromuscular transmission. Reduction or inhibtion of Alk function results in enhanced synaptic transmission. Activation of Alk conversely inhibits synaptic transmission. Restoration of wildtype postsynaptic Alk expression in Alk partial loss-of-function mutants rescues NMJ transmission phenotypes and confirms that postsynaptic Alk regulates NMJ transmission. The effects of impaired Alk signaling on neurotransmission are observed in the absence of associated changes in NMJ structure. Complete removal of Jeb in motor neurons, however, disrupts both presynaptic bouton architecture and postsynaptic differentiation. Non-physiologic activation of Alk signaling also negatively regulates NMJ growth. Activation of Jeb-Alk signaling triggers the Ras-MAP kinase cascade in both pre- and postsynaptic compartments. These novel roles for Jeb-Alk signaling in the modulation of synaptic function and structure have potential implications for recently reported Alk functions in human addiction, retention of spatial memory, cognitive dysfunction in neurofibromatosis and the pathogenesis of amyotrophic lateral sclerosis. PMID:22949158

  3. The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes.

    PubMed Central

    Spradling, A C; Stern, D; Beaton, A; Rhem, E J; Laverty, T; Mozden, N; Misra, S; Rubin, G M

    1999-01-01

    A fundamental goal of genetics and functional genomics is to identify and mutate every gene in model organisms such as Drosophila melanogaster. The Berkeley Drosophila Genome Project (BDGP) gene disruption project generates single P-element insertion strains that each mutate unique genomic open reading frames. Such strains strongly facilitate further genetic and molecular studies of the disrupted loci, but it has remained unclear if P elements can be used to mutate all Drosophila genes. We now report that the primary collection has grown to contain 1045 strains that disrupt more than 25% of the estimated 3600 Drosophila genes that are essential for adult viability. Of these P insertions, 67% have been verified by genetic tests to cause the associated recessive mutant phenotypes, and the validity of most of the remaining lines is predicted on statistical grounds. Sequences flanking >920 insertions have been determined to exactly position them in the genome and to identify 376 potentially affected transcripts from collections of EST sequences. Strains in the BDGP collection are available from the Bloomington Stock Center and have already assisted the research community in characterizing >250 Drosophila genes. The likely identity of 131 additional genes in the collection is reported here. Our results show that Drosophila genes have a wide range of sensitivity to inactivation by P elements, and provide a rationale for greatly expanding the BDGP primary collection based entirely on insertion site sequencing. We predict that this approach can bring >85% of all Drosophila open reading frames under experimental control. PMID:10471706

  4. Contribution of Drosophila TRPA1 to Metabolism.

    PubMed

    Lee, Jung-Eun; Kim, Yunjung; Kim, Kyoung Heon; Lee, Do Yup; Lee, Youngseok

    2016-01-01

    Transient receptor potential (TRP) cation channels are highly conserved in humans and insects. Some of these channels are expressed in internal organs and their functions remain incompletely understood. By direct knock-in of the GAL4 gene into the trpA1 locus in Drosophila, we identified the expression of this gene in the subesophageal ganglion (SOGs) region. In addition, the neurites present in the dorsal posterior region as well as the drosophila insulin-like peptide 2 (dILP2)-positive neurons send signals to the SOGs. The signal is sent to the crop, which is an enlarged organ of the esophagus and functions as a storage place for food in the digestive system. To systematically investigate the role of TRPA1 in metabolism, we applied non-targeted metabolite profiling analysis together with gas-chromatography/time-of-flight mass spectrometry, with an aim to identify a wide range of primary metabolites. We effectively captured distinctive metabolomic phenotypes and identified specific metabolic dysregulation triggered by TRPA1 mutation based on reconstructed metabolic network analysis. Primarily, the network analysis pinpointed the simultaneous down-regulation of intermediates in the methionine salvation pathway, in contrast to the synchronized up-regulation of a range of free fatty acids. The gene dosage-dependent dynamics of metabolite levels among wild-type, hetero- and homozygous mutants, and their coordinated metabolic modulation under multiple gene settings across five different genotypes confirmed the direct linkages of TRPA1 to metabolism. PMID:27055172

  5. Genetic analysis of glutamatergic function in Drosophila

    SciTech Connect

    Chase, B.A.; Kankel, D.R.

    1987-01-01

    Neurotransmitters are essential for communication between neurons and hence are vital in the overall integrative functioning of the nervous system. Previous work on acetylcholine metabolism in the fruit fly, Drosophila melanogaster, has also raised the possibility that transmitter metabolism may play a prominent role in either the achievement or maintenance of the normal structure of the central nervous system in this species. Unfortunately, acetylcholine is rather poorly characterized as a neurotransmitter in Drosophila; consequently, we have begun an analysis of the role of glutamate (probably the best characterized transmitter in this organism) in the formation and/or maintenance of nervous system structure. We present here the results of a series of preliminary analyses. To suggest where glutamatergic function may be localized, an examination of the spatial distribution of high affinity (/sup 3/H)-glutamate binding sites are presented. We present the results of an analysis of the spatial and temporal distribution of enzymatic activities thought to be important in the regulation of transmitter-glutamate pools (i.e., glutamate oxaloacetic transaminase, glutaminase, and glutamate dehydrogenase). To begin to examine whether mutations in any of these functions are capable of affecting glutamatergic activity, we present the results of an initial genetic analysis of one enzymatic function, glutamate oxaloacetic transaminase (GOT), chosen because of its differential distribution within the adult central nervous system and musculature.

  6. Loading Drosophila nerve terminals with calcium indicators.

    PubMed

    Rossano, Adam J; Macleod, Gregory T

    2007-01-01

    Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca(2+)-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca(2+) changes in nerve terminals. However, these indicators have limited sensitivity to Ca(2+) and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca(2+) changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca(2+) indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca(2+) to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca(2+)-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca(2+) dynamics. Dextran-conjugation helps prevent Ca(2+) indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults. PMID:18997898

  7. Localization and possible functions of Drosophila septins.

    PubMed Central

    Fares, H; Peifer, M; Pringle, J R

    1995-01-01

    The septins are a family of homologous proteins that were originally identified in Saccharomyces cerevisiae, where they are associated with the "neck filaments" and are involved in cytokinesis and other aspects of the organization of the cell surface. We report here the identification of Sep1, a Drosophila melanogaster septin, based on its homology to the yeast septins. The predicted Sep1 amino acid sequence is 35-42% identical to the known S. cerevisiae septins; 52% identical to Pnut, a second D. melanogaster septin; and 53-73% identical to the known mammalian septins. Sep1-specific antibodies have been used to characterize its expression and localization. The protein is concentrated at the leading edge of the cleavage furrows of dividing cells and cellularizing embryos, suggesting a role in furrow formation. Other aspects of Sep1 localization suggest roles not directly related to cytokinesis. For example, Sep1 exhibits orderly, cell-cycle-coordinated rearrangements within the cortex of syncytial blastoderm embryos and in the cells of post-gastrulation embryos; Sep1 is also concentrated at the leading edge of the epithelium during dorsal closure in the embryo, in the neurons of the embryonic nervous system, and at the baso-lateral surfaces of ovarian follicle cells. The distribution of Sep1 typically overlaps, but is distinct from, that of actin. Both immunolocalization and biochemical experiments show that Sep1 is intimately associated with Pnut, suggesting that the Drosophila septins, like those in yeast, function as part of a complex. Images PMID:8590810

  8. Recombineering homologous recombination constructs in Drosophila.

    PubMed

    Carreira-Rosario, Arnaldo; Scoggin, Shane; Shalaby, Nevine A; Williams, Nathan David; Hiesinger, P Robin; Buszczak, Michael

    2013-01-01

    The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner. PMID:23893070

  9. Cellular Mechanisms of Drosophila Heart Morphogenesis

    PubMed Central

    Vogler, Georg; Bodmer, Rolf

    2015-01-01

    Many of the major discoveries in the fields of genetics and developmental biology have been made using the fruit fly, Drosophila melanogaster. With regard to heart development, the conserved network of core cardiac transcription factors that underlies cardiogenesis has been studied in great detail in the fly, and the importance of several signaling pathways that regulate heart morphogenesis, such as Slit/Robo, was first shown in the fly model. Recent technological advances have led to a large increase in the genomic data available from patients with congenital heart disease (CHD). This has highlighted a number of candidate genes and gene networks that are potentially involved in CHD. To validate genes and genetic interactions among candidate CHD-causing alleles and to better understand heart formation in general are major tasks. The specific limitations of the various cardiac model systems currently employed (mammalian and fish models) provide a niche for the fly model, despite its evolutionary distance to vertebrates and humans. Here, we review recent advances made using the Drosophila embryo that identify factors relevant for heart formation. These underline how this model organism still is invaluable for a better understanding of CHD. PMID:26236710

  10. Microarray analysis of immune challenged Drosophila hemocytes.

    PubMed

    Johansson, Karin C; Metzendorf, Christoph; Söderhäll, Kenneth

    2005-04-15

    Insect hemocytes play multiple roles in immunity and carry out cellular responses like phagocytosis, encapsulation and melanization as well as producing humoral effector proteins in the first line of defense after injury and invasion of microorganisms. In this work, we used the Drosophila melanogaster hemocyte-like cell line mbn-2 and Affymetrix Drosophila GeneChips to investigate the transcriptome of a single type of immune competent tissue exposed to Gram-negative cell wall components (crude LPS) or high dose infection with live Escherichia coli. We found that gene expression profiles of both treatments overlap but show important differences in expression levels of several genes involved in immunity. In addition, cell morphology during infection was monitored and revealed distinct alterations in cell shape and adhesion. Presence of large numbers of bacteria also increased the number of cells taking on crystal cell fate. Taken together, our results indicate that hemocytes sense and respond differently to purified bacterial surface molecules and infection with live and actively growing bacteria both at the level of gene expression and in cell behavior. PMID:15777795

  11. The Eye Specification Network in Drosophila

    PubMed Central

    WEASNER, BRANDON P.; ANDERSON, JASON; KUMAR, JUSTIN P.

    2010-01-01

    One of the most exciting revelations in retinal biology is the realization that the molecules and mechanisms that regulate eye development have been conserved in all seeing animals including such diverse organisms as the fruit fly, mouse and man. The emerging commonality among mechanisms used in eye development allows for the use of model systems such as the fruit fly, Drosophila melanogaster, to provide key insights into the development and diseases of the mammalian eye. Eye specification in Drosophila is controlled, in part, by the concerted activities of eight nuclear proteins and several signal transduction cascades that together form a tightly woven regulatory network. Loss of function mutations in several components lead to the complete derailment of eye development while ectopic expression of threse genes in non-retinal tissues can direct the fates of these tissues towards eye formation. Here we will describe what is currently known about this remarkable regulatory cassettee highlight some of the outstanding questions that still need to be answered. PMID:25580038

  12. A dynamic deep sleep stage in Drosophila.

    PubMed

    van Alphen, Bart; Yap, Melvyn H W; Kirszenblat, Leonie; Kottler, Benjamin; van Swinderen, Bruno

    2013-04-17

    How might one determine whether simple animals such as flies sleep in stages? Sleep in mammals is a dynamic process involving different stages of sleep intensity, and these are typically associated with measurable changes in brain activity (Blake and Gerard, 1937; Rechtschaffen and Kales, 1968; Webb and Agnew, 1971). Evidence for different sleep stages in invertebrates remains elusive, even though it has been well established that many invertebrate species require sleep (Campbell and Tobler, 1984; Hendricks et al., 2000; Shaw et al., 2000; Sauer et al., 2003). Here we used electrophysiology and arousal-testing paradigms to show that the fruit fly, Drosophila melanogaster, transitions between deeper and lighter sleep within extended bouts of inactivity, with deeper sleep intensities after ∼15 and ∼30 min of inactivity. As in mammals, the timing and intensity of these dynamic sleep processes in flies is homeostatically regulated and modulated by behavioral experience. Two molecules linked to synaptic plasticity regulate the intensity of the first deep sleep stage. Optogenetic upregulation of cyclic adenosine monophosphate during the day increases sleep intensity at night, whereas loss of function of a molecule involved in synaptic pruning, the fragile-X mental retardation protein, increases sleep intensity during the day. Our results show that sleep is not homogenous in insects, and suggest that waking behavior and the associated synaptic plasticity mechanisms determine the timing and intensity of deep sleep stages in Drosophila. PMID:23595750

  13. Selective anticancer agents suppress aging in Drosophila.

    PubMed

    Danilov, Anton; Shaposhnikov, Mikhail; Plyusnina, Ekaterina; Kogan, Valeria; Fedichev, Peter; Moskalev, Alexey

    2013-09-01

    Mutations of the PI3K, TOR, iNOS, and NF-κB genes increase lifespan of model organisms and reduce the risk of some aging-associated diseases. We studied the effects of inhibitors of PI3K (wortmannin), TOR (rapamycin), iNOS (1400W), NF-κB (pyrrolidin dithiocarbamate and QNZ), and the combined effects of inhibitors: PI3K (wortmannin) and TOR (rapamycin), NF-κB (pyrrolidin dithiocarbamates) and PI3K (wortmannin), NF-κB (pyrrolidine dithiocarbamates) and TOR (rapamycin) on Drosophila melanogaster lifespan and quality of life (locomotor activity and fertility). Our data demonstrate that pharmacological inhibition of PI3K, TOR, NF-κB, and iNOS increases lifespan of Drosophila without decreasing quality of life. The greatest lifespan expanding effect was achieved by a combination of rapamycin (5 μM) and wortmannin (5 μM) (by 23.4%). The bioinformatic analysis (KEGG, REACTOME.PATH, DOLite, and GO.BP) showed the greatest aging-suppressor activity of rapamycin, consistent with experimental data. PMID:24096697

  14. Imaging fictive locomotor patterns in larval Drosophila

    PubMed Central

    Bayley, Timothy G.; Taylor, Adam L.; Berni, Jimena; Bate, Michael; Hedwig, Berthold

    2015-01-01

    We have established a preparation in larval Drosophila to monitor fictive locomotion simultaneously across abdominal and thoracic segments of the isolated CNS with genetically encoded Ca2+ indicators. The Ca2+ signals closely followed spiking activity measured electrophysiologically in nerve roots. Three motor patterns are analyzed. Two comprise waves of Ca2+ signals that progress along the longitudinal body axis in a posterior-to-anterior or anterior-to-posterior direction. These waves had statistically indistinguishable intersegmental phase delays compared with segmental contractions during forward and backward crawling behavior, despite being ∼10 times slower. During these waves, motor neurons of the dorsal longitudinal and transverse muscles were active in the same order as the muscle groups are recruited during crawling behavior. A third fictive motor pattern exhibits a left-right asymmetry across segments and bears similarities with turning behavior in intact larvae, occurring equally frequently and involving asymmetry in the same segments. Ablation of the segments in which forward and backward waves of Ca2+ signals were normally initiated did not eliminate production of Ca2+ waves. When the brain and subesophageal ganglion (SOG) were removed, the remaining ganglia retained the ability to produce both forward and backward waves of motor activity, although the speed and frequency of waves changed. Bilateral asymmetry of activity was reduced when the brain was removed and abolished when the SOG was removed. This work paves the way to studying the neural and genetic underpinnings of segmentally coordinated motor pattern generation in Drosophila with imaging techniques. PMID:26311188

  15. Selective anticancer agents suppress aging in Drosophila

    PubMed Central

    Danilov, Anton; Shaposhnikov, Mikhail; Plyusnina, Ekaterina; Kogan, Valeria; Fedichev, Peter; Moskalev, Alexey

    2013-01-01

    Mutations of the PI3K, TOR, iNOS, and NF-κB genes increase lifespan of model organisms and reduce the risk of some aging-associated diseases. We studied the effects of inhibitors of PI3K (wortmannin), TOR (rapamycin), iNOS (1400W), NF-κB (pyrrolidin dithiocarbamate and QNZ), and the combined effects of inhibitors: PI3K (wortmannin) and TOR (rapamycin), NF-κB (pyrrolidin dithiocarbamates) and PI3K (wortmannin), NF-κB (pyrrolidine dithiocarbamates) and TOR (rapamycin) on Drosophila melanogaster lifespan and quality of life (locomotor activity and fertility). Our data demonstrate that pharmacological inhibition of PI3K, TOR, NF-κB, and iNOS increases lifespan of Drosophila without decreasing quality of life. The greatest lifespan expanding effect was achieved by a combination of rapamycin (5 μM) and wortmannin (5 μM) (by 23.4%). The bioinformatic analysis (KEGG, REACTOME.PATH, DOLite, and GO.BP) showed the greatest aging-suppressor activity of rapamycin, consistent with experimental data. PMID:24096697

  16. Contribution of Drosophila TRPA1 to Metabolism

    PubMed Central

    Lee, Jung-Eun; Kim, Yunjung; Kim, Kyoung Heon

    2016-01-01

    Transient receptor potential (TRP) cation channels are highly conserved in humans and insects. Some of these channels are expressed in internal organs and their functions remain incompletely understood. By direct knock-in of the GAL4 gene into the trpA1 locus in Drosophila, we identified the expression of this gene in the subesophageal ganglion (SOGs) region. In addition, the neurites present in the dorsal posterior region as well as the drosophila insulin-like peptide 2 (dILP2)-positive neurons send signals to the SOGs. The signal is sent to the crop, which is an enlarged organ of the esophagus and functions as a storage place for food in the digestive system. To systematically investigate the role of TRPA1 in metabolism, we applied non-targeted metabolite profiling analysis together with gas-chromatography/time-of-flight mass spectrometry, with an aim to identify a wide range of primary metabolites. We effectively captured distinctive metabolomic phenotypes and identified specific metabolic dysregulation triggered by TRPA1 mutation based on reconstructed metabolic network analysis. Primarily, the network analysis pinpointed the simultaneous down-regulation of intermediates in the methionine salvation pathway, in contrast to the synchronized up-regulation of a range of free fatty acids. The gene dosage-dependent dynamics of metabolite levels among wild-type, hetero- and homozygous mutants, and their coordinated metabolic modulation under multiple gene settings across five different genotypes confirmed the direct linkages of TRPA1 to metabolism. PMID:27055172

  17. Peptidoglycan recognition by the Drosophila Imd pathway.

    PubMed

    Kaneko, Takashi; Golenbock, Douglas; Silverman, Neal

    2005-01-01

    The structural requirements for recognition of peptidoglycan (PGN) by PGRP-LC and activation of the Drosophila IMD pathway are not yet clear. In order to examine this question more carefully, the activity of peptidoglycan from different types of bacteria was compared in cell-based and whole animal assays. Drosophila S2* cells, but not adult flies, responded to Lys-type Micrococcus luteus PGN, but with significantly less potency compared to Dap-type Escherichia coli PGN, while intact Lys-type PGN from Staphylococcus aureus was inactive. After treatment with lysostaphin, which digests the cross-bridging peptides, S. aureus PGN weakly stimulated the IMD pathway, similar to M. luteus PGN. Further digestion with mutanolysin, which creates monomeric PGN fragments, abolished the activity of S. aureus PGN. On the other hand, monomeric E. coli PGN, generated by mutanolysin digestion, was still active but required different isoforms of PGRP-LC for recognition. Polymeric PGN required only PGRP-LCx, while monomeric E. coli PGN required both the PGRP-LCa and PGRP-LCx isoforms. These results suggest that the recognition by PGRP-LCx alone requires polymeric PGN, and that polymeric Dap-type PGN is a more potent PGRP-LCx agonist, compared to Lys-type PGN. These results also suggest that the heteromeric PGRP-LCa/LCx receptor complex recognizes monomeric Dap-type, but not Lys-type, PGN. PMID:16303095

  18. Functional Expression of Drosophila para Sodium Channels

    PubMed Central

    Warmke, Jeffrey W.; Reenan, Robert A.G.; Wang, Peiyi; Qian, Su; Arena, Joseph P.; Wang, Jixin; Wunderler, Denise; Liu, Ken; Kaczorowski, Gregory J.; Ploeg, Lex H.T. Van der; Ganetzky, Barry; Cohen, Charles J.

    1997-01-01

    The Drosophila para sodium channel α subunit was expressed in Xenopus oocytes alone and in combination with tipE, a putative Drosophila sodium channel accessory subunit. Coexpression of tipE with para results in elevated levels of sodium currents and accelerated current decay. Para/TipE sodium channels have biophysical and pharmacological properties similar to those of native channels. However, the pharmacology of these channels differs from that of vertebrate sodium channels: (a) toxin II from Anemonia sulcata, which slows inactivation, binds to Para and some mammalian sodium channels with similar affinity (Kd ≅ 10 nM), but this toxin causes a 100-fold greater decrease in the rate of inactivation of Para/TipE than of mammalian channels; (b) Para sodium channels are >10-fold more sensitive to block by tetrodotoxin; and (c) modification by the pyrethroid insecticide permethrin is >100-fold more potent for Para than for rat brain type IIA sodium channels. Our results suggest that the selective toxicity of pyrethroid insecticides is due at least in part to the greater affinity of pyrethroids for insect sodium channels than for mammalian sodium channels. PMID:9236205

  19. Protein interaction mapping: A Drosophila case study

    PubMed Central

    Formstecher, Etienne; Aresta, Sandra; Collura, Vincent; Hamburger, Alexandre; Meil, Alain; Trehin, Alexandra; Reverdy, Céline; Betin, Virginie; Maire, Sophie; Brun, Christine; Jacq, Bernard; Arpin, Monique; Bellaiche, Yohanns; Bellusci, Saverio; Benaroch, Philippe; Bornens, Michel; Chanet, Roland; Chavrier, Philippe; Delattre, Olivier; Doye, Valérie; Fehon, Richard; Faye, Gérard; Galli, Thierry; Girault, Jean-Antoine; Goud, Bruno; de Gunzburg, Jean; Johannes, Ludger; Junier, Marie-Pierre; Mirouse, Vincent; Mukherjee, Ashim; Papadopoulo, Dora; Perez, Franck; Plessis, Anne; Rossé, Carine; Saule, Simon; Stoppa-Lyonnet, Dominique; Vincent, Alain; White, Michael; Legrain, Pierre; Wojcik, Jérôme; Camonis, Jacques; Daviet, Laurent

    2005-01-01

    The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider (http://pim.hybrigenics.com), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps. PMID:15710747

  20. Imaging fictive locomotor patterns in larval Drosophila.

    PubMed

    Pulver, Stefan R; Bayley, Timothy G; Taylor, Adam L; Berni, Jimena; Bate, Michael; Hedwig, Berthold

    2015-11-01

    We have established a preparation in larval Drosophila to monitor fictive locomotion simultaneously across abdominal and thoracic segments of the isolated CNS with genetically encoded Ca(2+) indicators. The Ca(2+) signals closely followed spiking activity measured electrophysiologically in nerve roots. Three motor patterns are analyzed. Two comprise waves of Ca(2+) signals that progress along the longitudinal body axis in a posterior-to-anterior or anterior-to-posterior direction. These waves had statistically indistinguishable intersegmental phase delays compared with segmental contractions during forward and backward crawling behavior, despite being ∼10 times slower. During these waves, motor neurons of the dorsal longitudinal and transverse muscles were active in the same order as the muscle groups are recruited during crawling behavior. A third fictive motor pattern exhibits a left-right asymmetry across segments and bears similarities with turning behavior in intact larvae, occurring equally frequently and involving asymmetry in the same segments. Ablation of the segments in which forward and backward waves of Ca(2+) signals were normally initiated did not eliminate production of Ca(2+) waves. When the brain and subesophageal ganglion (SOG) were removed, the remaining ganglia retained the ability to produce both forward and backward waves of motor activity, although the speed and frequency of waves changed. Bilateral asymmetry of activity was reduced when the brain was removed and abolished when the SOG was removed. This work paves the way to studying the neural and genetic underpinnings of segmentally coordinated motor pattern generation in Drosophila with imaging techniques. PMID:26311188

  1. Thermosensory processing in the Drosophila brain.

    PubMed

    Liu, Wendy W; Mazor, Ofer; Wilson, Rachel I

    2015-03-19

    In Drosophila, just as in vertebrates, changes in external temperature are encoded by bidirectional opponent thermoreceptor cells: some cells are excited by warming and inhibited by cooling, whereas others are excited by cooling and inhibited by warming. The central circuits that process these signals are not understood. In Drosophila, a specific brain region receives input from thermoreceptor cells. Here we show that distinct genetically identified projection neurons (PNs) in this brain region are excited by cooling, warming, or both. The PNs excited by cooling receive mainly feed-forward excitation from cool thermoreceptors. In contrast, the PNs excited by warming ('warm-PNs') receive both excitation from warm thermoreceptors and crossover inhibition from cool thermoreceptors through inhibitory interneurons. Notably, this crossover inhibition elicits warming-evoked excitation, because warming suppresses tonic activity in cool thermoreceptors. This in turn disinhibits warm-PNs and sums with feed-forward excitation evoked by warming. Crossover inhibition could cancel non-thermal activity (noise) that is positively correlated among warm and cool thermoreceptor cells, while reinforcing thermal activity which is anti-correlated. Our results show how central circuits can combine signals from bidirectional opponent neurons to construct sensitive and robust neural codes. PMID:25739502

  2. Magnetoreception Regulates Male Courtship Activity in Drosophila

    PubMed Central

    Wu, Chia-Lin; Fu, Tsai-Feng; Chiang, Meng-Hsuan; Chang, Yu-Wei; Her, Jim-Long; Wu, Tony

    2016-01-01

    The possible neurological and biophysical effects of magnetic fields on animals is an area of active study. Here, we report that courtship activity of male Drosophila increases in a magnetic field and that this effect is regulated by the blue light-dependent photoreceptor cryptochrome (CRY). Naïve male flies exhibited significantly increased courtship activities when they were exposed to a ≥ 20-Gauss static magnetic field, compared with their behavior in the natural environment (0 Gauss). CRY-deficient flies, cryb and crym, did not show an increased courtship index in a magnetic field. RNAi-mediated knockdown of cry in cry-GAL4-positive neurons disrupted the increased male courtship activity in a magnetic field. Genetically expressing cry under the control of cry-GAL4 in the CRY-deficient flies restored the increase in male courtship index that occurred in a magnetic field. Interestingly, artificially activating cry-GAL4-expressing neurons, which include large ventral lateral neurons and small ventral lateral neurons, via expression of thermosensitive cation channel dTrpA1, also increased the male courtship index. This enhancement was abolished by the addition of the cry-GAL80 transgene. Our results highlight the phenomenon of increased male courtship activity caused by a magnetic field through CRY-dependent magnetic sensation in CRY expression neurons in Drosophila. PMID:27195955

  3. Dissection of larval CNS in Drosophila melanogaster.

    PubMed

    Hafer, Nathaniel; Schedl, Paul

    2006-12-01

    The central nervous system (CNS) of Drosophila larvae is complex and poorly understood. One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins. Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity. In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining. In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS. Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS. If the dissection is performed carefully the CNS will remain attached to the cuticle. We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides. We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS. The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise. PMID:18704179

  4. A tripartite synapse model in Drosophila.

    PubMed

    Danjo, Rie; Kawasaki, Fumiko; Ordway, Richard W

    2011-01-01

    Tripartite (three-part) synapses are defined by physical and functional interactions of glia with pre- and post-synaptic elements. Although tripartite synapses are thought to be of widespread importance in neurological health and disease, we are only beginning to develop an understanding of glial contributions to synaptic function. In contrast to studies of neuronal mechanisms, a significant limitation has been the lack of an invertebrate genetic model system in which conserved mechanisms of tripartite synapse function may be examined through large-scale application of forward genetics and genome-wide genetic tools. Here we report a Drosophila tripartite synapse model which exhibits morphological and functional properties similar to those of mammalian synapses, including glial regulation of extracellular glutamate, synaptically-induced glial calcium transients and glial coupling of synapses with tracheal structures mediating gas exchange. In combination with classical and cell-type specific genetic approaches in Drosophila, this model is expected to provide new insights into the molecular and cellular mechanisms of tripartite synapse function. PMID:21359186

  5. Molecular evolution of Drosophila metallothionein genes.

    PubMed

    Lange, B W; Langley, C H; Stephan, W

    1990-12-01

    The metallothionein genes of Drosophila melanogaster, Mtn and Mto, may play an important role in heavy metal detoxification. Several different tandem duplications of Mtn have been shown to increase cadmium and copper tolerance, as well as Mtn expression. In order to investigate the possibility of increased selection for duplications of these genes in natural populations exposed to high levels of heavy metals, we compared the frequencies of such duplications among flies collected from metal-contaminated and non-contaminated orchards in Pennsylvania, Tennessee and Georgia. Restriction enzyme analysis was used to screen 1666 wild third chromosomes for Mtn duplications and a subset (327) of these lines for Mto duplications. The frequency of pooled Mtn duplications found ranged from 0% to 20%, and was not significantly higher at the contaminated sites. No Mto duplications were identified. Estimates of sequence diversity at the Mtn locus among a subsample (92) of the duplication survey were obtained using four-cutter analysis. This analysis revealed a low level of polymorphism, consistent with both selection at the Mtn locus, and a fairly recent origin for the duplications. To further examine this hypothesis, we sequenced an Mtn allele of Drosophila simulans and measured the amount of nucleotide sequence divergence between D. simulans and its sibling species D. melanogaster. The levels of silent nucleotide polymorphism and divergence in the Mtn region were compared with those in the Adh region, using the neutrality test of R.R. Hudson, M. Kreitman and M. Aguadé. PMID:1981765

  6. A Drosophila metallophosphoesterase mediates deglycosylation of rhodopsin

    PubMed Central

    Cao, Jinguo; Li, Yi; Xia, Wenjing; Reddig, Keith; Hu, Wen; Xie, Wei; Li, Hong-Sheng; Han, Junhai

    2011-01-01

    Oligosaccharide chains of newly synthesized membrane receptors are trimmed and modified to optimize their trafficking and/or signalling before delivery to the cell surface. For most membrane receptors, the functional significance of oligosaccharide chain modification is unknown. During the maturation of Rh1 rhodopsin, a Drosophila light receptor, the oligosaccharide chain is trimmed extensively. Neither the functional significance of this modification nor the enzymes mediating this process are known. Here, we identify a dmppe (Drosophila metallophosphoesterase) mutant with incomplete deglycosylation of Rh1, and show that the retained oligosaccharide chain does not affect Rh1 localization or signalling. The incomplete deglycosylation, however, renders Rh1 more sensitive to endocytic degradation, and causes morphological and functional defects in photoreceptors of aged dmppe flies. We further demonstrate that the dMPPE protein functions as an Mn2+/Zn2+-dependent phosphoesterase and mediates in vivo dephosphorylation of α-Man-II. Most importantly, the dephosphorylated α-Man-II is required for the removal of the Rh1 oligosaccharide chain. These observations suggest that the glycosylation status of membrane proteins is controlled through phosphorylation/dephosphorylation, and that MPPE acts as the phosphoesterase in this regulation. PMID:21804530

  7. An investigation of nutrient-dependent mRNA translation in Drosophila larvae.

    PubMed

    Nagarajan, Sabarish; Grewal, Savraj S

    2014-01-01

    The larval period of the Drosophila life cycle is characterized by immense growth. In nutrient rich conditions, larvae increase in mass approximately two hundred-fold in five days. However, upon nutrient deprivation, growth is arrested. The prevailing view is that dietary amino acids drive this larval growth by activating the conserved insulin/PI3 kinase and Target of rapamycin (TOR) pathways and promoting anabolic metabolism. One key anabolic process is protein synthesis. However, few studies have attempted to measure mRNA translation during larval development or examine the signaling requirements for nutrient-dependent regulation. Our work addresses this issue. Using polysome analyses, we observed that starvation rapidly (within thirty minutes) decreased larval mRNA translation, with a maximal decrease at 6-18 hours. By analyzing individual genes, we observed that nutrient-deprivation led to a general reduction in mRNA translation, regardless of any starvation-mediated changes (increase or decrease) in total transcript levels. Although sugars and amino acids are key regulators of translation in animal cells and are the major macronutrients in the larval diet, we found that they alone were not sufficient to maintain mRNA translation in larvae. The insulin/PI3 kinase and TOR pathways are widely proposed as the main link between nutrients and mRNA translation in animal cells. However, we found that genetic activation of PI3K and TOR signaling, or regulation of two effectors - 4EBP and S6K - could not prevent the starvation-mediated translation inhibition. Similarly, we showed that the nutrient stress-activated eIF2α kinases, GCN2 and PERK, were not required for starvation-induced inhibition of translation in larvae. These findings indicate that nutrient control of mRNA translation in larvae is more complex than simply amino acid activation of insulin and TOR signaling. PMID:25305039

  8. An investigation of nutrient-dependent mRNA translation in Drosophila larvae

    PubMed Central

    Nagarajan, Sabarish; Grewal, Savraj S.

    2014-01-01

    ABSTRACT The larval period of the Drosophila life cycle is characterized by immense growth. In nutrient rich conditions, larvae increase in mass approximately two hundred-fold in five days. However, upon nutrient deprivation, growth is arrested. The prevailing view is that dietary amino acids drive this larval growth by activating the conserved insulin/PI3 kinase and Target of rapamycin (TOR) pathways and promoting anabolic metabolism. One key anabolic process is protein synthesis. However, few studies have attempted to measure mRNA translation during larval development or examine the signaling requirements for nutrient-dependent regulation. Our work addresses this issue. Using polysome analyses, we observed that starvation rapidly (within thirty minutes) decreased larval mRNA translation, with a maximal decrease at 6–18 hours. By analyzing individual genes, we observed that nutrient-deprivation led to a general reduction in mRNA translation, regardless of any starvation-mediated changes (increase or decrease) in total transcript levels. Although sugars and amino acids are key regulators of translation in animal cells and are the major macronutrients in the larval diet, we found that they alone were not sufficient to maintain mRNA translation in larvae. The insulin/PI3 kinase and TOR pathways are widely proposed as the main link between nutrients and mRNA translation in animal cells. However, we found that genetic activation of PI3K and TOR signaling, or regulation of two effectors – 4EBP and S6K – could not prevent the starvation-mediated translation inhibition. Similarly, we showed that the nutrient stress-activated eIF2α kinases, GCN2 and PERK, were not required for starvation-induced inhibition of translation in larvae. These findings indicate that nutrient control of mRNA translation in larvae is more complex than simply amino acid activation of insulin and TOR signaling. PMID:25305039

  9. Discovering the first tyrosine kinase

    PubMed Central

    Hunter, Tony

    2015-01-01

    In the middle of the 20th century, animal tumor viruses were heralded as possible models for understanding human cancer. By the mid-1970s, the molecular basis by which tumor viruses transform cells into a malignant state was beginning to emerge as the first viral genomic sequences were reported and the proteins encoded by their transforming genes were identified and characterized. This was a time of great excitement and rapid progress. In 1978, prompted by the discovery from Ray Erikson’s group that the Rous sarcoma virus (RSV) v-Src–transforming protein had an associated protein kinase activity specific for threonine, my group at the Salk Institute set out to determine whether the polyomavirus middle T-transforming protein had a similar kinase activity. Here, I describe the experiments that led to the identification of a kinase activity associated with middle T antigen and our serendipitous discovery that this activity was specific for tyrosine in vitro, and how this in turn led to the fortuitous observation that the v-Src–associated kinase activity was also specific for tyrosine. Our finding that v-Src increased the level of phosphotyrosine in cellular proteins in RSV-transformed cells confirmed that v-Src is a tyrosine kinase and transforms cells by phosphorylating proteins on tyrosine. My colleague Bart Sefton and I reported these findings in the March issue of PNAS in 1980. Remarkably, all of the experiments in this paper were accomplished in less than one month. PMID:26130799

  10. Associations of Yeasts with Spotted-Wing Drosophila (Drosophila suzukii; Diptera: Drosophilidae) in Cherries and Raspberries

    PubMed Central

    Hernández, Alejandro; Zalom, Frank G.

    2012-01-01

    A rich history of investigation documents various Drosophila-yeast mutualisms, suggesting that Drosophila suzukii similarly has an association with a specific yeast species or community. To discover candidate yeast species, yeasts were isolated from larval frass, adult midguts, and fruit hosts of D. suzukii. Terminal restriction fragment length polymorphism (TRFLP) technology and decimal dilution plating were used to identify and determine the relative abundance of yeast species present in fruit juice samples that were either infested with D. suzukii or not infested. Yeasts were less abundant in uninfested than infested samples. A total of 126 independent yeast isolates were cultivated from frass, midguts, and fruit hosts of D. suzukii, representing 28 species of yeasts, with Hanseniaspora uvarum predominating. This suggests an association between D. suzukii and H. uvarum that could be utilized for pest management of the highly pestiferous D. suzukii. PMID:22582060

  11. Associations of yeasts with spotted-wing Drosophila (Drosophila suzukii; Diptera: Drosophilidae) in cherries and raspberries.

    PubMed

    Hamby, Kelly A; Hernández, Alejandro; Boundy-Mills, Kyria; Zalom, Frank G

    2012-07-01

    A rich history of investigation documents various Drosophila-yeast mutualisms, suggesting that Drosophila suzukii similarly has an association with a specific yeast species or community. To discover candidate yeast species, yeasts were isolated from larval frass, adult midguts, and fruit hosts of D. suzukii. Terminal restriction fragment length polymorphism (TRFLP) technology and decimal dilution plating were used to identify and determine the relative abundance of yeast species present in fruit juice samples that were either infested with D. suzukii or not infested. Yeasts were less abundant in uninfested than infested samples. A total of 126 independent yeast isolates were cultivated from frass, midguts, and fruit hosts of D. suzukii, representing 28 species of yeasts, with Hanseniaspora uvarum predominating. This suggests an association between D. suzukii and H. uvarum that could be utilized for pest management of the highly pestiferous D. suzukii. PMID:22582060

  12. Modelling the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase on adenylate kinase.

    PubMed Central

    Bertrand, L; Vertommen, D; Depiereux, E; Hue, L; Rider, M H; Feytmans, E

    1997-01-01

    Simultaneous multiple alignment of available sequences of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealed several segments of conserved residues in the 2-kinase domain. The sequence of the kinase domain was also compared with proteins of known three-dimensional structure. No similarity was found between the kinase domain of 6-phosphofructo-2-kinase and 6-phosphofructo-1-kinase. This questions the modelling of the 2-kinase domain on bacterial 6-phosphofructo-1-kinase that has previously been proposed [Bazan, Fletterick and Pilkis (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646]. However, sequence similarities were found between the 2-kinase domain and several nucleotide-binding proteins, the most similar being adenylate kinase. A structural model of the 2-kinase domain based on adenylate kinase is proposed. It accommodates all the results of site-directed mutagenesis studies carried out to date on residues in the 2-kinase domain. It also allows residues potentially involved in catalysis and/or substrate binding to be predicted. PMID:9032445

  13. Exit from mitosis is regulated by Drosophila fizzy and the sequential destruction of cyclins A, B and B3.

    PubMed Central

    Sigrist, S; Jacobs, H; Stratmann, R; Lehner, C F

    1995-01-01

    While entry into mitosis is triggered by activation of cdc2 kinase, exit from mitosis requires inactivation of this kinase. Inactivation results from proteolytic degradation of the regulatory cyclin subunits during mitosis. At least three different cyclin types, cyclins A, B and B3, associate with cdc2 kinase in higher eukaryotes and are sequentially degraded in mitosis. We show here that mutations in the Drosophila gene fizzy (fzy) block the mitotic degradation of these cyclins. Moreover, expression of mutant cyclins (delta cyclins) lacking the destruction box motif required for mitotic degradation affects mitotic progression at distinct stages. Deltacyclin A results in a delay in metaphase, deltacyclin B in an early anaphase arrest and deltacyclin B3 in a late anaphase arrest, suggesting that mitotic progression beyond metaphase is ordered by the sequential degradation of these different cyclins. Coexpression of deltacyclins A, B and B3 allows a delayed separation of sister chromosomes, but interferes wit chromosome segregation to the poles. Mutations in fzy block both sister chromosome separation and segregation, indicating that fzy plays a crucial role in the metaphase/anaphase transition. Images PMID:7588612

  14. Nemo phosphorylates Eyes absent and enhances output from the Eya-Sine oculis transcriptional complex during Drosophila retinal determination

    PubMed Central

    Morillo, Santiago A.; Braid, Lorena R.; Verheyen, Esther M.; Rebay, Ilaria

    2013-01-01

    The retinal determination gene network comprises a collection of transcription factors that respond to multiple signaling inputs to direct Drosophila eye development. Previous genetic studies have shown that nemo (nmo), a gene encoding a proline-directed serine/threonine kinase, can promote retinal specification through interactions with the retinal determination gene network, although the molecular point of cross-talk was not defined. Here, we report that the Nemo kinase positively and directly regulates Eyes absent (Eya). Genetic assays show that Nmo catalytic activity enhances Eya-mediated ectopic eye formation and potentiates induction of the Eya-Sine oculis (So) transcriptional targets dachshund and lozenge. Biochemical analyses demonstrate that Nmo forms a complex with and phosphorylates Eya at two consensus mitogen-activated protein kinase (MAPK) phosphorylation sites. These same sites appear crucial for Nmo-mediated activation of Eya function in vivo. Thus, we propose that Nmo phosphorylation of Eya potentiates its transactivation function to enhance transcription of Eya-So target genes during eye specification and development. PMID:22394486

  15. The conserved Misshapen-Warts-Yorkie pathway acts in enteroblasts to regulate intestinal stem cells in Drosophila

    PubMed Central

    Li, Qi; Li, Shuangxi; Mana-Capelli, Sebastian; Roth Flach, Rachel J.; Danai, Laura V.; Amcheslavsky, Alla; Nie, Yingchao; Kaneko, Satoshi; Yao, Xiaohao; Chen, Xiaochu; Cotton, Jennifer L.; Mao, Junhao; McCollum, Dannel; Jiang, Jin; Czech, Michael P.; Xu, Lan; Ip, Y. Tony

    2014-01-01

    SUMMARY Similar to the mammalian intestine, the Drosophila adult midgut has resident stem cells that support growth and regeneration. How the niche regulates intestinal stem cell activity in both mammals and flies is not well understood. Here we show that the conserved germinal center protein kinase Misshapen restricts intestinal stem cell division by repressing the expression of the JAK-STAT pathway ligand Upd3 in differentiating enteroblasts. Misshapen, a distant relative to the prototypic Warts activating kinase Hippo, interacts with and activates Warts to negatively regulate the activity of Yorkie and the expression of Upd3. The mammalian Misshapen homolog MAP4K4 similarly interacts with LATS (Warts homolog) and promotes inhibition of YAP (Yorkie homolog). Together, this work reveals that the Misshapen-Warts-Yorkie pathway acts in enteroblasts to control niche signaling to intestinal stem cells. These findings also provide a model in which to study requirements for MAP4K4-related kinases in MST1/2-independent regulation of LATS and YAP. PMID:25453828

  16. Drosophila blood as a model system for stress sensing mechanisms.

    PubMed

    Shim, Jiwon

    2015-04-01

    The Drosophila lymph gland is the hematopoietic organ in which stem-like progenitors proliferate and give rise to myeloid-type blood cells. Mechanisms involved in Drosophila hematopoiesis are well established and known to be conserved in the vertebrate system. Recent studies in Drosophila lymph gland have provided novel insights into how external and internal stresses integrate into blood progenitor maintenance mechanisms and the control of blood cell fate decision. In this review, I will introduce a developmental overview of the Drosophila hematopoietic system, and recent understandings of how the system uses developmental signals not only for hematopoiesis but also as sensors for stress and environmental changes to elicit necessary blood responses. PMID:25560697

  17. FlyRNAi: the Drosophila RNAi screening center database

    PubMed Central

    Flockhart, Ian; Booker, Matthew; Kiger, Amy; Boutros, Michael; Armknecht, Susan; Ramadan, Nadire; Richardson, Kris; Xu, Andrew; Perrimon, Norbert; Mathey-Prevot, Bernard

    2006-01-01

    RNA interference (RNAi) has become a powerful tool for genetic screening in Drosophila. At the Drosophila RNAi Screening Center (DRSC), we are using a library of over 21 000 double-stranded RNAs targeting known and predicted genes in Drosophila. This library is available for the use of visiting scientists wishing to perform full-genome RNAi screens. The data generated from these screens are collected in the DRSC database () in a flexible format for the convenience of the scientist and for archiving data. The long-term goal of this database is to provide annotations for as many of the uncharacterized genes in Drosophila as possible. Data from published screens are available to the public through a highly configurable interface that allows detailed examination of the data and provides access to a number of other databases and bioinformatics tools. PMID:16381918

  18. Functional genomic analysis of the Drosophila immune response.

    PubMed

    Valanne, Susanna

    2014-01-01

    Drosophila melanogaster has been widely used as a model organism for over a century now, and also as an immunological research model for over 20 years. With the emergence of RNA interference (RNAi) in Drosophila as a robust tool to silence genes of interest, large-scale or genome-wide functional analysis has become a popular way of studying the Drosophila immune response in cell culture. Drosophila immunity is composed of cellular and humoral immunity mechanisms, and especially the systemic, humoral response pathways have been extensively dissected using the functional genomic approach. Although most components of the main immune pathways had already been found using traditional genetic screening techniques, important findings including pathway components, positive and negative regulators and modifiers have been made with RNAi screening. Additionally, RNAi screening has produced new information on host-pathogen interactions related to the pathogenesis of many microbial species. PMID:23707784

  19. Insights From Natural Host-Parasite Interactions: The Drosophila Model

    PubMed Central

    Keebaugh, Erin S.; Schlenke, Todd A.

    2013-01-01

    Immune responses against opportunistic pathogens have been extensively studied in Drosophila, leading to a detailed map of the genetics behind innate immunity networks including the Toll, Imd, Jak-Stat, and JNK pathways. However, immune mechanisms of other organisms, particularly plants, have primarily been investigated using natural pathogens. It was the use of natural pathogens in plant research that revealed the plant R/Avr system, a specialized immune response derived from antagonistic coevolution between plant immune proteins and their natural pathogens’ virulence proteins. Thus, we recommend that researchers begin to use natural Drosophila pathogens to identify novel immune mechanisms that may have arisen through antagonistic coevolution with common natural pathogens. In this review, we address the benefits of using natural pathogens in research, describe the known natural pathogens of Drosophila, and discuss exciting prospects for future research on select natural pathogens of Drosophila. PMID:23764256

  20. Drosophila as a Potential Model for Ocular Tumors.

    PubMed

    Bennett, Daimark; Lyulcheva, Ekaterina; Cobbe, Neville

    2015-04-01

    Drosophila has made many contributions to our understanding of cancer genes and mechanisms that have subsequently been validated in mammals. Despite anatomical differences between fly and human eyes, flies offer a tractable genetic model in which to dissect the functional importance of genetic lesions found to be affected in human ocular tumors. Here, we discuss different approaches for using Drosophila as a model for ocular cancer and how studies on ocular cancer genes in flies have begun to reveal potential strategies for therapeutic intervention. We also discuss recent developments in the use of Drosophila for drug discovery, which is coming to the fore as Drosophila models are becoming tailored to study tumor types found in the clinic. PMID:27172095

  1. Functional Gustatory Role of Chemoreceptors in Drosophila Wings.

    PubMed

    Raad, Hussein; Ferveur, Jean-François; Ledger, Neil; Capovilla, Maria; Robichon, Alain

    2016-05-17

    Neuroanatomical evidence argues for the presence of taste sensilla in Drosophila wings; however, the taste physiology of insect wings remains hypothetical, and a comprehensive link to mechanical functions, such as flight, wing flapping, and grooming, is lacking. Our data show that the sensilla of the Drosophila anterior wing margin respond to both sweet and bitter molecules through an increase in cytosolic Ca(2+) levels. Conversely, genetically modified flies presenting a wing-specific reduction in chemosensory cells show severe defects in both wing taste signaling and the exploratory guidance associated with chemodetection. In Drosophila, the chemodetection machinery includes mechanical grooming, which facilitates the contact between tastants and wing chemoreceptors, and the vibrations of flapping wings that nebulize volatile molecules as carboxylic acids. Together, these data demonstrate that the Drosophila wing chemosensory sensilla are a functional taste organ and that they may have a role in the exploration of ecological niches. PMID:27160896

  2. Genotoxic effects of cisplatin in somatic tissue of Drosophila melanogaster

    SciTech Connect

    Katz, A.J.

    1987-01-01

    Third instar larvae of Drosophila melanogaster transdihybrid for mwh and flr were exposed to varying concentrations of cisplatin by feeding on dry media wetted with aqueous solutions of the test compound. Larval feeding continued until pupation, and surviving transdihybrid adults were collected seven days following commencement of feeding. Wings of adults were removed and scored under 400X magnification for the presence of twin spots and single spots comprised of clones of cells possessing malformed wing hairs. Cisplatin was found to induce both twin spots and single spots, and significant linear concentration-response relationships were obtained with respect to the induction of all endpoints. This capacity to induce mitotic exchange in the somatic tissue of Drosophila compares well with the compound's reported ability to induce chromosome breaks in Drosophila germ cells. However, not all compounds possess similar genotoxic profiles in the somatic an germ tissue of Drosophila.

  3. Recombination and the frequency spectrum in Drosophila melanogaster and Drosophila simulans.

    PubMed

    Przeworski, M; Wall, J D; Andolfatto, P

    2001-03-01

    Most "tests of neutrality" assess whether particular data sets depart from the predictions of a standard neutral model with no recombination. For Drosophila, where nuclear polymorphism data routinely show evidence of genetic exchange, the assumption of no recombination is often unrealistic. In addition, while conservative, this assumption is made at the cost of a great loss in power. Perhaps as a result, tests of the frequency spectrum based on zero recombination suggest an adequate fit of Drosophila polymorphism data to the predictions of the standard neutral model. Here, we analyze the frequency spectrum of a large number of loci in Drosophila melanogaster and D. simulans using two summary statistics. We use an estimate of the population recombination rate based on a laboratory estimate of the rate of crossing over per physical length and an estimate of the species' effective population size. In contrast to previous studies, we find that roughly half of the loci depart from the predictions of the standard neutral model. The extent of the departure depends on the exact recombination rate, but the global pattern that emerges is robust. Interestingly, these departures from neutral expectations are not unidirectional. The large variance in outcomes may be due to a complex demographic history and inconsistent sampling, or to the pervasive action of natural selection. PMID:11230530

  4. Wolbachia from Drosophila incompta: just a hitchhiker shared by Drosophila in the New and Old World?

    PubMed

    Wallau, G L; da Rosa, M T; De Ré, F C; Loreto, E L S

    2016-08-01

    Wolbachia are intracellular endosymbionts that infect arthropods and filarial nematodes, occasionally causing a wide variety of modifications in host biology, such as male-killing and cytoplasmic incompatibility (CI), amongst others. This study assembled draft genomes for Wolbachia infecting Drosophila incompta, a species that uses flowers as exclusive breeding and feeding sites, in two distinct Brazilian populations. The absence of four genes involved in CI from this genome, together with literature reports of low frequencies of infected flies in wild populations that contain high mitogenome polymorphism, suggests that this bacterium does not induce CI in D. incompta. Phylogenomic analysis placed Wolbachia infecting D. incompta as closely related to the wMel strain which received such name since it was originally detected in Drosophila melanogaster. In addition, phylogenetic analysis using the Wolbachia surface protein gene and five genes used for multilocus sequence typing of Wolbachia found infecting Drosophila and other arthropod species of Old and New World displayed a complex evolutionary scenario involving recent horizontal transfer bursts in all major clades of Wolbachia pipens belonging to the supergroup A in both geographical regions. PMID:27122079

  5. Loss of Drosophila pheromone reverses its role in sexual communication in Drosophila suzukii

    PubMed Central

    Dekker, Teun; Revadi, Santosh; Mansourian, Suzan; Ramasamy, Sukanya; Lebreton, Sebastien; Becher, Paul G.; Angeli, Sergio; Rota-Stabelli, Omar; Anfora, Gianfranco

    2015-01-01

    The Drosophila pheromone cis-11-octadecenyl acetate (cVA) is used as pheromone throughout the melanogaster group and fulfils a primary role in sexual and social behaviours. Here, we found that Drosophila suzukii, an invasive pest that oviposits in undamaged ripe fruit, does not produce cVA. In fact, its production site, the ejaculatory bulb, is atrophied. Despite loss of cVA production, its receptor, Or67d, and cognate sensillum, T1, which are essential in cVA-mediated behaviours, were fully functional. However, T1 expression was dramatically reduced in D. suzukii, and the corresponding antennal lobe glomerulus, DA1, minute. Behavioural responses to cVA depend on the input balance of Or67d neurons (driving cVA-mediated behaviours) and Or65a neurons (inhibiting cVA-mediated behaviours). Accordingly, the shifted input balance in D. suzukii has reversed cVA's role in sexual behaviour: perfuming D. suzukii males with Drosophila melanogaster equivalents of cVA strongly reduced mating rates. cVA has thus evolved from a generic sex pheromone to a heterospecific signal that disrupts mating in D. suzukii, a saltational shift, mediated through offsetting the input balance that is highly conserved in congeneric species. This study underlines that dramatic changes in a species' sensory preference can result from rather ‘simple’ numerical shifts in underlying neural circuits. PMID:25716789

  6. Anomalies in the expression profile of interspecific hybrids of Drosophila melanogaster and Drosophila simulans.

    PubMed

    Ranz, José M; Namgyal, Kalsang; Gibson, Greg; Hartl, Daniel L

    2004-03-01

    When females of Drosophila melanogaster and males of Drosophila simulans are mated, the male progeny are inviable, whereas the female progeny display manifold malformations and are sterile. These abnormalities result from genetic incompatibilities accumulated since the time the lineages of the species diverged, and may have their origin in aberrant gene transcription. Because compensatory changes within species may obscure differences at the regulatory level in conventional comparisons of the expression profile between species, we have compared the gene-expression profile of hybrid females with those of females of the parental species in order to identify regulatory incompatibilities. In the hybrid females, we find abnormal levels of messenger RNA for a large fraction of the Drosophila transcriptome. These include a gross underexpression of genes preferentially expressed in females, accompanying gonadal atrophy. The hybrid females also show significant overexpression of male-biased genes, which we attribute to incompatibilities in the regulatory mechanisms that normally act to control the expression of these genes in females. The net result of the multiple incompatibilities is that the gene-expression profiles of the parental females are more similar to each other than either is to that of the hybrid. PMID:14962989

  7. Conservation of Olfactory Avoidance in Drosophila Species and Identification of Repellents for Drosophila suzukii

    PubMed Central

    Krause Pham, Christine; Ray, Anandasankar

    2015-01-01

    Flying insects use olfaction to navigate towards fruits in complex odor environments with remarkable accuracy. Some fruits change odor profiles substantially during ripening and related species can prefer different stages. In Drosophila species attractive odorants have been studied extensively, but little is understood about the role of avoidance pathways. In order to examine the role of the avoidance cue CO2 emitted from fruit on behavior of two species with different ripening stage preferences, we investigated the CO2-detection pathway in Drosophila melanogaster and Drosophila suzukii, a harmful pest of fruits. Avoidance to CO2 is not conserved in D. suzukii suggesting a behavioral adaptation that could facilitate attraction to younger fruit with higher CO2 emission levels. We investigated known innate avoidance pathways from five species at different evolutionary distances: D. melanogaster, D. yakuba, D. suzukii, D. pseudoobscura and D. virilis. Surprisingly, only DEET shows strong repellency across all species, whereas CO2, citronellal and ethyl 3-hydroxybutyrate show only limited conservation. These findings guide us to test recently discovered safe DEET substitutes, and we identify one that protects fruits from D. suzukii thus providing a new behavioral strategy for controlling agricultural pests. PMID:26098542

  8. Loss of Drosophila pheromone reverses its role in sexual communication in Drosophila suzukii.

    PubMed

    Dekker, Teun; Revadi, Santosh; Mansourian, Suzan; Ramasamy, Sukanya; Lebreton, Sebastien; Becher, Paul G; Angeli, Sergio; Rota-Stabelli, Omar; Anfora, Gianfranco

    2015-04-01

    The Drosophila pheromone cis-11-octadecenyl acetate (cVA) is used as pheromone throughout the melanogaster group and fulfils a primary role in sexual and social behaviours. Here, we found that Drosophila suzukii, an invasive pest that oviposits in undamaged ripe fruit, does not produce cVA. In fact, its production site, the ejaculatory bulb, is atrophied. Despite loss of cVA production, its receptor, Or67d, and cognate sensillum, T1, which are essential in cVA-mediated behaviours, were fully functional. However, T1 expression was dramatically reduced in D. suzukii, and the corresponding antennal lobe glomerulus, DA1, minute. Behavioural responses to cVA depend on the input balance of Or67d neurons (driving cVA-mediated behaviours) and Or65a neurons (inhibiting cVA-mediated behaviours). Accordingly, the shifted input balance in D. suzukii has reversed cVA's role in sexual behaviour: perfuming D. suzukii males with Drosophila melanogaster equivalents of cVA strongly reduced mating rates. cVA has thus evolved from a generic sex pheromone to a heterospecific signal that disrupts mating in D. suzukii, a saltational shift, mediated through offsetting the input balance that is highly conserved in congeneric species. This study underlines that dramatic changes in a species' sensory preference can result from rather 'simple' numerical shifts in underlying neural circuits. PMID:25716789

  9. Drosophila GRAIL: An intelligent system for gene recognition in Drosophila DNA sequences

    SciTech Connect

    Xu, Ying; Einstein, J.R.; Uberbacher, E.C.; Helt, G.; Rubin, G.

    1995-06-01

    An AI-based system for gene recognition in Drosophila DNA sequences was designed and implemented. The system consists of two main modules, one for coding exon recognition and one for single gene model construction. The exon recognition module finds a coding exon by recognition of its splice junctions (or translation start) and coding potential. The core of this module is a set of neural networks which evaluate an exon candidate for the possibility of being a true coding exon using the ``recognized`` splice junction (or translation start) and coding signals. The recognition process consists of four steps: generation of an exon candidate pool, elimination of improbable candidates using heuristic rules, candidate evaluation by trained neural networks, and candidate cluster resolution and final exon prediction. The gene model construction module takes as input the clustered exon candidates and builds a ``best`` possible single gene model using an efficient dynamic programming algorithm. 129 Drosophila sequences consisting of 441 coding exons including 216358 coding bases were extructed from GenBank and used to build statistical matrices and to train the neural networks. On this training set the system recognized 97% of the coding messages and predicted only 5% false messages. Among the ``correctly`` predicted exons, 68% match the actual exon exactly and 96% have at least one edge predicted correctly. On an independent test set consisting of 30 Drosophila sequences, the system recognized 96% of the coding messages and predicted 7% false messages.

  10. Conservation of Olfactory Avoidance in Drosophila Species and Identification of Repellents for Drosophila suzukii.

    PubMed

    Krause Pham, Christine; Ray, Anandasankar

    2015-01-01

    Flying insects use olfaction to navigate towards fruits in complex odor environments with remarkable accuracy. Some fruits change odor profiles substantially during ripening and related species can prefer different stages. In Drosophila species attractive odorants have been studied extensively, but little is understood about the role of avoidance pathways. In order to examine the role of the avoidance cue CO2 emitted from fruit on behavior of two species with different ripening stage preferences, we investigated the CO2-detection pathway in Drosophila melanogaster and Drosophila suzukii, a harmful pest of fruits. Avoidance to CO2 is not conserved in D. suzukii suggesting a behavioral adaptation that could facilitate attraction to younger fruit with higher CO2 emission levels. We investigated known innate avoidance pathways from five species at different evolutionary distances: D. melanogaster, D. yakuba, D. suzukii, D. pseudoobscura and D. virilis. Surprisingly, only DEET shows strong repellency across all species, whereas CO2, citronellal and ethyl 3-hydroxybutyrate show only limited conservation. These findings guide us to test recently discovered safe DEET substitutes, and we identify one that protects fruits from D. suzukii thus providing a new behavioral strategy for controlling agricultural pests. PMID:26098542

  11. Genomic and karyotypic variation in Drosophila parasitoids (Hymenoptera, Cynipoidea, Figitidae).

    PubMed

    Gokhman, Vladimir E; Johnston, J Spencer; Small, Chiyedza; Rajwani, Roma; Hanrahan, Shawn J; Govind, Shubha

    2011-01-01

    Drosophila melanogaster Meigen, 1830 has served as a model insect for over a century. Sequencing of the 11 additional Drosophila Fallen, 1823 species marks substantial progress in comparative genomics of this genus. By comparison, practically nothing is known about the genome size or genome sequences of parasitic wasps of Drosophila. Here, we present the first comparative analysis of genome size and karyotype structures of Drosophila parasitoids of the Leptopilina Förster, 1869 and Ganaspis Förster, 1869 species. The gametic genome size of Ganaspis xanthopoda (Ashmead, 1896) is larger than those of the three Leptopilina species studied. The genome sizes of all parasitic wasps studied here are also larger than those known for all Drosophila species. Surprisingly, genome sizes of these Drosophila parasitoids exceed the average value known for all previously studied Hymenoptera. The haploid chromosome number of both Leptopilina heterotoma (Thomson, 1862) and Leptopilina victoriae Nordlander, 1980 is ten. A chromosomal fusion appears to have produced a distinct karyotype for Leptopilina boulardi (Barbotin, Carton et Keiner-Pillault, 1979)(n = 9), whose genome size is smaller than that of wasps of the Leptopilina heterotoma clade. Like Leptopilina boulardi, the haploid chromosome number for Ganaspis xanthopoda is also nine. Our studies reveal a positive, but non linear, correlation between the genome size and total chromosome length in Drosophila parasitoids. These Drosophila parasitoids differ widely in their host range, and utilize different infection strategies to overcome host defense. Their comparative genomics, in relation to their exceptionally well-characterized hosts, will prove to be valuable for understanding the molecular basis of the host-parasite arms race and how such mechanisms shape the genetic structures of insectcommunities. PMID:24260630

  12. Suppression of Scant Identifies Endos as a Substrate of Greatwall Kinase and a Negative Regulator of Protein Phosphatase 2A in Mitosis

    PubMed Central

    Rangone, Hélène; Wegel, Eva; Gatt, Melanie K.; Yeung, Eirene; Flowers, Alexander; Debski, Janusz; Dadlez, Michal; Janssens, Veerle; Carpenter, Adelaide T. C.; Glover, David M.

    2011-01-01

    Protein phosphatase 2A (PP2A) plays a major role in dephosphorylating the targets of the major mitotic kinase Cdk1 at mitotic exit, yet how it is regulated in mitotic progression is poorly understood. Here we show that mutations in either the catalytic or regulatory twins/B55 subunit of PP2A act as enhancers of gwlScant, a gain-of-function allele of the Greatwall kinase gene that leads to embryonic lethality in Drosophila when the maternal dosage of the mitotic kinase Polo is reduced. We also show that heterozygous mutant endos alleles suppress heterozygous gwlScant; many more embryos survive. Furthermore, heterozygous PP2A mutations make females heterozygous for the strong mutation polo11 partially sterile, even in the absence of gwlScant. Heterozygosity for an endos mutation suppresses this PP2A/polo11 sterility. Homozygous mutation or knockdown of endos leads to phenotypes suggestive of defects in maintaining the mitotic state. In accord with the genetic interactions shown by the gwlScant dominant mutant, the mitotic defects of Endos knockdown in cultured cells can be suppressed by knockdown of either the catalytic or the Twins/B55 regulatory subunits of PP2A but not by the other three regulatory B subunits of Drosophila PP2A. Greatwall phosphorylates Endos at a single site, Ser68, and this is essential for Endos function. Together these interactions suggest that Greatwall and Endos act to promote the inactivation of PP2A-Twins/B55 in Drosophila. We discuss the involvement of Polo kinase in such a regulatory loop. PMID:21852956

  13. Functional properties of Drosophila inositol trisphosphate receptors.

    PubMed Central

    Swatton, J E; Morris, S A; Wissing, F; Taylor, C W

    2001-01-01

    The functional properties of the only inositol trisphosphate (IP(3)) receptor subtype expressed in Drosophila were examined in permeabilized S2 cells. The IP(3) receptors of S2 cells bound (1,4,5)IP(3) with high affinity (K(d)=8.5+/-1.1 nM), mediated positively co-operative Ca(2+) release from a thapsigargin-sensitive Ca(2+) store (EC(50)=75+/-4 nM, Hill coefficient=2.1+/-0.2), and they were recognized by an antiserum to a peptide conserved in all IP(3) receptor subtypes in the same way as mammalian IP(3) receptors. As with mammalian IP(3) receptors, (2,4,5)IP(3) (EC(50)=2.3+/-0.3 microM) and (4,5)IP(2) (EC(50) approx. 10 microM) were approx. 20- and 100-fold less potent than (1,4,5)IP(3). Adenophostin A, which is typically approx. 10-fold more potent than IP(3) at mammalian IP(3) receptors, was 46-fold more potent than IP(3) in S2 cells (EC(50)=1.67+/-0.07 nM). Responses to submaximal concentrations of IP(3) were quantal and IP(3)-evoked Ca(2+) release was biphasically regulated by cytosolic Ca(2+). Using rapid superfusion to examine the kinetics of IP(3)-evoked Ca(2+) release from S2 cells, we established that IP(3) (10 microM) maximally activated Drosophila IP(3) receptors within 400 ms. The activity of the receptors then slowly decayed (t(1/2)=2.03+/-0.07 s) to a stable state which had 47+/-1% of the activity of the maximally active state. We conclude that the single subtype of IP(3) receptor expressed in Drosophila has similar functional properties to mammalian IP(3) receptors and that analyses of IP(3) receptor function in this genetically tractable organism are therefore likely to contribute to understanding the roles of mammalian IP(3) receptors. PMID:11583592

  14. Drosophila larvae: Thermal ecology in changing environments

    NASA Astrophysics Data System (ADS)

    Wang, George

    Temperature affects almost all aspects of life. Although much work has been done to assess the impact of temperature on organismal performance, relatively little is known about how organisms behaviorally regulate temperature, how these behaviors effect population fitness, or how changing climate may interact with these behaviors. I explore these questions with the model system Drosophila larvae. Larvae are small, with a low thermal mass and limited capacity for physiological thermoregulation. Mortality is generally high in larvae, with large potential impacts on population growth rate. Thus behavioral thermoregulation in larvae should be of critical selective importance. I present a review of the current knowledge of Drosophila thermal preference. I describe quantifiable thermoregulatory behaviors ( TMV and TW) unique to larvae. I show interspecific variation of these behaviors in Drosophila melanogaster and several close relatives, and intraspecific variation between populations collected from different environments. I also investigate these behaviors in two mutant lines, ssa and biz, to investigate the genetic basis of these behaviors. I show that larval thermoregulatory systems are independent of those of adults. Further these thermoregulatory behaviors differ between two sister species, D. yakuba and D. santomea. Although these two species readily hybridize in laboratory conditions, very few hybrids are observed in the field. The surprising result that hybrids of D. yakuba and D. santomea seem to inherit TMV from D. yakuba suggests a novel extrinsic isolation mechanism between the two species. I explore how fitness is the result of the interaction between genetics and the environment. I utilize Monte Carlo simulation to show how non-linear norms of reaction generate variation in populations even in the absence of behavior or epigenetic evolutionary mechanisms. Finally I investigate the global distribution of temperatures in which these organisms exist using

  15. BOD1 Is Required for Cognitive Function in Humans and Drosophila.

    PubMed

    Esmaeeli-Nieh, Sahar; Fenckova, Michaela; Porter, Iain M; Motazacker, M Mahdi; Nijhof, Bonnie; Castells-Nobau, Anna; Asztalos, Zoltan; Weißmann, Robert; Behjati, Farkhondeh; Tzschach, Andreas; Felbor, Ute; Scherthan, Harry; Sayfati, Seyed Morteza; Ropers, H Hilger; Kahrizi, Kimia; Najmabadi, Hossein; Swedlow, Jason R; Schenck, Annette; Kuss, Andreas W

    2016-05-01

    Here we report a stop-mutation in the BOD1 (Biorientation Defective 1) gene, which co-segregates with intellectual disability in a large consanguineous family, where individuals that are homozygous for the mutation have no detectable BOD1 mRNA or protein. The BOD1 protein is required for proper chromosome segregation, regulating phosphorylation of PLK1 substrates by modulating Protein Phosphatase 2A (PP2A) activity during mitosis. We report that fibroblast cell lines derived from homozygous BOD1 mutation carriers show aberrant localisation of the cell cycle kinase PLK1 and its phosphatase PP2A at mitotic kinetochores. However, in contrast to the mitotic arrest observed in BOD1-siRNA treated HeLa cells, patient-derived cells progressed through mitosis with no apparent segregation defects but at an accelerated rate compared to controls. The relatively normal cell cycle progression observed in cultured cells is in line with the absence of gross structural brain abnormalities in the affected individuals. Moreover, we found that in normal adult brain tissues BOD1 expression is maintained at considerable levels, in contrast to PLK1 expression, and provide evidence for synaptic localization of Bod1 in murine neurons. These observations suggest that BOD1 plays a cell cycle-independent role in the nervous system. To address this possibility, we established two Drosophila models, where neuron-specific knockdown of BOD1 caused pronounced learning deficits and significant abnormalities in synapse morphology. Together our results reveal novel postmitotic functions of BOD1 as well as pathogenic mechanisms that strongly support a causative role of BOD1 deficiency in the aetiology of intellectual disability. Moreover, by demonstrating its requirement for cognitive function in humans and Drosophila we provide evidence for a conserved role of BOD1 in the development and maintenance of cognitive features. PMID:27166630

  16. TRP, TRPL and Cacophony Channels Mediate Ca2+ Influx and Exocytosis in Photoreceptors Axons in Drosophila

    PubMed Central

    Astorga, Guadalupe; Härtel, Steffen; Sanhueza, Magdalena; Bacigalupo, Juan

    2012-01-01

    In Drosophila photoreceptors Ca2+-permeable channels TRP and TRPL are the targets of phototransduction, occurring in photosensitive microvilli and mediated by a phospholipase C (PLC) pathway. Using a novel Drosophila brain slice preparation, we studied the distribution and physiological properties of TRP and TRPL in the lamina of the visual system. Immunohistochemical images revealed considerable expression in photoreceptors axons at the lamina. Other phototransduction proteins are also present, mainly PLC and protein kinase C, while rhodopsin is absent. The voltage-dependent Ca2+ channel cacophony is also present there. Measurements in the lamina with the Ca2+ fluorescent protein G-CaMP ectopically expressed in photoreceptors, revealed depolarization-induced Ca2+ increments mediated by cacophony. Additional Ca2+ influx depends on TRP and TRPL, apparently functioning as store-operated channels. Single synaptic boutons resolved in the lamina by FM4-64 fluorescence revealed that vesicle exocytosis depends on cacophony, TRP and TRPL. In the PLC mutant norpA bouton labeling was also impaired, implicating an additional modulation by this enzyme. Internal Ca2+ also contributes to exocytosis, since this process was reduced after Ca2+-store depletion. Therefore, several Ca2+ pathways participate in photoreceptor neurotransmitter release: one is activated by depolarization and involves cacophony; this is complemented by internal Ca2+ release and the activation of TRP and TRPL coupled to Ca2+ depletion of internal reservoirs. PLC may regulate the last two processes. TRP and TRPL would participate in two different functions in distant cellular regions, where they are opened by different mechanisms. This work sheds new light on the mechanism of neurotransmitter release in tonic synapses of non-spiking neurons. PMID:22952921

  17. A Systems-Level Interrogation Identifies Regulators of Drosophila Blood Cell Number and Survival

    PubMed Central

    Makhijani, Kalpana; Alexander, Brandy; Perrimon, Norbert; Brückner, Katja

    2015-01-01

    In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems. PMID:25749252

  18. Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila.

    PubMed

    Sousa-Nunes, Rita; Yee, Lih Ling; Gould, Alex P

    2011-03-24

    Many stem, progenitor and cancer cells undergo periods of mitotic quiescence from which they can be reactivated. The signals triggering entry into and exit from this reversible dormant state are not well understood. In the developing Drosophila central nervous system, multipotent self-renewing progenitors called neuroblasts undergo quiescence in a stereotypical spatiotemporal pattern. Entry into quiescence is regulated by Hox proteins and an internal neuroblast timer. Exit from quiescence (reactivation) is subject to a nutritional checkpoint requiring dietary amino acids. Organ co-cultures also implicate an unidentified signal from an adipose/hepatic-like tissue called the fat body. Here we provide in vivo evidence that Slimfast amino-acid sensing and Target of rapamycin (TOR) signalling activate a fat-body-derived signal (FDS) required for neuroblast reactivation. Downstream of this signal, Insulin-like receptor signalling and the Phosphatidylinositol 3-kinase (PI3K)/TOR network are required in neuroblasts for exit from quiescence. We demonstrate that nutritionally regulated glial cells provide the source of Insulin-like peptides (ILPs) relevant for timely neuroblast reactivation but not for overall larval growth. Conversely, ILPs secreted into the haemolymph by median neurosecretory cells systemically control organismal size but do not reactivate neuroblasts. Drosophila thus contains two segregated ILP pools, one regulating proliferation within the central nervous system and the other controlling tissue growth systemically. Our findings support a model in which amino acids trigger the cell cycle re-entry of neural progenitors via a fat-body-glia-neuroblasts relay. This mechanism indicates that dietary nutrients and remote organs, as well as local niches, are key regulators of transitions in stem-cell behaviour. PMID:21346761

  19. BOD1 Is Required for Cognitive Function in Humans and Drosophila

    PubMed Central

    Motazacker, M. Mahdi; Nijhof, Bonnie; Castells-Nobau, Anna; Asztalos, Zoltan; Weißmann, Robert; Behjati, Farkhondeh; Tzschach, Andreas; Felbor, Ute; Scherthan, Harry; Sayfati, Seyed Morteza; Ropers, H. Hilger.; Kahrizi, Kimia; Najmabadi, Hossein; Swedlow, Jason R.; Schenck, Annette; Kuss, Andreas W.

    2016-01-01

    Here we report a stop-mutation in the BOD1 (Biorientation Defective 1) gene, which co-segregates with intellectual disability in a large consanguineous family, where individuals that are homozygous for the mutation have no detectable BOD1 mRNA or protein. The BOD1 protein is required for proper chromosome segregation, regulating phosphorylation of PLK1 substrates by modulating Protein Phosphatase 2A (PP2A) activity during mitosis. We report that fibroblast cell lines derived from homozygous BOD1 mutation carriers show aberrant localisation of the cell cycle kinase PLK1 and its phosphatase PP2A at mitotic kinetochores. However, in contrast to the mitotic arrest observed in BOD1-siRNA treated HeLa cells, patient-derived cells progressed through mitosis with no apparent segregation defects but at an accelerated rate compared to controls. The relatively normal cell cycle progression observed in cultured cells is in line with the absence of gross structural brain abnormalities in the affected individuals. Moreover, we found that in normal adult brain tissues BOD1 expression is maintained at considerable levels, in contrast to PLK1 expression, and provide evidence for synaptic localization of Bod1 in murine neurons. These observations suggest that BOD1 plays a cell cycle-independent role in the nervous system. To address this possibility, we established two Drosophila models, where neuron-specific knockdown of BOD1 caused pronounced learning deficits and significant abnormalities in synapse morphology. Together our results reveal novel postmitotic functions of BOD1 as well as pathogenic mechanisms that strongly support a causative role of BOD1 deficiency in the aetiology of intellectual disability. Moreover, by demonstrating its requirement for cognitive function in humans and Drosophila we provide evidence for a conserved role of BOD1 in the development and maintenance of cognitive features. PMID:27166630

  20. Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila

    PubMed Central

    Sen, Aditya; Kalvakuri, Sreehari; Bodmer, Rolf; Cox, Rachel T.

    2015-01-01

    ABSTRACT Loss of mitochondrial function often leads to neurodegeneration and is thought to be one of the underlying causes of neurodegenerative diseases such as Parkinson's disease (PD). However, the precise events linking mitochondrial dysfunction to neuronal death remain elusive. PTEN-induced putative kinase 1 (PINK1) and Parkin (Park), either of which, when mutated, are responsible for early-onset PD, mark individual mitochondria for destruction at the mitochondrial outer membrane. The specific molecular pathways that regulate signaling between the nucleus and mitochondria to sense mitochondrial dysfunction under normal physiological conditions are not well understood. Here, we show that Drosophila Clueless (Clu), a highly conserved protein required for normal mitochondrial function, can associate with Translocase of the outer membrane (TOM) 20, Porin and PINK1, and is thus located at the mitochondrial outer membrane. Previously, we found that clu genetically interacts with park in Drosophila female germ cells. Here, we show that clu also genetically interacts with PINK1, and our epistasis analysis places clu downstream of PINK1 and upstream of park. In addition, Clu forms a complex with PINK1 and Park, further supporting that Clu links mitochondrial function with the PINK1-Park pathway. Lack of Clu causes PINK1 and Park to interact with each other, and clu mutants have decreased mitochondrial protein levels, suggesting that Clu can act as a negative regulator of the PINK1-Park pathway. Taken together, these results suggest that Clu directly modulates mitochondrial function, and that Clu's function contributes to the PINK1-Park pathway of mitochondrial quality control. PMID:26035866