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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. Tanshinone IIA enhances bystander cell killing of cancer cells expressing Drosophila melanogaster deoxyribonucleoside kinase in nuclei and mitochondria.

    PubMed

    Jiang, Haiyang; Zhao, Lei; Dong, Xiaoshen; He, Anning; Zheng, Caiwei; Johansson, Magnus; Karlsson, Anna; Zheng, Xinyu

    2015-09-01

    Heterologous expression of the Drosophila melanogaster multi-substrate deoxyribonucleoside kinase (Dm-dNK) increases the sensitivity of cancer cells to several cytotoxic nucleoside analogs. Thus, it may be used as a suicide gene in combined gene/chemotherapy treatment of cancer. To further characterize this potential suicide gene, we constructed two retroviral vectors that enabled the expression of Dm-dNK in cancer cells. One vector harbored the wild‑type enzyme that localized to the nucleus. The other vector harbored a mitochondrial localized mutant enzyme that was constructed by deleting the nuclear localization signal and fusing it to a mitochondrial import signal of cytochrome c oxidase. A thymidine kinase-deficient osteosarcoma cell line was transduced with the recombinant viruses. The sensitivity and bystander cell killing in the presence of pyrimidine nucleoside analogs (E)-5-(2-bromovinyl)‑2'‑deoxyuridine and 1-β-D-arabinofuranosylthymine were investigated. Tanshinone IIA is a constituent of Danshen; a traditional Chinese medicine used in the treatment of cardiovascular diseases. This study also looked at the influence of Tanshinone IIA on the bystander effect and the underlying mechanisms. We showed that sensitivity of the osteosarcoma cell line to the nucleoside analogs and the efficiency of bystander cell killing were independent of the subcellular localization of Dm-dNK. The enhanced effect of tanshinone IIA on the bystander effect was related to the increased expression of Cx43 and Cx26.

  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. Dominant Mutations of Drosophila Map Kinase Kinase and Their Activities in Drosophila and Yeast Map Kinase Cascades

    PubMed Central

    Lim, Y. M.; Tsuda, L.; Inoue, Y. H.; Irie, K.; Adachi-Yamada, T.; Hata, M.; Nishi, Y.; Matsumoto, K.; Nishida, Y.

    1997-01-01

    Eight alleles of Dsor1 encoding a Drosophila homologue of mitogen-activated protein (MAP) kinase kinase were obtained as dominant suppressors of the MAP kinase kinase kinase D-raf. These Dsor1 alleles themselves showed no obvious phenotypic consequences nor any effect on the viability of the flies, although they were highly sensitive to upstream signals and strongly interacted with gain-of-function mutations of upstream factors. They suppressed mutations for receptor tyrosine kinases (RTKs); torso (tor), sevenless (sev) and to a lesser extent Drosophila EGF receptor (DER). Furthermore, the Dsor1 alleles showed no significant interaction with gain-of-function mutations of DER. The observed difference in activity of the Dsor1 alleles among the RTK pathways suggests Dsor1 is one of the components of the pathway that regulates signal specificity. Expression of Dsor1 in budding yeast demonstrated that Dsor1 can activate yeast MAP kinase homologues if a proper activator of Dsor1 is coexpressed. Nucleotide sequencing of the Dsor1 mutant genes revealed that most of the mutations are associated with amino acid changes at highly conserved residues in the kinase domain. The results suggest that they function as suppressors due to increased reactivity to upstream factors. PMID:9136016

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

    PubMed

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

    2008-09-01

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

  6. Structure of the kinase domain of Gilgamesh from Drosophila melanogaster.

    PubMed

    Han, Ni; Chen, CuiCui; Shi, Zhubing; Cheng, Dianlin

    2014-04-01

    The CK1 family kinases regulate multiple cellular aspects and play important roles in Wnt/Wingless and Hedgehog signalling. The kinase domain of Drosophila Gilgamesh isoform I (Gilgamesh-I), a homologue of human CK1-γ, was purified and crystallized. Crystals of methylated Gilgamesh-I kinase domain with a D210A mutation diffracted to 2.85 Å resolution and belonged to space group P43212, with unit-cell parameters a = b = 52.025, c = 291.727 Å. The structure of Gilgamesh-I kinase domain, which was determined by molecular replacement, has conserved catalytic elements and an active conformation. Structural comparison indicates that an extended loop between the α1 helix and the β4 strand exists in the Gilgamesh-I kinase domain. This extended loop may regulate the activity and function of Gilgamesh-I.

  7. Structure of the kinase domain of Gilgamesh from Drosophila melanogaster

    PubMed Central

    Han, Ni; Chen, CuiCui; Shi, Zhubing; Cheng, Dianlin

    2014-01-01

    The CK1 family kinases regulate multiple cellular aspects and play important roles in Wnt/Wingless and Hedgehog signalling. The kinase domain of Drosophila Gilgamesh isoform I (Gilgamesh-I), a homologue of human CK1-γ, was purified and crystallized. Crystals of methylated Gilgamesh-I kinase domain with a D210A mutation diffracted to 2.85 Å resolution and belonged to space group P43212, with unit-cell parameters a = b = 52.025, c = 291.727 Å. The structure of Gilgamesh-I kinase domain, which was determined by molecular replacement, has conserved catalytic elements and an active conformation. Structural comparison indicates that an extended loop between the α1 helix and the β4 strand exists in the Gilgamesh-I kinase domain. This extended loop may regulate the activity and function of Gilgamesh-I. PMID:24699734

  8. Biological phosphorylation of an Unnatural Base Pair (UBP) using a Drosophila melanogaster deoxynucleoside kinase (DmdNK) mutant

    PubMed Central

    Daugherty, Ashley B.; Yang, Zunyi; Shaw, Ryan; Dong, Mengxing; Lutz, Stefan; Benner, Steven A.

    2017-01-01

    One research goal for unnatural base pair (UBP) is to replicate, transcribe and translate them in vivo. Accordingly, the corresponding unnatural nucleoside triphosphates must be available at sufficient concentrations within the cell. To achieve this goal, the unnatural nucleoside analogues must be phosphorylated to the corresponding nucleoside triphosphates by a cascade of three kinases. The first step is the monophosphorylation of unnatural deoxynucleoside catalyzed by deoxynucleoside kinases (dNK), which is generally considered the rate limiting step because of the high specificity of dNKs. Here, we applied a Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) to the phosphorylation of an UBP (a pyrimidine analogue (6-amino-5-nitro-3-(1’-b-d-2’-deoxyribofuranosyl)-2(1H)-pyridone, Z) and its complementary purine analogue (2-amino-8-(1’-b-d-2’-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, P). The results showed that DmdNK could efficiently phosphorylate only the dP nucleoside. To improve the catalytic efficiency, a DmdNK-Q81E mutant was created based on rational design and structural analyses. This mutant could efficiently phosphorylate both dZ and dP nucleoside. Structural modeling indicated that the increased efficiency of dZ phosphorylation by the DmdNK-Q81E mutant might be related to the three additional hydrogen bonds formed between E81 and the dZ base. Overall, this study provides a groundwork for the biological phosphorylation and synthesis of unnatural base pair in vivo. PMID:28323896

  9. Conserved family of glycerol kinase loci in Drosophila melanogaster

    PubMed Central

    Martinez Agosto, Julian A.; McCabe, Edward R.B.

    2009-01-01

    Glycerol kinase (GK) is an enzyme that catalyzes the formation of glycerol 3-phosphate from ATP and glycerol, the rate-limiting step in glycerol utilization. We analyzed the genome of the model organism Drosophila melanogaster and identified five GK orthologs, including two loci with sequence homology to the mammalian Xp21 GK protein. Using a combination of sequence analysis and evolutionary comparisons of orthologs between species, we characterized functional domains in the protein required for GK activity. Our findings include additional conserved domains that suggest novel nuclear and mitochondrial functions for glycerol kinase in apoptosis and transcriptional regulation. Investigation of GK function in Drosophila will inform us about the role of this enzyme in development and will provide us with a tool to examine genetic modifiers of human metabolic disorders. PMID:16545593

  10. Pantothenate kinase-associated neurodegeneration: insights from a Drosophila model.

    PubMed

    Wu, Zhihao; Li, Chenghua; Lv, Shan; Zhou, Bing

    2009-10-01

    Pantothenate-Kinase-Associated-Neurodegeneration (PKAN) is a devastating disease, resulting from mutations in pantothenate kinase 2 (PANK2), one of the four human pantothenate kinase genes (PANK1-4). Interestingly, PanK2 appears to be the only mitochondria-targeted human PanK. It is unknown whether the mitochondria-targeted PanK is associated with any unique function, nor whether PKAN is due solely to the loss of pantothenate kinase activity. Drosophila PANK [fumble (fbl)] encodes several isoforms of pantothenate kinase products, one of which localizes to mitochondria and the others cytosol. fbl flies exhibit many characteristic features reminiscent of PKAN patients. Various forms of Drosophila fbl and human PANK2 were introduced into fbl flies to study their in vivo functions. Only mitochondria-targeted Fbl or human PanK2 was able to rescue fbl mutation, with the rescuing ability sensitive to the expression level of the transgene. Transgenic lines with low expression of normal Fbl or PanK2 displayed similar phenotypes as PANK2 mutant transgenic flies. These PanK2 mutants all showed reduced and phenotype severity-correlated in vitro pantothenate kinase activities. Amazingly, cytosolic PanK3 and PanK4 could mostly, but not fully, rescue fbl defects except the male sterility. Therefore, fbl appears to be the orthologue of human PANK2, and PanK2 is functionally more potent than PanK3 and PanK4 in vivo. We suggest that mitochondria-located pantothenate kinase is required to achieve the maximal enzymatic activity to fulfill the most challenging task such as maintaining male fertility and optimal neuronal functions, and PKAN features are mainly due to the reduction of the total cellular pantothenate kinase activity in the most susceptible regions.

  11. Ack kinase regulates CTP synthase filaments during Drosophila oogenesis.

    PubMed

    Strochlic, Todd I; Stavrides, Kevin P; Thomas, Sam V; Nicolas, Emmanuelle; O'Reilly, Alana M; Peterson, Jeffrey R

    2014-11-01

    The enzyme CTP synthase (CTPS) dynamically assembles into macromolecular filaments in bacteria, yeast, Drosophila, and mammalian cells, but the role of this morphological reorganization in regulating CTPS activity is controversial. During Drosophila oogenesis, CTPS filaments are transiently apparent in ovarian germline cells during a period of intense genomic endoreplication and stockpiling of ribosomal RNA. Here, we demonstrate that CTPS filaments are catalytically active and that their assembly is regulated by the non-receptor tyrosine kinase DAck, the Drosophila homologue of mammalian Ack1 (activated cdc42-associated kinase 1), which we find also localizes to CTPS filaments. Egg chambers from flies deficient in DAck or lacking DAck catalytic activity exhibit disrupted CTPS filament architecture and morphological defects that correlate with reduced fertility. Furthermore, ovaries from these flies exhibit reduced levels of total RNA, suggesting that DAck may regulate CTP synthase activity. These findings highlight an unexpected function for DAck and provide insight into a novel pathway for the developmental control of an essential metabolic pathway governing nucleotide biosynthesis.

  12. Roles of receptor tyrosine kinases in Drosophila development.

    PubMed

    Shilo, B Z

    1992-08-01

    Communication between cells is a fundamental component of development and morphogenesis. Identification of the molecules mediating cell-cell communication is crucial for elucidation of the molecular basis of these processes. Receptor tyrosine kinases (RTKs) appear to play a central role in this context by transmitting into cells information dictating their fate. The functions of RTKs in Drosophila are extremely diverse, and include maternal determination of embryonic polarity (torso and torpedo), determination of neuroblast identity (faint little ball), and guidance of tracheal cell migration in the embryo (breathless). During compound eye development, RTKs affect the number of photoreceptor clusters (Ellipse) and the determination of photoreceptor R7 identity (sevenless). The phenotypes of mutations in RTK loci serve as a starting point for understanding processes dictating cell identity at the level of the whole organism. Recently, they have also begun to provide a basis for selection of second-site suppressor mutations, encoding additional elements in their signal transduction pathway. Common themes between the functions, regulation, and signal transduction pathways of Drosophila RTKs are drawn.

  13. 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.

  14. The Drosophila rolled locus encodes a MAP kinase required in the sevenless signal transduction pathway.

    PubMed Central

    Biggs, W H; Zavitz, K H; Dickson, B; van der Straten, A; Brunner, D; Hafen, E; Zipursky, S L

    1994-01-01

    Mitogen-activated protein (MAP) kinases have been proposed to play a critical role in receptor tyrosine kinase (RTK)-mediated signal transduction pathways. Although genetic and biochemical studies of RTK pathways in Caenorhabditis elegans, Drosophila melanogaster and mammals have revealed remarkable similarities, a genetic requirement for MAP kinases in RTK signaling has not been established. During retinal development in Drosophila, the sevenless (Sev) RTK is required for development of the R7 photoreceptor cell. Components of the signal transduction pathway activated by Sev in the R7 precursor include proteins encoded by the gap1, drk, Sos, ras1 and raf loci. In this report we present evidence that a Drosophila MAP kinase, ERK-A, is encoded by the rolled locus and is required downstream of raf in the Sev signal transduction pathway. Images PMID:8157002

  15. 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

  16. Transgene expression of Drosophila melanogaster nucleoside kinase reverses mitochondrial thymidine kinase 2 deficiency.

    PubMed

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

    2013-02-15

    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.

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

    PubMed Central

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

    1991-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  19. 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.

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

    PubMed

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

    2007-06-25

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

  1. 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.

  2. 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.

  3. Mutation in the Jak kinase JH2 domain hyperactivates Drosophila and mammalian Jak-Stat pathways.

    PubMed Central

    Luo, H; Rose, P; Barber, D; Hanratty, W P; Lee, S; Roberts, T M; D'Andrea, A D; Dearolf, C R

    1997-01-01

    The Jak (Janus) family of nonreceptor tyrosine kinases plays a critical role in cytokine signal transduction pathways. In Drosophila melanogaster, the dominant hop(Tum-l) mutation in the Hop Jak kinase causes leukemia-like and other developmental defects. Previous studies have suggested that the Hop(Tum-l) protein might be a hyperactive kinase. Here, we report on the new dominant mutation hop(T42), which causes abnormalities that are similar to but more extreme than those caused by hop(Tum-l). We determined that Hop(T42) contains a glutamic acid-to-lysine substitution at amino acid residue 695 (E695K). This residue occurs in the JH2 (kinase-like) domain and is conserved among all Jak family members. We determined that Hop(Tum-1) and Hop(T42) both hyperphosphorylated and hyperactivated D-Stat when overexpressed in Drosophila cells. Moreover, we found that the hop(T42) phenotype was partially rescued by a reduction of wild-type D-stat activity. Finally, generation of the corresponding E695K mutation in murine Jak2 resulted in increased autophosphorylation and increased activation of Stat5 in COS cells. These results demonstrate that the mutant Hop proteins do indeed have increased tyrosine kinase activity, that the mutations hyperactivate the Hop-D-Stat pathway, and that Drosophila is a relevant system for the functional dissection of mammalian Jak-Stat pathways. Finally, we propose a model for the role of the Hop-D-Stat pathway in Drosophila hematopoiesis. PMID:9032284

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

    PubMed

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

    2012-01-01

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

  5. Functions and Mechanisms of Receptor Tyrosine Kinase Torso Signaling: Lessons From Drosophila Embryonic Terminal Development

    PubMed Central

    Li, Willis X.

    2011-01-01

    The Torso receptor tyrosine kinase (RTK) is required for cell fate specification in the terminal regions (head and tail) of the early Drosophila embryo. Torso contains a split tyrosine kinase domain and belongs to the type III subgroup of the RTK superfamily that also includes the platelet-derived growth factor receptors, stem cell or steel factor receptor c-Kit proto-oncoprotein, colony-stimulating factor-1 receptor, and vascular endothelial growth factor receptor. The Torso pathway has been a model system for studying RTK signal transduction. Genetic and biochemical studies of Torso signaling have provided valuable insights into the biological functions and mechanisms of RTK signaling during early Drosophila embryogenesis. PMID:15704136

  6. Inhibition of Polo kinase by BI2536 affects centriole separation during Drosophila male meiosis.

    PubMed

    Riparbelli, Maria G; Gottardo, Marco; Glover, David M; Callaini, Giuliano

    2014-01-01

    Pharmacological inhibition of Drosophila Polo kinase with BI2536 has allowed us to re-examine the requirements for Polo during Drosophila male gametogenesis. BI2536-treated spermatocytes persisted in a pro-metaphase state without dividing and had condensed chromosomes that did not separate. Centrosomes failed to recruit γ-tubulin and centrosomin (Cnn) and were not associated with microtubule arrays that were abnormal and did not form proper bipolar spindles. Centrioles, which usually separate during the anaphase of the first meiosis, remained held together in a V-shaped configuration suggesting that Polo kinase regulates the proteolysis that breaks centriole linkage to ensure their disengagement. Despite these defects spermatid differentiation proceeds, leading to axoneme formation.

  7. Inhibition of Polo kinase by BI2536 affects centriole separation during Drosophila male meiosis

    PubMed Central

    Riparbelli, Maria G; Gottardo, Marco; Glover, David M; Callaini, Giuliano

    2014-01-01

    Pharmacological inhibition of Drosophila Polo kinase with BI2536 has allowed us to re-examine the requirements for Polo during Drosophila male gametogenesis. BI2536-treated spermatocytes persisted in a pro-metaphase state without dividing and had condensed chromosomes that did not separate. Centrosomes failed to recruit γ-tubulin and centrosomin (Cnn) and were not associated with microtubule arrays that were abnormal and did not form proper bipolar spindles. Centrioles, which usually separate during the anaphase of the first meiosis, remained held together in a V-shaped configuration suggesting that Polo kinase regulates the proteolysis that breaks centriole linkage to ensure their disengagement. Despite these defects spermatid differentiation proceeds, leading to axoneme formation. PMID:24802643

  8. Natural variation in Drosophila melanogaster diapause due to the insulin-regulated PI3-kinase

    PubMed Central

    Williams, Karen D.; Busto, Macarena; Suster, Maximiliano L.; So, Anthony K.-C.; Ben-Shahar, Yehuda; Leevers, Sally J.; Sokolowski, Marla B.

    2006-01-01

    This study links natural variation in a Drosophila melanogaster overwintering strategy, diapause, to the insulin-regulated phosphatidylinositol 3-kinase (PI3-kinase) gene, Dp110. Variation in diapause, a reproductive arrest, was associated with Dp110 by using Dp110 deletions and genomic rescue fragments in transgenic flies. Deletions of Dp110 increased the proportion of individuals in diapause, whereas expression of Dp110 in the nervous system, but not including the visual system, decreased it. The roles of phosphatidylinositol 3-kinase for both diapause in D. melanogaster and dauer formation in Caenorhabditis elegans suggest a conserved role for this kinase in both reproductive and developmental arrests in response to environmental stresses. PMID:17043223

  9. Combining Genetic Perturbations and Proteomics to Examine Kinase-Phosphatase Networks in Drosophila Embryos

    PubMed Central

    Sopko, Richelle; Foos, Marianna; Vinayagam, Arunachalam; Zhai, Bo; Binari, Richard; Hu, Yanhui; Randklev, Sakara; Perkins, Lizabeth A.; Gygi, Steven P.; Perrimon, Norbert

    2014-01-01

    Summary Connecting phosphorylation events to kinases and phosphatases is key to understanding the molecular organization and signaling dynamics of networks. We have generated a validated set of transgenic RNA-interference reagents for knockdown and characterization of all protein kinases and phosphatases present during early Drosophila melanogaster development. These genetic tools enable collection of sufficient quantities of embryos depleted of single gene products for proteomics. As a demonstration of an application of the collection, we have used multiplexed isobaric-labeling for quantitative proteomics to derive global phosphorylation signatures associated with kinase-depleted embryos, in order to systematically link phosphosites with relevant kinases. We demonstrate how this strategy uncovers kinase consensus motifs and prioritizes phosphoproteins for kinase target validation. We validate this approach by providing auxiliary evidence for Wee kinase-directed regulation of the chromatin regulator Stonewall. Further, we show how correlative phosphorylation at the site level can indicate function, as exemplified by Sterile20-like kinase-dependent regulation of Stat92E. PMID:25284370

  10. Src kinases and ERK activate distinct responses to Stitcher receptor tyrosine kinase signaling during wound healing in Drosophila.

    PubMed

    Tsarouhas, Vasilios; Yao, Liqun; Samakovlis, Christos

    2014-04-15

    Metazoans have evolved efficient mechanisms for epidermal repair and survival following injury. Several cellular responses and key signaling molecules that are involved in wound healing have been identified in Drosophila, but the coordination of cytoskeletal rearrangements and the activation of gene expression during barrier repair are poorly understood. The Ret-like receptor tyrosine kinase (RTK) Stitcher (Stit, also known as Cad96Ca) regulates both re-epithelialization and transcriptional activation by Grainy head (Grh) to induce restoration of the extracellular barrier. Here, we describe the immediate downstream effectors of Stit signaling in vivo. Drk (Downstream of receptor kinase) and Src family tyrosine kinases bind to the same docking site in the Stit intracellular domain. Drk is required for the full activation of transcriptional responses but is dispensable for re-epithelialization. By contrast, Src family kinases (SFKs) control both the assembly of a contractile actin ring at the wound periphery and Grh-dependent activation of barrier-repair genes. Our analysis identifies distinct pathways mediating injury responses and reveals an RTK-dependent activation mode for Src kinases and their central functions during epidermal wound healing in vivo.

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

    PubMed

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

    2011-08-05

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

  12. Activation of a Drosophila Janus kinase (JAK) causes hematopoietic neoplasia and developmental defects.

    PubMed Central

    Harrison, D A; Binari, R; Nahreini, T S; Gilman, M; Perrimon, N

    1995-01-01

    In mammals, many cytokines and growth factors stimulate members of the Janus kinase (JAK) family to transduce signals for the proliferation and differentiation of various cell types, particularly in hematopoietic lineages. Mutations in the Drosophila hopscotch (hop) gene, which encodes a JAK, also cause proliferative defects. Loss-of-function alleles result in lethality and underproliferation of diploid tissues of the larva. A dominant gain-of-function allele, Tumorous-lethal (hopTum-l), leads to formation of melanotic tumors and hypertrophy of the larval lymph glands, the hematopoietic organs. We show that a single amino acid change in Hop is associated with the hopTum-l mutation. Overexpression of either wild-type hop or hopTum-l in the larval lymph glands causes melanotic tumors and lymph gland hypertrophy indistinguishable from the original hopTum-l mutation. In addition, overexpression of Hop in other tissues of the larva leads to pattern defects in the adult or to lethality. Finally, overexpression of either hop or hopTum-l in Drosophila cell culture results in tyrosine phosphorylation of Hop protein. However, overexpression of hopTum-l results in greater phosphorylation than overexpression of the wild-type. We conclude that hopTum-l encodes a hyperactive Hop kinase and that overactivity of Hop in lymph glands causes malignant neoplasia of Drosophila blood cells. Images PMID:7796812

  13. Drosophila Citron Kinase Is Required for the Final Steps of CytokinesisD⃞

    PubMed Central

    Naim, Valeria; Imarisio, Sara; Di Cunto, Ferdinando; Gatti, Maurizio; Bonaccorsi, Silvia

    2004-01-01

    The mechanisms underlying completion of cytokinesis are still poorly understood. Here, we show that the Drosophila orthologue of mammalian Citron kinases is essential for the final events of the cytokinetic process. Flies bearing mutations in the Drosophila citron kinase (dck) gene were defective in both neuroblast and spermatocyte cytokinesis. In both cell types, early cytokinetic events such as central spindle assembly and contractile ring formation were completely normal. Moreover, cytokinetic rings constricted normally, leading to complete furrow ingression. However late telophases of both cell types displayed persistent midbodies associated with disorganized F actin and anillin structures. Similar defects were observed in dck RNA interference (RNAi) telophases, which, in addition to abnormal F actin and anillin rings, also displayed aberrant membrane protrusions at the cleavage site. Together, these results indicate that mutations in the dck gene result in morphologically abnormal intercellular bridges and in delayed resolution of these structures, suggesting that the wild-type function of dck is required for abscission at the end of cytokinesis. The phenotype of Dck-depleted cells is different from those observed in most Drosophila cytokinesis mutants but extraordinarily similar to that caused by anillin RNAi, suggesting that Dck and anillin are in the same pathway for completion of cytokinesis. PMID:15371536

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

    PubMed

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

    2008-12-30

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

  15. Chemical reactivity analysis of deoxyribonucleosides and deoxyribonucleoside analogues (NRTIs): a first-principles density functional approach.

    PubMed

    Kumar, Vipin; Kishor, Shyam; Ramaniah, Lavanya M

    2012-08-01

    The structures, energetics, as well as several important chemical parameters, of antiretroviral drugs - nucleoside reverse transcriptase inhibitors (NRTIs) - and natural deoxyribonucleosides in both neutral, and positively and negatively charged states, are investigated. These studies are carried out within the frame work of first-principles density-functional theory (DFT), using the Becke-Lee-Yang-Parr (BLYP) generalized gradient corrections to the local spin density approximation exchange and correlation energy, norm-conserving pseudopotentials and a plane-wave expansion of Kohn-Sham orbitals. Conceptual DFT is used to determine global and local chemical reactivity parameters. Our results are in good agreement with the best available experiments to date. The variation in the bond lengths and bond angles on cation formation indicates that the electron is lost from the base part of these molecules. Further, the presence of the deoxyribose sugar moiety lowers their ionization potential and increases their electron affinity, in comparison to the isolated DNA base. The effectiveness of the drug action in terminating the viral DNA chain, is explained using the global reactivity parameters, by comparing the reactivities of the drug molecules with those of the competing deoxyribonucleosides. The widely followed clinical practice, of avoiding the simultaneous administration of certain drugs, is also explained from the hardness and softness parameters. For most of the drug molecules, our study validates the generally accepted wisdom, that monophosphorylation is the crucial reaction step in the phosphorylation reaction in DNA nucleotide synthesis.

  16. Activated Cdc42 kinase regulates Dock localization in male germ cells during Drosophila spermatogenesis.

    PubMed

    Abdallah, Abbas M; Zhou, Xin; Kim, Christine; Shah, Kushani K; Hogden, Christopher; Schoenherr, Jessica A; Clemens, James C; Chang, Henry C

    2013-06-15

    Deregulation of the non-receptor tyrosine kinase ACK1 (Activated Cdc42-associated kinase) correlates with poor prognosis in cancers and has been implicated in promoting metastasis. To further understand its in vivo function, we have characterized the developmental defects of a null mutation in Drosophila Ack, which bears a high degree of sequence similarity to mammalian ACK1 but lacks a CRIB domain. We show that Ack, while not essential for viability, is critical for sperm formation. This function depends on Ack tyrosine kinase activity and is required cell autonomously in differentiating male germ cells at or after the spermatocyte stage. Ack associates predominantly with endocytic clathrin sites in spermatocytes, but disruption of Ack function has no apparent effect on clathrin localization and receptor-mediated internalization of Boss (Bride of sevenless) protein in eye discs. Instead, Ack is required for the subcellular distribution of Dock (dreadlocks), the Drosophila homolog of the SH2- and SH3-containing adaptor protein Nck. Moreover, Dock forms a complex with Ack, and the localization of Dock in male germ cells depends on its SH2 domain. Together, our results suggest that Ack-dependent tyrosine phosphorylation recruits Dock to promote sperm differentiation. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila.

    PubMed

    Montagne, Jacques; Lecerf, Caroline; Parvy, Jean-Philippe; Bennion, Janis M; Radimerski, Thomas; Ruhf, Marie-Laure; Zilbermann, Frederic; Vouilloz, Nicole; Stocker, Hugo; Hafen, Ernst; Kozma, Sara C; Thomas, George

    2010-05-06

    S6 kinases (S6Ks) act to integrate nutrient and insulin signaling pathways and, as such, function as positive effectors in cell growth and organismal development. However, they also have been shown to play a key role in limiting insulin signaling and in mediating the autophagic response. To identify novel regulators of S6K signaling, we have used a Drosophila-based, sensitized, gain-of-function genetic screen. Unexpectedly, one of the strongest enhancers to emerge from this screen was the nuclear receptor (NR), Drosophila hormone receptor 3 (DHR3), a critical constituent in the coordination of Drosophila metamorphosis. Here we demonstrate that DHR3, through dS6K, also acts to regulate cell-autonomous growth. Moreover, we show that the ligand-binding domain (LBD) of DHR3 is essential for mediating this response. Consistent with these findings, we have identified an endogenous DHR3 isoform that lacks the DBD. These results provide the first molecular link between the dS6K pathway, critical in controlling nutrient-dependent growth, and that of DHR3, a major mediator of ecdysone signaling, which, acting together, coordinate metamorphosis.

  18. The JIL-1 Kinase Affects Telomere Expression in the Different Telomere Domains of Drosophila

    PubMed Central

    Silva-Sousa, Rute; Casacuberta, Elena

    2013-01-01

    In Drosophila, the non-LTR retrotransposons HeT-A, TART and TAHRE build a head-to-tail array of repetitions that constitute the telomere domain by targeted transposition at the end of the chromosome whenever needed. As a consequence, Drosophila telomeres have the peculiarity to harbor the genes in charge of telomere elongation. Understanding telomere expression is important in Drosophila since telomere homeostasis depends in part on the expression of this genomic compartment. We have recently shown that the essential kinase JIL-1 is the first positive regulator of the telomere retrotransposons. JIL-1 mediates chromatin changes at the promoter of the HeT-A retrotransposon that are necessary to obtain wild type levels of expression of these telomere transposons. With the present study, we show how JIL-1 is also needed for the expression of a reporter gene embedded in the telomere domain. Our analysis, using different reporter lines from the telomere and subtelomere domains of different chromosomes, indicates that JIL-1 likely acts protecting the telomere domain from the spreading of repressive chromatin from the adjacent subtelomere domain. Moreover, the analysis of the 4R telomere suggests a slightly different chromatin structure at this telomere. In summary, our results strongly suggest that the action of JIL-1 depends on which telomere domain, which chromosome and which promoter is embedded in the telomere chromatin. PMID:24244743

  19. Drosophila Abelson kinase mediates cell invasion and proliferation via two distinct MAPK pathways

    PubMed Central

    Singh, Jaskirat; Aaronson, Stuart A.; Mlodzik, Marek

    2010-01-01

    The Abelson (Abl) family of non-receptor tyrosine kinases plays important role in cell morphogenesis, motility and proliferation. Although the function of Abl has been extensively studied in leukemia, its role in epithelial cell invasion remains obscure. Using the Drosophila wing epithelium as an in-vivo model system, we demonstrate that overexpression (activation) of Drosophila Abl (dAbl) causes loss of epithelial apical/basal cell polarity and secretion of matrix metalloproteinases, resulting in a cellular invasion and apoptosis. Our in vivo data indicate that dAbl acts downstream of the Src kinases, which are known regulators of cell adhesion and invasion. Downstream of dAbl, Rac GTPases activate two distinct MAPK pathways: JNK signaling (required for cell invasion and apoptosis) and ERK signaling (inducing cell proliferation). Activated Abl also increases the activity of Src members through a positive feedback loop leading to signal amplification. Thus targeting Src-Abl, using available dual inhibitors, could be of therapeutic importance in tumor cell metastasis. PMID:20453880

  20. fumble encodes a pantothenate kinase homolog required for proper mitosis and meiosis in Drosophila melanogaster.

    PubMed Central

    Afshar, K; Gönczy, P; DiNardo, S; Wasserman, S A

    2001-01-01

    A number of fundamental processes comprise the cell division cycle, including spindle formation, chromosome segregation, and cytokinesis. Our current understanding of these processes has benefited from the isolation and analysis of mutants, with the meiotic divisions in the male germline of Drosophila being particularly well suited to the identification of the required genes. We show here that the fumble (fbl) gene is required for cell division in Drosophila. We find that dividing cells in fbl-deficient testes exhibit abnormalities in bipolar spindle organization, chromosome segregation, and contractile ring formation. Cytological analysis of larval neuroblasts from null mutants reveals a reduced mitotic index and the presence of polyploid cells. Molecular analysis demonstrates that fbl encodes three protein isoforms, all of which contain a domain with high similarity to the pantothenate kinases of A. nidulans and mouse. The largest Fumble isoform is dispersed in the cytoplasm during interphase, concentrates around the spindle at metaphase, and localizes to the spindle midbody at telophase. During early embryonic development, the protein localizes to areas of membrane deposition and/or rearrangement, such as the metaphase and cellularization furrows. Given the role of pantothenate kinase in production of Coenzyme A and in phospholipid biosynthesis, this pattern of localization is suggestive of a role for fbl in membrane synthesis. We propose that abnormalities in synthesis and redistribution of membranous structures during the cell division cycle underlie the cell division defects in fbl mutant cells. PMID:11238410

  1. ATM kinase inhibition in glial cells activates the innate immune response and causes neurodegeneration in Drosophila.

    PubMed

    Petersen, Andrew J; Rimkus, Stacey A; Wassarman, David A

    2012-03-13

    To investigate the mechanistic basis for central nervous system (CNS) neurodegeneration in the disease ataxia-telangiectasia (A-T), we analyzed flies mutant for the causative gene A-T mutated (ATM). ATM encodes a protein kinase that functions to monitor the genomic integrity of cells and control cell cycle, DNA repair, and apoptosis programs. Mutation of the C-terminal amino acid in Drosophila ATM inhibited the kinase activity and caused neuron and glial cell death in the adult brain and a reduction in mobility and longevity. These data indicate that reduced ATM kinase activity is sufficient to cause neurodegeneration in A-T. ATM kinase mutant flies also had elevated expression of innate immune response genes in glial cells. ATM knockdown in glial cells, but not neurons, was sufficient to cause neuron and glial cell death, a reduction in mobility and longevity, and elevated expression of innate immune response genes in glial cells, indicating that a non-cell-autonomous mechanism contributes to neurodegeneration in A-T. Taken together, these data suggest that early-onset CNS neurodegeneration in A-T is similar to late-onset CNS neurodegeneration in diseases such as Alzheimer's in which uncontrolled inflammatory response mediated by glial cells drives neurodegeneration.

  2. 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

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

    PubMed

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

    2015-09-15

    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.

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

    PubMed

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

    2009-02-01

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

  5. Two distinct transmembrane serine/threonine kinases from Drosophila melanogaster form an activin receptor complex.

    PubMed Central

    Wrana, J L; Tran, H; Attisano, L; Arora, K; Childs, S R; Massagué, J; O'Connor, M B

    1994-01-01

    A transmembrane protein serine/threonine kinase, Atr-I, that is structurally related to receptors for members of the transforming growth factor-beta (TGF-beta) family has been cloned from Drosophila melanogaster. The spacing of extracellular cysteines and the cytoplasmic domain of Atr-I resemble most closely those of the recently described mammalian type I receptors for TGF-beta and activin. When expressed alone in test cells, Atr-I is unable to bind TGF-beta, activin, or bone morphogenetic protein 2. However, Atr-I binds activin efficiently when coexpressed with the distantly related Drosophila activin receptor Atr-II, with which it forms a heteromeric complex. Atr-I can also bind activin in concert with mammalian activin type II receptors. Two alternative forms of Atr-I have been identified that differ in an ectodomain region encompassing the cysteine box motif characteristic of receptors in this family. Comparison of Atr-I with other type I receptors reveals the presence of a characteristic 30-amino-acid domain immediately upstream of the kinase region in all these receptors. This domain, of unknown function, contains a repeated Gly-Ser sequence and is therefore referred to as the GS domain. Maternal Atr-I transcripts are abundant in the oocyte and widespread during embryo development and in the imaginal discs of the larva. The structural properties, binding specificity, and dependence on type II receptors define Atr-I as an activin type I receptor from D. melanogaster. These results indicate that the heteromeric kinase structure is a general feature of this receptor family. Images PMID:8289834

  6. 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…

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

    ERIC Educational Resources Information Center

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

    2004-01-01

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

  8. 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.

  9. Glycogen synthase kinase-3/Shaggy mediates ethanol-induced excitotoxic cell death of Drosophila olfactory neurons

    PubMed Central

    French, Rachael L.; Heberlein, Ulrike

    2009-01-01

    It has long been known that heavy alcohol consumption leads to neuropathology and neuronal death. While the response of neurons to an ethanol insult is strongly influenced by genetic background, the underlying mechanisms are poorly understood. Here, we show that even a single intoxicating exposure to ethanol causes non-cell-autonomous apoptotic death specifically of Drosophila olfactory neurons, which is accompanied by a loss of a behavioral response to the smell of ethanol and a blackening of the third antennal segment. The Drosophila homolog of glycogen synthase kinase-3 (GSK-3)β, Shaggy, is required for ethanol-induced apoptosis. Consistent with this requirement, the GSK-3β inhibitor lithium protects against the neurotoxic effects of ethanol, indicating the possibility for pharmacological intervention in cases of alcohol-induced neurodegeneration. Ethanol-induced death of olfactory neurons requires both their neural activity and functional NMDA receptors. This system will allow the investigation of the genetic and molecular basis of ethanol-induced apoptosis in general and provide an understanding of the molecular role of GSK-3β in programmed cell death. PMID:19923438

  10. 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

  11. Molecular cloning and biochemical characterization of a Drosophila phosphatidylinositol-specific phosphoinositide 3-kinase.

    PubMed

    Linassier, C; MacDougall, L K; Domin, J; Waterfield, M D

    1997-02-01

    Molecular, biochemical and genetic characterization of phosphoinositide 3-kinases (PI3Ks) have identified distinct classes of enzymes involved in processes mediated by activation of cell-surface receptors and in constitutive intracellular protein trafficking events. The latter process appears to involve a PtdIns-specific PI3K first described in yeast as a mutant, vps34, defective in the sorting of newly synthesized proteins from the Golgi to the vacuole. We have identified a representative member of each class of PI3Ks in Drosophila using a PCR-based approach. In the present paper we describe the molecular cloning of a PI3K from Drosophila, P13K_59F, that shows sequence similarity to Vps34. PI3K_59F encodes a protein of 108 kDa co-linear with Vps34 homologues, and with three regions of sequence similarity to other PI3Ks. Biochemical characterization of the enzyme, by expression of the complete coding sequence as a glutathione S-transferase fusion protein in Sf9 cells, demonstrates that PI3K_59F is a PtdIns-specific PI3K that can utilize either Mg2+ or Mn2+. This activity is sensitive to inhibition both by non-ionic detergent (Nonidet P40) and by wortmannin (IC50 10 nM). PI3K_59F, therefore, conserves both the structural and biochemical properties of the Vps34 class of enzymes.

  12. Deoxyribonucleoside-modified squaraines as near-IR viscosity sensors.

    PubMed

    Zhang, Yuanwei; Yue, Xiling; Kim, Bosung; Yao, Sheng; Belfield, Kevin D

    2014-06-10

    Deoxyribonucleoside-modified squaraines were synthesized by Sonogashira coupling reactions using an unsymmetrical, terminal alkynylated benzothiazolium squaraine dye. These non-natural nucleosides exhibited fluorescent 'turn-on' properties in viscous conditions with an enhancement of >300-fold. The viscosity-dependent fluorescence enhancement was attributed to a combination of hampering both molecular aggregation and intramolecular bond rotation of the squaraine probe. Fluorescence microscopy allowed visualization of highly viscous regions during various stages of cellular mitosis.

  13. The Drosophila Shark tyrosine kinase is required for embryonic dorsal closure

    PubMed Central

    Fernandez, Rafael; Takahashi, Fumitaka; Liu, Zhao; Steward, Ruth; Stein, David; Stanley, E. Richard

    2000-01-01

    Dorsal closure (DC) in the Drosophila embryo requires the coordinated interaction of two different functional domains of the epidermal cell layer—the leading edge (LE) and the lateral epidermis. In response to activation of a conserved c-Jun amino-terminal kinase (JNK) signaling module, the dorsal-most layer of cells, which constitute the LE of the stretching epithelial sheet, secrete Dpp, a member of the TGFβ superfamily. Dpp and other LE cell-derived signaling molecules stimulate the bilateral dorsal elongation of cells of the dorsolateral epidermis over the underlaying amnioserosa and the eventual fusion of their LEs along the dorsal midline. We have found that flies bearing a Shark tyrosine kinase gene mutation, shark1, exhibit a DC-defective phenotype. Dpp fails to be expressed in shark1 mutant LE cells. Consistent with these observations, epidermal-specific reconstitution of shark function or overexpression of an activated form of c-Jun in the shark1 mutant background, rescues the DC defect. Thus, Shark regulates the JNK signaling pathway leading to Dpp expression in LE cells. Furthermore, constitutive activation of the Dpp pathway throughout the epidermis fails to rescue the shark1 DC defect, suggesting that Shark may function in additional pathways in the LE and/or lateral epithelium. PMID:10716948

  14. Genetic analysis of rolled, which encodes a Drosophila mitogen-activated protein kinase.

    PubMed Central

    Lim, Y M; Nishizawa, K; Nishi, Y; Tsuda, L; Inoue, Y H; Nishida, Y

    1999-01-01

    Genetic and molecular characterization of the dominant suppressors of D-raf(C110) on the second chromosome identified two gain-of-function alleles of rolled (rl), which encodes a mitogen-activated protein (MAP) kinase in Drosophila. One of the alleles, rl(Su23), was found to bear the same molecular lesion as rl(Sem), which has been reported to be dominant female sterile. However, rl(Su23) and the current stock of rl(Sem) showed only a weak dominant female sterility. Detailed analyses of the rl mutations demonstrated moderate dominant activities of these alleles in the Torso (Tor) signaling pathway, which explains the weak dominant female sterility observed in this study. The dominant rl mutations failed to suppress the terminal class maternal-effect mutations, suggesting that activation of Rl is essential, but not sufficient, for Tor signaling. Involvement of rl in cell proliferation was also demonstrated by clonal analysis. Branching and integration of signals in the MAP kinase cascade is discussed. PMID:10511556

  15. The tyrosine kinase Stitcher activates Grainy head and epidermal wound healing in Drosophila.

    PubMed

    Wang, Shenqiu; Tsarouhas, Vasilios; Xylourgidis, Nikos; Sabri, Nafiseh; Tiklová, Katarína; Nautiyal, Naumi; Gallio, Marco; Samakovlis, Christos

    2009-07-01

    Epidermal injury initiates a cascade of inflammation, epithelial remodelling and integument repair at wound sites. The regeneration of the extracellular barrier and damaged tissue repair rely on the precise orchestration of epithelial responses triggered by the injury. Grainy head (Grh) transcription factors induce gene expression to crosslink the extracellular barrier in wounded flies and mice. However, the activation mechanisms and functions of Grh factors in re-epithelialization remain unknown. Here we identify stitcher (stit), a new Grh target in Drosophila melanogaster. stit encodes a Ret-family receptor tyrosine kinase required for efficient epidermal wound healing. Live imaging analysis reveals that Stit promotes actin cable assembly during wound re-epithelialization. Stit activation also induces extracellular signal-regulated kinase (ERK) phosphorylation along with the Grh-dependent expression of stit and barrier repair genes at the wound sites. The transcriptional stimulation of stit on injury triggers a positive feedback loop increasing the magnitude of epithelial responses. Thus, Stit activation upon wounding coordinates cytoskeletal rearrangements and the level of Grh-mediated transcriptional wound responses.

  16. AMP-activated protein kinase has diet-dependent and -independent roles in Drosophila oogenesis.

    PubMed

    Laws, Kaitlin M; Drummond-Barbosa, Daniela

    2016-12-01

    Multiple aspects of organismal physiology influence the number and activity of stem cells and their progeny, including nutritional status. Previous studies demonstrated that Drosophila germline stem cells (GSCs), follicle stem cells (FSCs), and their progeny sense and respond to diet via complex mechanisms involving many systemic and local signals. AMP-activated protein kinase, or AMPK, is a highly conserved regulator of energy homeostasis known to be activated under low cellular energy conditions; however, its role in the ovarian response to diet has not been investigated. Here, we describe nutrient-dependent and -independent requirements for AMPK in Drosophila oogenesis. We found that AMPK is cell autonomously required for the slow down in GSC and follicle cell proliferation that occurs on a poor diet. Similarly, AMPK activity is necessary in the germline for the degeneration of vitellogenic stages in response to nutrient deprivation. In contrast, AMPK activity is not required within the germline to modulate its growth. Instead, AMPK acts in follicle cells to negatively regulate their growth and proliferation, thereby indirectly limiting the size of the underlying germline cyst within developing follicles. Paradoxically, AMPK is required for GSC maintenance in well-fed flies (when AMPK activity is presumably at its lowest), suggesting potentially important roles for basal AMPK activity in specific cell types. Finally, we identified a nutrient-independent, developmental role for AMPK in cyst encapsulation by follicle cells. These results uncover specific AMPK requirements in multiple cell types in the ovary and suggest that AMPK can function outside of its canonical nutrient-sensing role in specific developmental contexts.

  17. 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

  18. Tousled-like kinase mediated a new type of cell death pathway in Drosophila

    PubMed Central

    Zhang, Y; Cai, R; Zhou, R; Li, Y; Liu, L

    2016-01-01

    Programmed cell death (PCD) has an important role in sculpting organisms during development. However, much remains to be learned about the molecular mechanism of PCD. We found that ectopic expression of tousled-like kinase (tlk) in Drosophila initiated a new type of cell death. Furthermore, the TLK-induced cell death is likely to be independent of the canonical caspase pathway and other known caspase-independent pathways. Genetically, atg2 RNAi could rescue the TLK-induced cell death, and this function of atg2 was likely distinct from its role in autophagy. In the developing retina, loss of tlk resulted in reduced PCD in the interommatidial cells (IOCs). Similarly, an increased number of IOCs was present in the atg2 deletion mutant clones. However, double knockdown of tlk and atg2 by RNAi did not have a synergistic effect. These results suggested that ATG2 may function downstream of TLK. In addition to a role in development, tlk and atg2 RNAi could rescue calcium overload-induced cell death. Together, our results suggest that TLK mediates a new type of cell death pathway that occurs in both development and calcium cytotoxicity. PMID:26088162

  19. A GFP trap study uncovers the functions of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis.

    PubMed

    Nerusheva, O O; Dorogova, N V; Gubanova, N V; Yudina, O S; Omelyanchuk, L V

    2009-05-01

    The function of the gene gilgamesh (89B9-12) encoding a casein kinase in Drosophila spermatogenesis was studied. The chimeric Gilgamesh-GFP protein in spermatocytes is cortically located. In the polar and apolar spermatocytes, it concentrates at the terminal ends of the fusome, the organelle that passes through the system of ring canals of the spermatocyte cyst. At the stage of spermatid elongation, the protein associates with the nucleus. A spot of the highest Gilgamesh-GFP concentration in the nucleus co-localizes with gamma-tubulin in the basal body. At later stages, Gilgamesh is localized to the individualization complex (IC), leaving the nuclei somewhat before the IC investment cones, as detected by actin binding. The sterile mutation due to the gilgamesh gene leads to the phenotype of scattered nuclei and altered structure of actin cones in the individualizing spermatid cyst. Ultrastructural evidence confirmed defective spermatid individualization due to the mutation. The phylogenetic origin of the protein, and the connection between vesicular trafficking and spermatid individualization, are discussed.

  20. Tousled-like kinase mediated a new type of cell death pathway in Drosophila.

    PubMed

    Zhang, Y; Cai, R; Zhou, R; Li, Y; Liu, L

    2016-01-01

    Programmed cell death (PCD) has an important role in sculpting organisms during development. However, much remains to be learned about the molecular mechanism of PCD. We found that ectopic expression of tousled-like kinase (tlk) in Drosophila initiated a new type of cell death. Furthermore, the TLK-induced cell death is likely to be independent of the canonical caspase pathway and other known caspase-independent pathways. Genetically, atg2 RNAi could rescue the TLK-induced cell death, and this function of atg2 was likely distinct from its role in autophagy. In the developing retina, loss of tlk resulted in reduced PCD in the interommatidial cells (IOCs). Similarly, an increased number of IOCs was present in the atg2 deletion mutant clones. However, double knockdown of tlk and atg2 by RNAi did not have a synergistic effect. These results suggested that ATG2 may function downstream of TLK. In addition to a role in development, tlk and atg2 RNAi could rescue calcium overload-induced cell death. Together, our results suggest that TLK mediates a new type of cell death pathway that occurs in both development and calcium cytotoxicity.

  1. 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

  2. Drosophila S6 Kinase Like Inhibits Neuromuscular Junction Growth by Downregulating the BMP Receptor Thickveins

    PubMed Central

    Zhao, Guoli; Wu, Yingga; Du, Li; Li, Wenhua; Xiong, Ying; Yao, Aiyu; Wang, Qifu; Zhang, Yong Q.

    2015-01-01

    Synaptic connections must be precisely controlled to ensure proper neural circuit formation. In Drosophila melanogaster, bone morphogenetic protein (BMP) promotes growth of the neuromuscular junction (NMJ) by binding and activating the BMP ligand receptors wishful thinking (Wit) and thickveins (Tkv) expressed in motor neurons. We report here that an evolutionally conserved, previously uncharacterized member of the S6 kinase (S6K) family S6K like (S6KL) acts as a negative regulator of BMP signaling. S6KL null mutants were viable and fertile but exhibited more satellite boutons, fewer and larger synaptic vesicles, larger spontaneous miniature excitatory junctional potential (mEJP) amplitudes, and reduced synaptic endocytosis at the NMJ terminals. Reducing the gene dose by half of tkv in S6KL mutant background reversed the NMJ overgrowth phenotype. The NMJ phenotypes of S6KL mutants were accompanied by an elevated level of Tkv protein and phosphorylated Mad, an effector of the BMP signaling pathway, in the nervous system. In addition, Tkv physically interacted with S6KL in cultured S2 cells. Furthermore, knockdown of S6KL enhanced Tkv expression, while S6KL overexpression downregulated Tkv in cultured S2 cells. This latter effect was blocked by the proteasome inhibitor MG132. Our results together demonstrate for the first time that S6KL regulates synaptic development and function by facilitating proteasomal degradation of the BMP receptor Tkv. PMID:25748449

  3. 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.

  4. Characterization of PINK1 (PTEN-induced putative kinase 1) mutations associated with Parkinson disease in mammalian cells and Drosophila.

    PubMed

    Song, Saera; Jang, Seoyeon; Park, Jeehye; Bang, Sunhoe; Choi, Sekyu; Kwon, Kyum-Yil; Zhuang, Xiaoxi; Kim, Eunjoon; Chung, Jongkyeong

    2013-02-22

    Mutations in PINK1 (PTEN-induced putative kinase 1) are tightly linked to autosomal recessive Parkinson disease (PD). Although more than 50 mutations in PINK1 have been discovered, the role of these mutations in PD pathogenesis remains poorly understood. Here, we characterized 17 representative PINK1 pathogenic mutations in both mammalian cells and Drosophila. These mutations did not affect the typical cleavage patterns and subcellular localization of PINK1 under both normal and damaged mitochondria conditions in mammalian cells. However, PINK1 mutations in the kinase domain failed to translocate Parkin to mitochondria and to induce mitochondrial aggregation. Consistent with the mammalian data, Drosophila PINK1 mutants with mutations in the kinase domain (G426D and L464P) did not genetically interact with Parkin. Furthermore, PINK1-null flies expressing the transgenic G426D mutant displayed defective phenotypes with increasing age, whereas L464P mutant-expressing flies exhibited the phenotypes at an earlier age. Collectively, these results strongly support the hypothesis that the kinase activity of PINK1 is essential for its function and for regulating downstream Parkin functions in mitochondria. We believe that this study provides the basis for understanding the molecular and physiological functions of various PINK1 mutations and provides insights into the pathogenic mechanisms of PINK1-linked PD.

  5. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) regulates TOR signaling and cell growth during Drosophila development

    PubMed Central

    Gupta, Amit; Toscano, Sarah; Trivedi, Deepti; Jones, David R.; Mathre, Swarna; Clarke, Jonathan H.; Divecha, Nullin; Raghu, Padinjat

    2013-01-01

    During development, Drosophila larvae undergo a dramatic increase in body mass wherein nutritional and developmental cues are transduced into growth through the activity of complex signaling pathways. Class I phosphoinositide 3-kinases have an established role in this process. In this study we identify Drosophila phosphatidylinositol 5-phosphate 4-kinase (dPIP4K) as a phosphoinositide kinase that regulates growth during larval development. Loss-of-function mutants in dPIP4K show reduced body weight and prolonged larval development, whereas overexpression of dPIP4K results both in an increase in body weight and shortening of larval development. The growth defect associated with dPIP4K loss of function is accompanied by a reduction in the average cell size of larval endoreplicative tissues. Our findings reveal that these phenotypes are underpinned by changes in the signaling input into the target of rapamycin (TOR) signaling complex and changes in the activity of its direct downstream target p70 S6 kinase. Together, these results define dPIP4K activity as a regulator of cell growth and TOR signaling during larval development. PMID:23530222

  6. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) regulates TOR signaling and cell growth during Drosophila development.

    PubMed

    Gupta, Amit; Toscano, Sarah; Trivedi, Deepti; Jones, David R; Mathre, Swarna; Clarke, Jonathan H; Divecha, Nullin; Raghu, Padinjat

    2013-04-09

    During development, Drosophila larvae undergo a dramatic increase in body mass wherein nutritional and developmental cues are transduced into growth through the activity of complex signaling pathways. Class I phosphoinositide 3-kinases have an established role in this process. In this study we identify Drosophila phosphatidylinositol 5-phosphate 4-kinase (dPIP4K) as a phosphoinositide kinase that regulates growth during larval development. Loss-of-function mutants in dPIP4K show reduced body weight and prolonged larval development, whereas overexpression of dPIP4K results both in an increase in body weight and shortening of larval development. The growth defect associated with dPIP4K loss of function is accompanied by a reduction in the average cell size of larval endoreplicative tissues. Our findings reveal that these phenotypes are underpinned by changes in the signaling input into the target of rapamycin (TOR) signaling complex and changes in the activity of its direct downstream target p70 S6 kinase. Together, these results define dPIP4K activity as a regulator of cell growth and TOR signaling during larval development.

  7. A genetic screen for modifiers of a kinase suppressor of Ras-dependent rough eye phenotype in Drosophila.

    PubMed Central

    Therrien, M; Morrison, D K; Wong, A M; Rubin, G M

    2000-01-01

    kinase suppressor of Ras (ksr) encodes a putative protein kinase that by genetic criteria appears to function downstream of RAS in multiple receptor tyrosine kinase (RTK) pathways. While biochemical evidence suggests that the role of KSR is closely linked to the signal transduction mechanism of the MAPK cascade, the precise molecular function of KSR remains unresolved. To further elucidate the role of KSR and to identify proteins that may be required for KSR function, we conducted a dominant modifier screen in Drosophila based on a KSR-dependent phenotype. Overexpression of the KSR kinase domain in a subset of cells during Drosophila eye development blocks photoreceptor cell differentiation and results in the external roughening of the adult eye. Therefore, mutations in genes functioning with KSR might modify the KSR-dependent phenotype. We screened approximately 185,000 mutagenized progeny for dominant modifiers of the KSR-dependent rough eye phenotype. A total of 15 complementation groups of Enhancers and four complementation groups of Suppressors were derived. Ten of these complementation groups correspond to mutations in known components of the Ras1 pathway, demonstrating the ability of the screen to specifically identify loci critical for Ras1 signaling and further confirming a role for KSR in Ras1 signaling. In addition, we have identified 4 additional complementation groups. One of them corresponds to the kismet locus, which encodes a putative chromatin remodeling factor. The relevance of these loci with respect to the function of KSR and the Ras1 pathway in general is discussed. PMID:11063697

  8. Reduction of thionucleosides - A prebiotic pathway to deoxyribonucleosides

    NASA Technical Reports Server (NTRS)

    Patel, A. D.; Schrier, W. H.; Hrncir, M. A.; Nagyvary, J. J.

    1981-01-01

    A mechanism is proposed for the prebiotic synthesis of deoxyribonucleotides and possible nucleic acid analogs from ribonucleotides by a pathway involving 2'-thio-2'-deoxyribonucleosides. The mechanism is supported by laboratory experiments in which 2'-thio-2'-deoxycytidine was synthesized from anhydro arabinosyl cytosine in dithiophosphate and CS2. The subsequent reduction of the thio-analogs has been achieved with ferrous ion, and photochemically. It is noted that the proposed pathway for prebiotic deoxyribonucleotide synthesis is in harmony with the Principle of Continuity, as both the proposed and present pathways rely on the reduction of a 2' functional group.

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

    PubMed

    Baldinger, Tina; Gossen, Manfred

    2009-01-01

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

  10. Glycerol Hypersensitivity in a Drosophila Model for Glycerol Kinase Deficiency Is Affected by Mutations in Eye Pigmentation Genes

    PubMed Central

    Wightman, Patrick J.; Jackson, George R.; Dipple, Katrina M.

    2012-01-01

    Glycerol kinase plays a critical role in metabolism by converting glycerol to glycerol 3-phosphate in an ATP dependent reaction. In humans, glycerol kinase deficiency results in a wide range of phenotypic variability; patients can have severe metabolic and CNS abnormalities, while others possess hyperglycerolemia and glyceroluria with no other apparent phenotype. In an effort to help understand the pathogenic mechanisms underlying the phenotypic variation, we have created a Drosophila model for glycerol kinase deficiency by RNAi targeting of dGyk (CG18374) and dGK (CG7995). As expected, RNAi flies have reduced glycerol kinase RNA expression, reduced phosphorylation activity and elevated glycerol levels. Further investigation revealed these flies to be hypersensitive to fly food supplemented with glycerol. Due to the hygroscopic nature of glycerol, we predict glycerol hypersensitivity is a result of greater susceptibility to desiccation, suggesting glycerol kinase to play an important role in desiccation resistance in insects. To evaluate a role for genetic modifier loci in determining severity of the glycerol hypersensitivity observed in knockdown flies, we performed a preliminary screen of lethal transposon insertion mutant flies using a glycerol hypersensitive survivorship assay. We demonstrate that this type of screen can identify both enhancer and suppressor genetic loci of glycerol hypersensitivity. Furthermore, we found that the glycerol hypersensitivity phenotype can be enhanced or suppressed by null mutations in eye pigmentation genes. Taken together, our data suggest proteins encoded by eye pigmentation genes play an important role in desiccation resistance and that eye pigmentation genes are strong modifiers of the glycerol hypersensitive phenotype identified in our Drosophila model for glycerol kinase deficiency. PMID:22427807

  11. LRRK2 kinase activity mediates toxic interactions between genetic mutation and oxidative stress in a Drosophila model: suppression by curcumin.

    PubMed

    Yang, Dejun; Li, Tianxia; Liu, Zhaohui; Arbez, Nicolas; Yan, Jianqun; Moran, Timothy H; Ross, Christopher A; Smith, Wanli W

    2012-09-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. The pathogenesis of PD is believed to involve both genetic susceptibility and environmental factors. Mutations in Leucine-rich repeat kinase 2 (LRRK2) cause genetic forms of PD, and the LRRK2 locus contributes to sporadic PD. Environmental toxins are believed to act in part by causing oxidative stress. Here we employed cell and Drosophila models to investigate the interaction between LRRK2 genetic mutations and oxidative stress. We found that H(2)O(2) increased LRRK2 kinase activity and enhanced LRRK2 cell toxicity in cultured cells and mouse primary cortical neurons. Furthermore, a sub-toxic dose of H(2)O(2) significantly shortened the survival of LRRK2 transgenic flies and augmented LRRK2-induced locomotor defects and dopamine neuron loss. Treatment with a LRRK2 kinase inhibitor (GW5074) or an anti-oxidant (curcumin) significantly suppressed these PD-like phenotypes in flies. Moreover, curcumin significantly reduced LRRK2 kinase activity and the levels of oxidized proteins, and thus acted as not only an antioxidant but also a LRRK2 kinase inhibitor. These results indicate that LRRK2 genetic alterations can interact with oxidative stress, converging on a pathogenic pathway that may be related to PD. These studies also identified curcumin as a LRRK2 kinase inhibitor that may be a useful candidate for LRRK2-linked PD intervention.

  12. Genetic analysis of a Drosophila neural cell adhesion molecule: interaction of fasciclin I and Abelson tyrosine kinase mutations.

    PubMed

    Elkins, T; Zinn, K; McAllister, L; Hoffmann, F M; Goodman, C S

    1990-02-23

    Drosophila fasciclin I is a homophilic cell adhesion molecule expressed in the developing embryo on the surface of a subset of fasciculating CNS axons, all PNS axons, and some nonneuronal cells. We have identified protein-null mutations in the fasciclin I (fas I) gene, and show that these mutants are viable and do not display gross defects in nervous system morphogenesis. The Drosophila Abelson (abl) proto-oncogene homolog encodes a cytoplasmic tyrosine kinase that is expressed during embryogenesis primarily in developing CNS axons; abl mutants show no gross defects in CNS morphogenesis. However, embryos doubly mutant for fas I and abl display major defects in CNS axon pathways, particularly in the commissural tracts where expression of these two proteins normally overlaps. The double mutant shows a clear defect in growth cone guidance; for example, the RP1 growth cone (normally fas I positive) does not follow its normal path across the commissure.

  13. 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

  14. The torpedo (DER) receptor tyrosine kinase is required at multiple times during Drosophila embryogenesis.

    PubMed

    Clifford, R; Schüpbach, T

    1992-07-01

    The torpedo (DER) gene of Drosophila, which encodes a receptor tyrosine kinase of the EGF receptor subfamily, is essential for oogenesis, embryogenesis and imaginal disc development. To gain insight into the nature of the signals transduced by the torpedo product, we have characterized the gene's loss-of-function phenotype in the embryo. Through the induction of germline clones, we provide a genetic demonstration that maternal torpedo product does not contribute to zygotic development. Thus, the embryonic lethal phenotypes examined accurately reflect the consequences of eliminating all gene activity from the zygote. Temperature-shift experiments with the conditional allele topIF26 show that torpedo is required at two distinct times during embryonic development: the gene is first needed for germband retraction and for the production of anterior, posterior and ventral cuticle, then later for the secretion of ventral denticles. Since denticle formation can be severely disrupted in topIF26 animals without affecting cuticle production, the early and late requirements for torpedo appear to be functionally unrelated. torpedo, therefore, is required at multiple times in the development of the ventral epidermis, and may transduce qualitatively different signals. Since the early requirement for torpedo correlates with the first visible defect in embryonic development, increased cell death in the amnioserosa, cephalic ectoderm and ventral epidermis, the abnormalities in cuticle production and germband shortening seen in the mutant may be secondary consequences of a primary defect in cell viability. Given that the onset of cell death in torpedo embryos is not preceded by any obvious defects in mitogenesis, the establishment of cell identities or the maintenance of gene expression, it is possible that torpedo transduces a signal necessary for cell survival per se during early embryogenesis. During late embryogenesis, torpedo may mediate the reception of a second signal which

  15. Directed evolution of an orthogonal nucleoside analog kinase via fluorescence-activated cell sorting.

    PubMed

    Liu, Lingfeng; Li, Yongfeng; Liotta, Dennis; Lutz, Stefan

    2009-07-01

    Nucleoside analogs (NAs) represent an important category of prodrugs for the treatment of viral infections and cancer, yet the biological potency of many analogs is compromised by their inefficient activation through cellular 2'-deoxyribonucleoside kinases (dNKs). We herein report the directed evolution and characterization of an orthogonal NA kinase for 3'-deoxythymidine (ddT), using a new FACS-based screening protocol in combination with a fluorescent analog of ddT. Four rounds of random mutagenesis and DNA shuffling of Drosophila melanogaster 2'-deoxynucleoside kinase, followed by FACS analysis, yielded an orthogonal ddT kinase with a 6-fold higher activity for the NA and a 20-fold k(cat)/K(M) preference for ddT over thymidine, an overall 10,000-fold change in substrate specificity. The contributions of individual amino acid substitutions in the ddT kinase were evaluated by reverse engineering, enabling a detailed structure-function analysis to rationalize the observed changes in performance. Based on our results, kinase engineering with fluorescent NAs and FACS should prove a highly versatile method for evolving selective kinase:NA pairs and for studying fundamental aspects of the structure-function relationship in dNKs.

  16. 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. Copyright © 2015 by the Genetics Society of America.

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

    PubMed Central

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

    2015-01-01

    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

  18. The Meiotic Recombination Checkpoint Suppresses NHK-1 Kinase to Prevent Reorganisation of the Oocyte Nucleus in Drosophila

    PubMed Central

    Lancaster, Oscar M.; Breuer, Manuel; Cullen, C. Fiona; Ito, Takashi; Ohkura, Hiroyuki

    2010-01-01

    The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular pathway by which this occurs remains to be identified. Here we show that the conserved kinase NHK-1 (Drosophila Vrk-1) is a crucial meiotic regulator controlled by the meiotic checkpoint. An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation. Rather, we find unrepaired DNA breaks formed during recombination suppress NHK-1 activity (inferred from the phosphorylation level of one of its substrates) through the meiotic checkpoint. Additionally DNA breaks induced by X-rays in cultured cells also suppress NHK-1 kinase activity. Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes. Therefore we propose that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired. PMID:21060809

  19. Drosophila p53-related protein kinase is required for PI3K/TOR pathway-dependent growth.

    PubMed

    Ibar, Consuelo; Cataldo, Vicente F; Vásquez-Doorman, Constanza; Olguín, Patricio; Glavic, Alvaro

    2013-03-01

    Cell growth and proliferation are pivotal for final organ and body size definition. p53-related protein kinase (Bud32/PRPK) has been identified as a protein involved in proliferation through its effects on transcription in yeast and p53 stabilization in human cell culture. However, the physiological function of Bud32/PRPK in metazoans is not well understood. In this work, we have analyzed the role of PRPK in Drosophila development. Drosophila PRPK is expressed in every tissue analyzed and is required to support proliferation and cell growth. The Prpk knockdown animals show phenotypes similar to those found in mutants for positive regulators of the PI3K/TOR pathway. This pathway has been shown to be fundamental for animal growth, transducing the hormonal and nutritional status into the protein translation machinery. Functional interactions have established that Prpk operates as a transducer of the PI3K/TOR pathway, being essential for TOR kinase activation and for the regulation of its targets (S6K and 4E-BP, autophagy and bulk endocytosis). This suggests that Prpk is crucial for stimulating the basal protein biosynthetic machinery in response to insulin signaling and to changes in nutrient availability.

  20. Inhibitors of LRRK2 kinase attenuate neurodegeneration and Parkinson-like phenotypes in Caenorhabditis elegans and Drosophila Parkinson's disease models

    PubMed Central

    Liu, Zhaohui; Hamamichi, Shusei; Dae Lee, Byoung; Yang, Dejun; Ray, Arpita; Caldwell, Guy A.; Caldwell, Kim A.; Dawson, Ted M.; Smith, Wanli W.; Dawson, Valina L.

    2011-01-01

    Mutations in leucine-rich repeat kinase 2 (LRRK2) have been identified as a genetic cause of familial Parkinson's disease (PD) and have also been found in the more common sporadic form of PD, thus positioning LRRK2 as important in the pathogenesis of PD. Biochemical studies of the disease-causing mutants of LRRK2 implicates an enhancement of kinase activity as the basis of neuronal toxicity and thus possibly the pathogenesis of PD due to LRRK2 mutations. Previously, a chemical library screen identified inhibitors of LRRK2 kinase activity. Here, two of these inhibitors, GW5074 and sorafenib, are shown to protect against G2019S LRRK2-induced neurodegeneration in vivo in Caenorhabditis elegans and in Drosophila. These findings indicate that increased kinase activity of LRRK2 is neurotoxic and that inhibition of LRRK2 activity can have a disease-modifying effect. This suggests that inhibition of LRRK2 holds promise as a treatment for PD. PMID:21768216

  1. Biochemical retrosynthesis of 2'-deoxyribonucleosides from glucose, acetaldehyde, and a nucleobase.

    PubMed

    Horinouchi, Nobuyuki; Ogawa, Jun; Kawano, Takako; Sakai, Takafumi; Saito, Kyota; Matsumoto, Seiichiro; Sasaki, Mie; Mikami, Yoichi; Shimizu, Sakayu

    2006-08-01

    2'-Deoxyribonucleosides are important as building blocks for the synthesis of antisense drugs, antiviral nucleosides, and 2'-deoxyribonucleotides for polymerase chain reaction. The microbial production of 2'-deoxyribonucleosides from simple materials, glucose, acetaldehyde, and a nucleobase, through the reverse reactions of 2'-deoxyribonucleoside degradation and the glycolytic pathway, was investigated. The glycolytic pathway of baker's yeast yielded fructose 1,6-diphosphate from glucose using the energy of adenosine 5'-triphosphate generated from adenosine 5'-monophosphate through alcoholic fermentation with the yeast. Fructose 1,6-diphosphate was further transformed to 2-deoxyribose 5-phosphate in the presence of acetaldehyde by deoxyriboaldolase-expressing Escherichia coli cells via D-glyceraldehyde 3-phosphate. E. coli transformants expressing phosphopentomutase and nucleoside phosphorylase produced 2'-deoxyribonucleosides from 2-deoxyribose 5-phosphate and a nucleobase via 2-deoxyribose 1-phosphate through the reverse reactions of 2'-deoxyribonucleoside degradation. Coupling of the glycolytic pathway and deoxyriboaldolase-catalyzing reaction efficiently supplied 2-deoxyribose 5-phosphate, which is a key intermediate for 2'-deoxyribonucleoside synthesis. 2'-Deoxyinosine (9.9 mM) was produced from glucose, acetaldehyde, and adenine through three-step reactions via fructose 1,6-diphosphate and then 2-deoxyribose 5-phosphate, the molar yield as to glucose being 17.8%.

  2. Expression of the PI4P 5-kinase Drosophila homologue skittles in the germline suggests a role in spermatogenesis and oogenesis.

    PubMed

    Knirr, Stefan; Santel, Ansgar; Renkawitz-Pohl, R

    1997-07-01

     We have isolated the Drosophila gene skittles (sktl) which shows homology to members of a novel family of phosphatidylinositol-4-phosphate 5-kinases, including the gene product encoded by the human STM-7.I gene which has been assigned to the neurodegenerative disorder Friedreichs ataxia. In situ hybridization reveals sktl expression during oogenesis and spermatogenesis.

  3. The Ral/Exocyst Effector Complex Counters c-Jun N-Terminal Kinase-Dependent Apoptosis in Drosophila melanogaster▿ †

    PubMed Central

    Balakireva, Maria; Rossé, Carine; Langevin, Johanna; Chien, Yu-chen; Gho, Michel; Gonzy-Treboul, Geneviève; Voegeling-Lemaire, Stéphanie; Aresta, Sandra; Lepesant, Jean-Antoine; Bellaiche, Yohanns; White, Michael; Camonis, Jacques

    2006-01-01

    Ral GTPase activity is a crucial cell-autonomous factor supporting tumor initiation and progression. To decipher pathways impacted by Ral, we have generated null and hypomorph alleles of the Drosophila melanogaster Ral gene. Ral null animals were not viable. Reduced Ral expression in cells of the sensory organ lineage had no effect on cell division but led to postmitotic cell-specific apoptosis. Genetic epistasis and immunofluorescence in differentiating sensory organs suggested that Ral activity suppresses c-Jun N-terminal kinase (JNK) activation and induces p38 mitogen-activated protein (MAP) kinase activation. HPK1/GCK-like kinase (HGK), a MAP kinase kinase kinase kinase that can drive JNK activation, was found as an exocyst-associated protein in vivo. The exocyst is a Ral effector, and the epistasis between mutants of Ral and of msn, the fly ortholog of HGK, suggest the functional relevance of an exocyst/HGK interaction. Genetic analysis also showed that the exocyst is required for the execution of Ral function in apoptosis. We conclude that in Drosophila Ral counters apoptotic programs to support cell fate determination by acting as a negative regulator of JNK activity and a positive activator of p38 MAP kinase. We propose that the exocyst complex is Ral executioner in the JNK pathway and that a cascade from Ral to the exocyst to HGK would be a molecular basis of Ral action on JNK. PMID:17000765

  4. Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila.

    PubMed

    Jeon, Mili; Scott, Matthew P; Zinn, Kai

    2012-06-15

    The respiratory (tracheal) system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. We previously described a unique embryonic tracheal tubulogenesis phenotype caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr) tyrosine kinase (TK). Ptp10D physically interacts with Egfr. Here we demonstrate that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, we show that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. We also show that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways.

  5. Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila

    PubMed Central

    Jeon, Mili; Scott, Matthew P.; Zinn, Kai

    2012-01-01

    Summary The respiratory (tracheal) system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. We previously described a unique embryonic tracheal tubulogenesis phenotype caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr) tyrosine kinase (TK). Ptp10D physically interacts with Egfr. Here we demonstrate that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, we show that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. We also show that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways. PMID:23213447

  6. Long term expression of Drosophila melanogaster nucleoside kinase in thymidine kinase 2-deficient mice with no lethal effects caused by nucleotide pool imbalances.

    PubMed

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

    2014-11-21

    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.

  7. Establishment of the Muscle-Tendon Junction During Thorax Morphogenesis in Drosophila Requires the Rho-Kinase.

    PubMed

    Vega-Macaya, Franco; Manieu, Catalina; Valdivia, Mauricio; Mlodzik, Marek; Olguín, Patricio

    2016-11-01

    The assembly of the musculoskeletal system in Drosophila relies on the integration of chemical and mechanical signaling between the developing muscles with ectodermal cells specialized as "tendon cells." Mechanical tension generated at the junction of flight muscles and tendon cells of the notum epithelium is required for muscle morphogenesis, and is balanced by the epithelium in order to not deform. We report that Drosophila Rho kinase (DRok) is necessary in tendon cells to assemble stable myotendinous junctions (MTJ), which are required for muscle morphogenesis and survival. In addition, DRok is required in tendon cells to maintain epithelial shape and cell orientation in the notum, independently of chascon (chas). Loss of DRok function in tendon cells results in mis-orientation of tendon cell extensions and abnormal accumulation of Thrombospondin and βPS-integrin, which may cause abnormal myotendinous junction formation and muscle morphogenesis. This role does not depend exclusively on nonmuscular Myosin-II activation (Myo-II), indicating that other DRok targets are key in this process. We propose that DRok function in tendon cells is key to promote the establishment of MTJ attachment and to balance mechanical tension generated at the MTJ by muscle compaction. Copyright © 2016 by the Genetics Society of America.

  8. 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.

  9. The Enzymatic Activity of Drosophila AWD/NDP Kinase Is Necessary but Not Sufficient for Its Biological Function

    PubMed

    Xu; Liu; Deng; Timmons; Hersperger; Steeg; Veron; Shearn

    1996-08-01

    The Drosophila abnormal wing discs (awd) gene encodes the subunit of a protein that has nucleoside diphosphate kinase (NDP kinase) activity. Null mutations of the awd gene cause lethality after puparium formation. Larvae homozygous for such mutations have small imaginal discs, lymph glands, and brain lobes. Neither the imaginal discs nor the ovaries from such null mutant larvae are capable of further growth or normal differentiation when transplanted into suitable host larvae. This null mutant phenotype can be entirely rescued by one copy of a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA. Tissue-specific expression of AWD protein from this rescue transgene is identical to tissue-specific expression of beta-galactosidase from a reporter transgene that has the same regulatory region fused to the bacterial lac Z gene. However, this rescue transgene or reporter transgene expression pattern is only a subset of the endogenous pattern of expression detected by either in situ hybridization or immunohistochemistry. This suggests that awd is normally expressed in some tissues where it is not required. The null mutant phenotype cannot be rescued at all by a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA with a mutation that eliminates NDP kinase activity by replacement of the active site histidine with alanine. This suggests that the enzymatic activity of the AWD protein is necessary for its biological function. The human genes nm23-H1 and nm23-H2 encode NDP kinase A and B subunits, respectively. The protein subunit encoded by either human nm23 gene is 78% identical to that encoded by the Drosophila awd gene. Transgenes that have the 750-bp awd upstream regulatory DNA fused to human nm23-H2 cDNA but not to nm23-H1 cDNA can rescue the imaginal disc phenotype and the zygotic lethality caused by homozygosis for an awd null mutation as efficiently as an awd transgene. However, rescue of female

  10. 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

  11. The octopamine receptor OAMB mediates ovulation via Ca2+/calmodulin-dependent protein kinase II in the Drosophila oviduct epithelium.

    PubMed

    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

  12. 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

  13. Phosphorylation of the Drosophila transient receptor potential ion channel is regulated by the phototransduction cascade and involves several protein kinases and phosphatases.

    PubMed

    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.

  14. Casein kinase I epsilon does not rescue double-time function in Drosophila despite evolutionarily conserved roles in the circadian clock.

    PubMed

    Sekine, Tatsumori; Yamaguchi, Terumi; Hamano, Kunikatsu; Young, Michael W; Shimoda, Masami; Saez, Lino

    2008-02-01

    Double-time (dbt) is a casein kinase gene involved in cell survival, proliferation, and circadian rhythms in the fruit fly, Drosophila melanogaster. Genetic and biochemical studies have shown that dbt and its mammalian ortholog casein kinase I epsilon (hckI epsilon) regulate the circadian phosphorylation of period (per), thus controlling per subcellular localization and stability. Mutations in these kinases can shorten the circadian period in both mammals and Drosophila. Since similar activities in circadian clock have been described for these kinases, we investigated whether the expression of mammalian casein kinase I can replace the activity of dbt in flies. Global expression of the full-length dbt rescued lethality of the null mutant dbt revVIII and rescued flies showed normal locomotor activity rhythms. Global expression of dbt also restored the locomotor activity rhythm of the arrhythmic genotype, dbt ar/dbt revVIII. In contrast, global expression of hckI epsilon or hckI alpha did not rescue lethality or locomotor activity of dbt mutants. Furthermore dbt overexpression in wild-type clock cells had only a small effect on period length, whereas hckI epsilon expression in clock cells greatly lengthened period to ~30.5 hours and increased the number of arrhythmic flies. These results indicate that hckI epsilon cannot replace the activity of dbt in flies despite the high degree of similarity in primary sequence and kinase function. Moreover, expression of hck Iepsilon in flies appears to interfere with dbt activity. Thus, caution should be used in interpreting assays that measure activity of mammalian casein kinase mutants in Drosophila, or that employ vertebrate CKI in studies of dPER phosphorylations.

  15. Dual roles for the Drosophila PI 4-kinase four wheel drive in localizing Rab11 during cytokinesis.

    PubMed

    Polevoy, Gordon; Wei, Ho-Chun; Wong, Raymond; Szentpetery, Zsofia; Kim, Yeun Ju; Goldbach, Philip; Steinbach, Sarah K; Balla, Tamas; Brill, Julie A

    2009-12-14

    Successful completion of cytokinesis relies on addition of new membrane, and requires the recycling endosome regulator Rab11, which localizes to the midzone. Despite the critical role of Rab11 in this process, little is known about the formation and composition of Rab11-containing organelles. Here, we identify the phosphatidylinositol (PI) 4-kinase III beta four wheel drive (Fwd) as a key regulator of Rab11 during cytokinesis in Drosophila melanogaster spermatocytes. We show Fwd is required for synthesis of PI 4-phosphate (PI4P) on Golgi membranes and for formation of PI4P-containing secretory organelles that localize to the midzone. Fwd binds and colocalizes with Rab11 on Golgi membranes, and is required for localization of Rab11 in dividing cells. A kinase-dead version of Fwd also binds Rab11 and partially restores cytokinesis to fwd mutant flies. Moreover, activated Rab11 partially suppresses loss of fwd. Our data suggest Fwd plays catalytic and noncatalytic roles in regulating Rab11 during cytokinesis.

  16. A histone code in meiosis: the histone kinase, NHK-1, is required for proper chromosomal architecture in Drosophila oocytes

    PubMed Central

    Ivanovska, Irena; Khandan, Tulasi; Ito, Takashi; Orr-Weaver, Terry L.

    2005-01-01

    To promote faithful propagation of the genetic material during sexual reproduction, meiotic chromosomes undergo specialized morphological changes that ensure accurate segregation of homologous chromosomes. The molecular mechanisms that establish the meiotic chromosomal structures are largely unknown. We describe a mutation in a recently identified Histone H2A kinase, nhk-1, in Drosophila that leads to female sterility due to defects in the formation of the meiotic chromosomal structures. The metaphase I arrest and the karyosome, a critical prophase I chromosomal structure, require nucleosomal histone kinase-1 (NHK-1) function. The defects are a result of failure to disassemble the synaptonemal complex and to load condensin onto the mutant chromosomes. Embryos laid by nhk-1-/- mutant females arrest with aberrant polar bodies and mitotic spindles, revealing that mitosis is affected as well. We analyzed the role of Histone H2A phosphorylation with respect to the histone code hypothesis and found that it is required for acetylation of Histone H3 and Histone H4 in meiosis. These studies reveal a critical role for histone modifications in chromosome dynamics in meiosis and mitosis. PMID:16230526

  17. Both synchronous and asynchronous muscle isoforms of projectin (the Drosophila bent locus product) contain functional kinase domains

    PubMed Central

    1995-01-01

    In Drosophila, the large muscle protein, projectin, has very different localizations in synchronous and asynchronous muscles, suggesting that projectin has different functions in different muscle types. The multiple projectin isoforms are encoded by a single gene; however they differ significantly in size (as detected by gel mobility) and show differences in some peptide fragments, presumably indicating alternative splicing or termination. We now report additional sequence of the projectin gene, showing a kinase domain and flanking regions highly similar to equivalent regions of twitchin, including a possible autoinhibitory region. In spite of apparent differences in function, all isoforms of projectin have the kinase domain and all are capable of autophosphorylation in vitro. The projectin gene is in polytene region 102C/D where the bentD phenotype maps. The recessive lethality of bentD is associated with a breakpoint that removes sequence of the projectin kinase domain. We find that different alleles of the highly mutable recessive lethal complementation group, l(4)2, also have defects in different parts of the projectin sequence, both NH2-terminal and COOH- terminal to the bentD breakpoint. These alleles are therefore renamed as alleles of the bent locus. Adults heterozygous for projectin mutations show little, if any, effect of one defective gene copy, but homozygosity for any of the defects is lethal. The times of death can vary with allele. Some alleles kill the embryos, others are larval lethal. These molecular studies begin to explain why genetic studies suggested that l(4)2 was a complex (or pseudoallelic) locus. PMID:7844153

  18. 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

  19. β-Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP-activated protein kinase-dependent increase in autophagy.

    PubMed

    Yang, Si; Long, Li-Hong; Li, Di; Zhang, Jian-Kang; Jin, Shan; Wang, Fang; Chen, Jian-Guo

    2015-12-01

    Previous studies have demonstrated that AMP-activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc-51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β-guanidinopropionic acid (β-GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β-GPA and aging remains elusive. In this study, we hypothesized that feeding β-GPA to adult Drosophila produces the lifespan extension via activation of AMPK-dependent autophagy. It was found that dietary administration of β-GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule-associated protein 1A/1B-light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β-GPA treatment, indicating that autophagic activity plays a role in the effect of β-GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β-GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β-GPA treatment significantly elevated the expression of phospho-T172-AMPK levels, while inhibition of AMPK by either AMPK-RNAi or compound C significantly attenuated the expression of autophagy-related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β-GPA can induce an extension of the lifespan of Drosophila via AMPK-Atg1-autophagy signaling pathway. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  20. Spatial Reorganization of the Endoplasmic Reticulum during Mitosis Relies on Mitotic Kinase Cyclin A in the Early Drosophila Embryo

    PubMed Central

    Bergman, Zane J.; Mclaurin, Justin D.; Eritano, Anthony S.; Johnson, Brittany M.; Sims, Amanda Q.; Riggs, Blake

    2015-01-01

    Mitotic cyclin-dependent kinase with their cyclin partners (cyclin:Cdks) are the master regulators of cell cycle progression responsible for regulating a host of activities during mitosis. Nuclear mitotic events, including chromosome condensation and segregation have been directly linked to Cdk activity. However, the regulation and timing of cytoplasmic mitotic events by cyclin:Cdks is poorly understood. In order to examine these mitotic cytoplasmic events, we looked at the dramatic changes in the endoplasmic reticulum (ER) during mitosis in the early Drosophila embryo. The dynamic changes of the ER can be arrested in an interphase state by inhibition of either DNA or protein synthesis. Here we show that this block can be alleviated by micro-injection of Cyclin A (CycA) in which defined mitotic ER clusters gathered at the spindle poles. Conversely, micro-injection of Cyclin B (CycB) did not affect spatial reorganization of the ER, suggesting CycA possesses the ability to initiate mitotic ER events in the cytoplasm. Additionally, RNAi-mediated simultaneous inhibition of all 3 mitotic cyclins (A, B and B3) blocked spatial reorganization of the ER. Our results suggest that mitotic ER reorganization events rely on CycA and that control and timing of nuclear and cytoplasmic events during mitosis may be defined by release of CycA from the nucleus as a consequence of breakdown of the nuclear envelope. PMID:25689737

  1. MASK, a large ankyrin repeat and KH domain-containing protein involved in Drosophila receptor tyrosine kinase signaling.

    PubMed

    Smith, Rachel K; Carroll, Pamela M; Allard, John D; Simon, Michael A

    2002-01-01

    The receptor tyrosine kinases Sevenless (SEV) and the Epidermal growth factor receptor (EGFR) are required for the proper development of the Drosophila eye. The protein tyrosine phosphatase Corkscrew (CSW) is a common component of many RTK signaling pathways, and is required for signaling downstream of SEV and EGFR. In order to identify additional components of these signaling pathways, mutations that enhanced the phenotype of a dominant negative form of Corkscrew were isolated. This genetic screen identified the novel signaling molecule MASK, a large protein that contains two blocks of ankyrin repeats as well as a KH domain. MASK genetically interacts with known components of these RTK signaling pathways. In the developing eye imaginal disc, loss of MASK function generates phenotypes similar to those generated by loss of other components of the SEV and EGFR pathways. These phenotypes include compromised photoreceptor differentiation, cell survival and proliferation. Although MASK is localized predominantly in the cellular cytoplasm, it is not absolutely required for MAPK activation or nuclear translocation. Based on our results, we propose that MASK is a novel mediator of RTK signaling, and may act either downstream of MAPK or transduce signaling through a parallel branch of the RTK pathway.

  2. 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

  3. 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. Copyright © 2011 EMBO Molecular Medicine.

  4. PI3 kinase signaling is involved in Aβ-induced memory loss in Drosophila

    PubMed Central

    Chiang, Hsueh-Cheng; Wang, Lei; Xie, Zuolei; Yau, Alice; Zhong, Yi

    2010-01-01

    Multiple intracellular signals are altered in Alzheimer's disease brain tissues, including the PI3K/Akt pathway. However, the pathological relevance of such alterations is poorly understood. In vitro studies yield results that seem to be consistent with the conventional perception in which an up-regulation of the cell survival pathway, PI3K pathway, is protective in Alzheimer's disease pathogenesis. The current in vivo genetic approach, however, reveals that inhibition of the PI3K pathway leads to rescuing of the β-amyloid peptide (Aβ)-induced memory loss in the Drosophila brain. We began our inquiry into the molecular basis of this memory loss by studying Aβ42-induced enhancement of long-term depression. We found that long-term depression is restored to a normal level through inhibition of PI3K activity. Aβ42-induced PI3K hyperactivity is directly confirmed by immunostaining of the PI3K phosphorylation targets, phospholipids. Such observations lead to the following demonstration that Aβ42-induced memory loss can be rescued through genetic silencing or pharmacological inhibition of PI3K functions. Our data suggest that Aβ42 stimulates PI3K, which in turn causes memory loss in association with an increase in accumulation of Aβ42 aggregates. PMID:20351282

  5. PI3 kinase signaling is involved in Abeta-induced memory loss in Drosophila.

    PubMed

    Chiang, Hsueh-Cheng; Wang, Lei; Xie, Zuolei; Yau, Alice; Zhong, Yi

    2010-04-13

    Multiple intracellular signals are altered in Alzheimer's disease brain tissues, including the PI3K/Akt pathway. However, the pathological relevance of such alterations is poorly understood. In vitro studies yield results that seem to be consistent with the conventional perception in which an up-regulation of the cell survival pathway, PI3K pathway, is protective in Alzheimer's disease pathogenesis. The current in vivo genetic approach, however, reveals that inhibition of the PI3K pathway leads to rescuing of the beta-amyloid peptide (Abeta)-induced memory loss in the Drosophila brain. We began our inquiry into the molecular basis of this memory loss by studying Abeta42-induced enhancement of long-term depression. We found that long-term depression is restored to a normal level through inhibition of PI3K activity. Abeta42-induced PI3K hyperactivity is directly confirmed by immunostaining of the PI3K phosphorylation targets, phospholipids. Such observations lead to the following demonstration that Abeta42-induced memory loss can be rescued through genetic silencing or pharmacological inhibition of PI3K functions. Our data suggest that Abeta42 stimulates PI3K, which in turn causes memory loss in association with an increase in accumulation of Abeta42 aggregates.

  6. 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

  7. cGMP-Dependent Protein Kinase Inhibition Extends the Upper Temperature Limit of Stimulus-Evoked Calcium Responses in Motoneuronal Boutons of Drosophila melanogaster Larvae

    PubMed Central

    Dawson-Scully, Ken

    2016-01-01

    While the mammalian brain functions within a very narrow range of oxygen concentrations and temperatures, the fruit fly, Drosophila melanogaster, has employed strategies to deal with a much wider range of acute environmental stressors. The foraging (for) gene encodes the cGMP-dependent protein kinase (PKG), has been shown to regulate thermotolerance in many stress-adapted species, including Drosophila, and could be a potential therapeutic target in the treatment of hyperthermia in mammals. Whereas previous thermotolerance studies have looked at the effects of PKG variation on Drosophila behavior or excitatory postsynaptic potentials at the neuromuscular junction (NMJ), little is known about PKG effects on presynaptic mechanisms. In this study, we characterize presynaptic calcium ([Ca2+]i) dynamics at the Drosophila larval NMJ to determine the effects of high temperature stress on synaptic transmission. We investigated the neuroprotective role of PKG modulation both genetically using RNA interference (RNAi), and pharmacologically, to determine if and how PKG affects presynaptic [Ca2+]i dynamics during hyperthermia. We found that PKG activity modulates presynaptic neuronal Ca2+ responses during acute hyperthermia, where PKG activation makes neurons more sensitive to temperature-induced failure of Ca2+ flux and PKG inhibition confers thermotolerance and maintains normal Ca2+ dynamics under the same conditions. Targeted motoneuronal knockdown of PKG using RNAi demonstrated that decreased PKG expression was sufficient to confer thermoprotection. These results demonstrate that the PKG pathway regulates presynaptic motoneuronal Ca2+ signaling to influence thermotolerance of presynaptic function during acute hyperthermia. PMID:27711243

  8. cGMP-Dependent Protein Kinase Inhibition Extends the Upper Temperature Limit of Stimulus-Evoked Calcium Responses in Motoneuronal Boutons of Drosophila melanogaster Larvae.

    PubMed

    Krill, Jennifer L; Dawson-Scully, Ken

    2016-01-01

    While the mammalian brain functions within a very narrow range of oxygen concentrations and temperatures, the fruit fly, Drosophila melanogaster, has employed strategies to deal with a much wider range of acute environmental stressors. The foraging (for) gene encodes the cGMP-dependent protein kinase (PKG), has been shown to regulate thermotolerance in many stress-adapted species, including Drosophila, and could be a potential therapeutic target in the treatment of hyperthermia in mammals. Whereas previous thermotolerance studies have looked at the effects of PKG variation on Drosophila behavior or excitatory postsynaptic potentials at the neuromuscular junction (NMJ), little is known about PKG effects on presynaptic mechanisms. In this study, we characterize presynaptic calcium ([Ca2+]i) dynamics at the Drosophila larval NMJ to determine the effects of high temperature stress on synaptic transmission. We investigated the neuroprotective role of PKG modulation both genetically using RNA interference (RNAi), and pharmacologically, to determine if and how PKG affects presynaptic [Ca2+]i dynamics during hyperthermia. We found that PKG activity modulates presynaptic neuronal Ca2+ responses during acute hyperthermia, where PKG activation makes neurons more sensitive to temperature-induced failure of Ca2+ flux and PKG inhibition confers thermotolerance and maintains normal Ca2+ dynamics under the same conditions. Targeted motoneuronal knockdown of PKG using RNAi demonstrated that decreased PKG expression was sufficient to confer thermoprotection. These results demonstrate that the PKG pathway regulates presynaptic motoneuronal Ca2+ signaling to influence thermotolerance of presynaptic function during acute hyperthermia.

  9. 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

  10. Drosophila Jun kinase regulates expression of decapentaplegic via the ETS-domain protein Aop and the AP-1 transcription factor DJun during dorsal closure.

    PubMed

    Riesgo-Escovar, J R; Hafen, E

    1997-07-01

    During Drosophila embryogenesis, ectodermal cells of the lateral epithelium stretch in a coordinated fashion to internalize the amnioserosa cells and close the embryo dorsally. This process, dorsal closure, requires two signaling pathways: the Drosophila Jun-amino-terminal kinase (DJNK) pathway and the Dpp pathway. We have identified mutations in DJun and show that DJNK controls dorsal closure by activating DJun and inactivating the ETS repressor Aop/Yan by phosphorylation. DJun and Aop regulate dpp expression in the most dorsal row of cells. Secreted Dpp then instructs more ventrally located cells to stretch. Our results provide a causal link between the DJNK and Dpp pathways during dorsal closure. Interestingly, in vertebrates, transforming growth factor-beta and c-Jun regulate collagenase gene expression during wound healing, a process that also involves the closing of an epithelial sheath.

  11. A protein related to p21-activated kinase (PAK) that is involved in neurogenesis in the Drosophila adult central nervous system.

    PubMed

    Melzig, J; Rein, K H; Schäfer, U; Pfister, H; Jäckle, H; Heisenberg, M; Raabe, T

    1998-11-05

    Brains are organized by the developmental processes generating them. The embryonic neurogenic phase of Drosophila melanogaster has been studied in detail at the genetic, cellular and molecular level. In contrast, much of what is known of postembryonic brain development has been gathered by neuroanatomical and gene expression studies. The molecular mechanisms underlying cellular diversity and structural organisation in the adult brain, such as the establishment of the correct neuroblast number, the spatial and temporal control of neuroblast proliferation, cell fate determination, and the generation of the precise pattern of neuronal connectivity, are largely unknown. In a screen for viable mutations affecting adult central brain structures, we isolated the mushroom bodies tiny (mbt) gene of Drosophila, which encodes a protein related to p21-activated kinase (PAK). We show that mutations in mbt primarily interfere with the generation or survival of the intrinsic cells (Kenyon cells) of the mushroom body, a paired neuropil structure in the adult brain involved in learning and memory.

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

    PubMed

    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

    2013-03-26

    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.

  13. Distinct effects of Abelson kinase mutations on myocytes and neurons in dissociated Drosophila embryonic cultures: mimicking of high temperature.

    PubMed

    Liu, Lijuan; Wu, Chun-Fang

    2014-01-01

    Abelson tyrosine kinase (Abl) is known to regulate axon guidance, muscle development, and cell-cell interaction in vivo. The Drosophila primary culture system offers advantages in exploring the cellular mechanisms mediated by Abl with utilizing various experimental manipulations. Here we demonstrate that single-embryo cultures exhibit stage-dependent characteristics of cellular differentiation and developmental progression in neurons and myocytes, as well as nerve-muscle contacts. In particular, muscle development critically depends on the stage of dissociated embryos. In wild-type (WT) cultures derived from embryos before stage 12, muscle cells remained within cell clusters and were rarely detected. Interestingly, abundant myocytes were spotted in Abl mutant cultures, exhibiting enhanced myocyte movement and fusion, as well as neuron-muscle contacts even in cultures dissociated from younger, stage 10 embryos. Notably, Abl myocytes frequently displayed well-expanded lamellipodia. Conversely, Abl neurons were characterized with fewer large veil-like lamellipodia, but instead had increased numbers of filopodia and darker nodes along neurites. These distinct phenotypes were equally evident in both homo- and hetero-zygous cultures (Abl/Abl vs. Abl/+) of different alleles (Abl(1) and Abl(4) ) indicating dominant mutational effects. Strikingly, in WT cultures derived from stage 10 embryos, high temperature (HT) incubation promoted muscle migration and fusion, partially mimicking the advanced muscle development typical of Abl cultures. However, HT enhanced neuronal growth with increased numbers of enlarged lamellipodia, distinct from the characteristic Abl neuronal morphology. Intriguingly, HT incubation also promoted Abl lamellipodia expansion, with a much greater effect on nerve cells than muscle. Our results suggest that Abl is an essential regulator for myocyte and neuron development and that high-temperature incubation partially mimics the faster muscle development

  14. Drosophila Aurora B Kinase Is Required for Histone H3 Phosphorylation and Condensin Recruitment during Chromosome Condensation and to Organize the Central Spindle during Cytokinesis

    PubMed Central

    Giet, Régis; Glover, David M.

    2001-01-01

    Aurora/Ipl1-related kinases are a conserved family of enzymes that have multiple functions during mitotic progression. Although it has been possible to use conventional genetic analysis to dissect the function of aurora, the founding family member in Drosophila (Glover, D.M., M.H. Leibowitz, D.A. McLean, and H. Parry. 1995. Cell. 81:95–105), the lack of mutations in a second aurora-like kinase gene, aurora B, precluded this approach. We now show that depleting Aurora B kinase using double-stranded RNA interference in cultured Drosophila cells results in polyploidy. aurora B encodes a passenger protein that associates first with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. Cells depleted of the Aurora B kinase show only partial chromosome condensation at mitosis. This is associated with a reduction in levels of the serine 10 phosphorylated form of histone H3 and a failure to recruit the Barren condensin protein onto chromosomes. These defects are associated with abnormal segregation resulting from lagging chromatids and extensive chromatin bridging at anaphase, similar to the phenotype of barren mutants (Bhat, M.A., A.V. Philp, D.M. Glover, and H.J. Bellen. 1996. Cell. 87:1103–1114.). The majority of treated cells also fail to undertake cytokinesis and show a reduced density of microtubules in the central region of the spindle. This is accompanied by a failure to correctly localize the Pavarotti kinesin-like protein, essential for this process. We discuss these conserved functions of Aurora B kinase in chromosome transmission and cytokinesis. PMID:11266459

  15. 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.

  16. 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.

  17. 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 Central

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

    2015-01-01

    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

  18. The Drosophila TIPE family member Sigmar interacts with the Ste20-like kinase Misshapen and modulates JNK signaling, cytoskeletal remodeling and autophagy.

    PubMed

    Chittaranjan, Suganthi; Xu, Jing; Kuzyk, Michael; Dullat, Harpreet K; Wilton, James; DeVorkin, Lindsay; Lebovitz, Chandra; Morin, Gregg B; Marra, Marco A; Gorski, Sharon M

    2015-04-02

    TNFAIP8 and other mammalian TIPE family proteins have attracted increased interest due to their associations with disease-related processes including oncogenic transformation, metastasis, and inflammation. The molecular and cellular functions of TIPE family proteins are still not well understood. Here we report the molecular and genetic characterization of the Drosophila TNFAIP8 homolog, CG4091/sigmar. Previous gene expression studies revealed dynamic expression of sigmar in larval salivary glands prior to histolysis. Here we demonstrate that in sigmar loss-of-function mutants, the salivary glands are morphologically abnormal with defects in the tubulin network and decreased autophagic flux. Sigmar localizes subcellularly to microtubule-containing projections in Drosophila S2 cells, and co-immunoprecipitates with the Ste20-like kinase Misshapen, a regulator of the JNK pathway. Further, the Drosophila TNF ligand Eiger can induce sigmar expression, and sigmar loss-of-function leads to altered localization of pDJNK in salivary glands. Together, these findings link Sigmar to the JNK pathway, cytoskeletal remodeling and autophagy activity during salivary gland development, and provide new insights into TIPE family member function.

  19. The Drosophila TIPE family member Sigmar interacts with the Ste20-like kinase Misshapen and modulates JNK signaling, cytoskeletal remodeling and autophagy

    PubMed Central

    Chittaranjan, Suganthi; Xu, Jing; Kuzyk, Michael; Dullat, Harpreet K.; Wilton, James; DeVorkin, Lindsay; Lebovitz, Chandra; Morin, Gregg B.; Marra, Marco A.; Gorski, Sharon M.

    2015-01-01

    TNFAIP8 and other mammalian TIPE family proteins have attracted increased interest due to their associations with disease-related processes including oncogenic transformation, metastasis, and inflammation. The molecular and cellular functions of TIPE family proteins are still not well understood. Here we report the molecular and genetic characterization of the Drosophila TNFAIP8 homolog, CG4091/sigmar. Previous gene expression studies revealed dynamic expression of sigmar in larval salivary glands prior to histolysis. Here we demonstrate that in sigmar loss-of-function mutants, the salivary glands are morphologically abnormal with defects in the tubulin network and decreased autophagic flux. Sigmar localizes subcellularly to microtubule-containing projections in Drosophila S2 cells, and co-immunoprecipitates with the Ste20-like kinase Misshapen, a regulator of the JNK pathway. Further, the Drosophila TNF ligand Eiger can induce sigmar expression, and sigmar loss-of-function leads to altered localization of pDJNK in salivary glands. Together, these findings link Sigmar to the JNK pathway, cytoskeletal remodeling and autophagy activity during salivary gland development, and provide new insights into TIPE family member function. PMID:25836674

  20. 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

  1. Disruption of Glycerol Metabolism by RNAi Targeting of Genes Encoding Glycerol Kinase Results in a Range of Phenotype Severity in Drosophila

    PubMed Central

    Wightman, Patrick J.; Jackson, George R.; Dipple, Katrina M.

    2013-01-01

    In Drosophila, RNAi targeting of either dGyk or dGK can result in two alternative phenotypes: adult glycerol hypersensitivity or larval lethality. Here we compare these two phenotypes at the level of glycerol kinase (GK) phosphorylation activity, dGyk and dGK-RNA expression, and glycerol levels. We found both phenotypes exhibit reduced but similar levels of GK phosphorylation activity. Reduced RNA expression levels of dGyk and dGK corresponded with RNAi progeny that developed into glycerol hypersensitive adult flies. However, quantification of dGyk/dGK expression levels for the larval lethality phenotype revealed unexpected levels possibly due to a compensatory mechanism between dGyk and dGK or RNAi inhibition. The enzymatic role of glycerol kinase converts glycerol to glycerol 3-phosphate. As expected, elevated glycerol levels were observed in larvae that went on to develop into glycerol hypersensitive adults. Interestingly, larvae that died before eclosion revealed extremely low glycerol levels. Further characterization identified a wing phenotype that is enhanced by a dGpdh null mutation, indicating disrupted glycerol metabolism underlies the wing phenotype. In humans, glycerol kinase deficiency (GKD) exhibits a wide range of phenotypic variation with no obvious genotype-phenotype correlations. Additionally, disease severity often does not correlate with GK phosphorylation activity. It is intriguing that both human GKD patients and our GKD Drosophila model show a range of phenotype severity. Additionally, the lack of correlation between GK phosphorylation and dGyk/dGK-RNA expression with phenotypic severity suggests further study including understanding the alternative functions of the GK protein, could provide insights into the complex pathogenic mechanism observed in human GKD patients. PMID:24039719

  2. Human Biodistribution and Radiation Dosimetry of (18)F-Clofarabine, a PET Probe Targeting the Deoxyribonucleoside Salvage Pathway.

    PubMed

    Barrio, Martin J; Spick, Claudio; Radu, Caius G; Lassmann, Michael; Eberlein, Uta; Allen-Auerbach, Martin; Schiepers, Christiaan; Slavik, Roger; Czernin, Johannes; Herrmann, Ken

    2017-03-01

    (18)F-clofarabine, a nucleotide purine analog, is a substrate for deoxycytidine kinase (dCK), a key enzyme in the deoxyribonucleoside salvage pathway. (18)F-clofarabine might be used to measure dCK expression and thus serve as a predictive biomarker for tumor responses to dCK-dependent prodrugs or small-molecule dCK inhibitors, respectively. As a prerequisite for clinical translation, we determined the human whole-body and organ dosimetry of (18)F-clofarabine. Methods: Five healthy volunteers were injected intravenously with 232.4 ± 1.5 MBq of (18)F-clofarabine. Immediately after tracer injection, a dynamic scan of the entire chest was acquired for 30 min. This was followed by 3 static whole-body scans at 45, 90, and 135 min after tracer injection. Regions of interest were drawn around multiple organs on the CT scan and copied to the PET scans. Organ activity was determined and absorbed dose was estimated with OLINDA/EXM software. Results: The urinary bladder (critical organ), liver, kidney, and spleen exhibited the highest uptake. For an activity of 250 MBq, the absorbed doses in the bladder, liver, kidney, and spleen were 58.5, 6.6, 6.3, and 4.3 mGy, respectively. The average effective dose coefficient was 5.1 mSv. Conclusion: Our results hint that (18)F-clofarabine can be used safely in humans to measure tissue dCK expression. Future studies will determine whether (18)F-clofarabine may serve as a predictive biomarker for responses to dCK-dependent prodrugs or small-molecule dCK inhibitors.

  3. Mutations in CG8878, a Novel Putative Protein Kinase, Enhance P Element Dependent Silencing (PDS) and Position Effect Variegation (PEV) in Drosophila melanogaster

    PubMed Central

    McCracken, Allen; Locke, John

    2014-01-01

    Genes in multicellular organisms are expressed as part of a developmental program that is largely dependent on self-perpetuating higher-order chromatin states. The mechanism of establishing and maintaining these epigenetic events is well studied in Drosophila. The first known example of an epigenetic effect was that of (PEV) in Drosophila, which has been shown to be due to gene silencing via heterochromatin formation. We are investigating a process similar to Position Effect Variegation (PEV) using a mini-w transgene, called Pci, inserted in the upstream regulatory region of ci. The mini-white+ transgene in Pci is expressed throughout the adult eye; however, when other P or KP elements are present, a variegated eye phenotype results indicating random w+ silencing during development. This P element dependent silencing (PDS) can be modified by the haplo-suppressors/triplo-enhancers, Su(var)205 and Su(var)3–7, indicating that these heterochromatic modifiers also act dose dependently in PDS. Here we use a spontaneous derivative mutation of Pci called PciE1 (E1) that variegates like PDS in the absence of P elements, presumably due to an adjacent gypsy element insertion, to screen for second-site modifier mutations that enhance variable silencing of white+ in E1. We isolated 7 mutations in CG8878, an essential gene, that enhance the E1 variegated phenotype. CG8878, a previously uncharacterized gene, potentially encodes a serine/threonine kinase whose closest Drosophila paralogue, ballchen (nhk-1), phosphorylates histones. These mutant alleles enhance both PDS at E1 and Position Effect Variegation (PEV) at wm4, indicating a previously unknown common silencing mechanism between the two. PMID:24614804

  4. A cGMP-Dependent Protein Kinase Gene, foraging, Modifies Habituation-Like Response Decrement of the Giant Fiber Escape Circuit in Drosophila

    PubMed Central

    Engel, Jeff E.; Xie, Xian-Jin; Sokolowski, Marla B.; Wu, Chun-Fang

    2000-01-01

    The Drosophila giant fiber jump-and-flight escape response is a model for genetic analysis of both the physiology and the plasticity of a sensorimotor behavioral pathway. We previously established the electrically induced giant fiber response in intact tethered flies as a model for habituation, a form of nonassociative learning. Here, we show that the rate of stimulus-dependent response decrement of this neural pathway in a habituation protocol is correlated with PKG (cGMP-Dependent Protein Kinase) activity and foraging behavior. We assayed response decrement for natural and mutant rover and sitter alleles of the foraging (for) gene that encodes a Drosophila PKG. Rover larvae and adults, which have higher PKG activities, travel significantly farther while foraging than sitters with lower PKG activities. Response decrement was most rapid in genotypes previously shown to have low PKG activities and sitter-like foraging behavior. We also found differences in spontaneous recovery (the reversal of response decrement during a rest from stimulation) and a dishabituation-like phenomenon (the reversal of response decrement evoked by a novel stimulus). This electrophysiological study in an intact animal preparation provides one of the first direct demonstrations that PKG can affect plasticity in a simple learning paradigm. It increases our understanding of the complex interplay of factors that can modulate the sensitivity of the giant fiber escape response, and it defines a new adult-stage phenotype of the foraging locus. Finally, these results show that behaviorally relevant neural plasticity in an identified circuit can be influenced by a single-locus genetic polymorphism existing in a natural population of Drosophila. PMID:11040266

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

    PubMed

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

    2014-11-01

    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.

  6. A sensitized genetic screen to identify novel regulators and components of the Drosophila janus kinase/signal transducer and activator of transcription pathway.

    PubMed Central

    Bach, Erika A; Vincent, Stephane; Zeidler, Martin P; Perrimon, Norbert

    2003-01-01

    The JAK/STAT pathway exerts pleiotropic effects on a wide range of developmental processes in Drosophila. Four key components have been identified: Unpaired, a secreted ligand; Domeless, a cytokine-like receptor; Hopscotch, a JAK kinase; and Stat92E, a STAT transcription factor. The identification of additional components and regulators of this pathway remains an important issue. To this end, we have generated a transgenic line where we misexpress the upd ligand in the developing Drosophila eye. GMR-upd transgenic animals have dramatically enlarged eye-imaginal discs and compound eyes that are normally patterned. We demonstrate that the enlarged-eye phenotype is a result of an increase in cell number, and not cell volume, and arises from additional mitoses in larval eye discs. Thus, the GMR-upd line represents a system in which the proliferation and differentiation of eye precursor cells are separable. Removal of one copy of stat92E substantially reduces the enlarged-eye phenotype. We performed an F1 deficiency screen to identify dominant modifiers of the GMR-upd phenotype. We have identified 9 regions that enhance this eye phenotype and two specific enhancers: C-terminal binding protein and Daughters against dpp. We also identified 20 regions that suppress GMR-upd and 13 specific suppressors: zeste-white 13, pineapple eye, Dichaete, histone 2A variant, headcase, plexus, kohtalo, crumbs, hedgehog, decapentaplegic, thickveins, saxophone, and Mothers against dpp. PMID:14668372

  7. DISCO interacting protein 2 determines direction of axon projection under the regulation of c-Jun N-terminal kinase in the Drosophila mushroom body.

    PubMed

    Nitta, Yohei; Sugie, Atsushi

    2017-04-08

    Precisely controlled axon guidance for complex neuronal wiring is essential for appropriate neuronal function. c-Jun N-terminal kinase (JNK) was found to play a role in axon guidance recently as well as in cell proliferation, protection and apoptosis. In spite of many genetic and molecular studies on these biological processes regulated by JNK, how JNK regulates axon guidance accurately has not been fully explained thus far. To address this question, we use the Drosophila mushroom body (MB) as a model since the α/β axons project in two distinct directions. Here we show that DISCO interacting protein 2 (DIP2) is required for the accurate direction of axonal guidance. DIP2 expression is under the regulation of Basket (Bsk), the Drosophila homologue of JNK. We additionally found that the Bsk/DIP2 pathway is independent from the AP-1 transcriptional factor complex pathway, which is directly activated by Bsk. In conclusion, our findings revealed DIP2 as a novel effector downstream of Bsk modulating the direction of axon projection.

  8. Role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila melanogaster wing imaginal disc.

    PubMed

    Mattila, Jaakko; Omelyanchuk, Leonid; Kyttälä, Satu; Turunen, Heikki; Nokkala, Seppo

    2005-01-01

    When a fragment of a Drosophila imaginal disc is cultured in growth permissive conditions, it either regenerates the missing structures or duplicates the pattern present in the fragment. This kind of pattern regulation is known to be epimorphic, i.e. the new pattern is generated by proliferation in a specialized tissue called the blastema. Pattern regulation is accompanied by the healing of the cut surfaces restoring the continuous epithelia. Wound healing has been considered to be the inductive signal to commence regenerative cell divisions. Although the general outlines of the proliferation dynamics in a regenerating imaginal disc blastema have been well studied, little is known about the mechanisms driving cells into the regenerative cell cycles. In this study, we have investigated the role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila wing imaginal disc. By utilizing in vivo and in vitro culturing of incised and fragmented discs, we have been able to visualize the dynamics in cellular architecture and gene expression involved in the healing and regeneration process. Our results directly show that homotypic wound healing is not a prerequisite for regenerative cell divisions. We also show that JNK signaling participates in imaginal disc wound healing and is regulated by the physical dynamics of the process, as well as in recruiting cells into the regenerative cell cycles. A model describing the determination of blastema size is discussed.

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

    PubMed

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

    1998-12-01

    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.

  10. 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

  11. Leucine-Rich Repeat Kinase 2 interacts with Parkin, DJ-1 and PINK-1 in a Drosophila melanogaster model of Parkinson's disease.

    PubMed

    Venderova, Katerina; Kabbach, Ghassan; Abdel-Messih, Elizabeth; Zhang, Yi; Parks, Robin J; Imai, Yuzuru; Gehrke, Stephan; Ngsee, Johnny; Lavoie, Matthew J; Slack, Ruth S; Rao, Yong; Zhang, Zhuohua; Lu, Bingwei; Haque, M Emdadul; Park, David S

    2009-11-15

    Mutations in the LRRK2 gene are the most common genetic cause of familial Parkinson's disease (PD). However, its physiological and pathological functions are unknown. Therefore, we generated several independent Drosophila lines carrying WT or mutant human LRRK2 (mutations in kinase, COR or LRR domains, resp.). Ectopic expression of WT or mutant LRRK2 in dopaminergic neurons caused their significant loss accompanied by complex age-dependent changes in locomotor activity. Overall, the ubiquitous expression of LRRK2 increased lifespan and fertility of the flies. However, these flies were more sensitive to rotenone. LRRK2 expression in the eye exacerbated retinal degeneration. Importantly, in double transgenic flies, various indices of the eye and dopaminergic survival were modified in a complex fashion by a concomitant expression of PINK1, DJ-1 or Parkin. This evidence suggests a genetic interaction between these PD-relevant genes.

  12. Mechanisms of Allosteric Activation and Inhibition of the Deoxyribonucleoside Triphosphate Triphosphohydrolase from Enterococcus faecalis*♦

    PubMed Central

    Vorontsov, Ivan I.; Wu, Ying; DeLucia, Maria; Minasov, George; Mehrens, Jennifer; Shuvalova, Ludmilla; Anderson, Wayne F.; Ahn, Jinwoo

    2014-01-01

    EF1143 from Enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase), converting dNTPs into the deoxyribonucleosides and triphosphate. The dNTPase activity of EF1143 is regulated by canonical dNTPs, which simultaneously act as substrates and activity modulators. Previous crystal structures of apo-EF1143 and the protein bound to both dGTP and dATP suggested allosteric regulation of its enzymatic activity by dGTP binding at four identical allosteric sites. However, whether and how other canonical dNTPs regulate the enzyme activity was not defined. Here, we present the crystal structure of EF1143 in complex with dGTP and dTTP. The new structure reveals that the tetrameric EF1143 contains four additional secondary allosteric sites adjacent to the previously identified dGTP-binding primary regulatory sites. Structural and enzyme kinetic studies indicate that dGTP binding to the first allosteric site, with nanomolar affinity, is a prerequisite for substrate docking and hydrolysis. Then, the presence of a particular dNTP in the second site either enhances or inhibits the dNTPase activity of EF1143. Our results provide the first mechanistic insight into dNTP-mediated regulation of dNTPase activity. PMID:24338016

  13. An Investigation of 2′-Deoxyribonucleoside Cyanoboranes in Mice for Therapeutic Safety

    PubMed Central

    Hall, Iris H.; Burnham, Bruce S.; Rajendran, K. G.; Chang, J.-J.; Sood, Anup; Spielvogel, Bernard

    1994-01-01

    A standard acute toxicity study was undertaken to assess 2′-deoxyribonucleoside cyanoboranes for therapeutic safety. 2′-Deoxyribonucleoside cyanoboranes and related derivatives were nontoxic at doses required for anti-neoplastic and hypolipidemic activities. At higher doses (50 and 100 mg/kg/day IP for 7 days), all treated animals survived with slight reductions in total body weight and small decrements in daily food consumption. No clinical chemistry value was elevated to a magnitude suggesting onset of organ specific toxicity. However, agents appeared to modulate subpopulations of white blood cells, i.e., more lymphocytes than PMNs were present in blood from treated animals as determined by differential cell counts. This modulation is correlated with increases in granulomatous foci in the spleen and mesentery of treated animals after 7 days. The kidney was damaged only by Compound 5 at 50 and 100 mg/kg/day; Compound 5 had the most potent anti-neoplastic activity. The compounds demonstrated no in vitro toxicity against human HCT-8 ileum cells. LD50 values were greater than 1000 mg/kg, IP, for all compounds. PMID:18476214

  14. A Drosophila homolog of cyclase-associated proteins collaborates with the Abl tyrosine kinase to control midline axon pathfinding.

    PubMed

    Wills, Zachary; Emerson, Mark; Rusch, Jannette; Bikoff, Jay; Baum, Buzz; Perrimon, Norbert; Van Vactor, David

    2002-11-14

    We demonstrate that Drosophila capulet (capt), a homolog of the adenylyl cyclase-associated protein that binds and regulates actin in yeast, associates with Abl in Drosophila cells, suggesting a functional relationship in vivo. We find a robust and specific genetic interaction between capt and Abl at the midline choice point where the growth cone repellent Slit functions to restrict axon crossing. Genetic interactions between capt and slit support a model where Capt and Abl collaborate as part of the repellent response. Further support for this model is provided by genetic interactions that both capt and Abl display with multiple members of the Roundabout receptor family. These studies identify Capulet as part of an emerging pathway linking guidance signals to regulation of cytoskeletal dynamics and suggest that the Abl pathway mediates signals downstream of multiple Roundabout receptors.

  15. 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

  16. 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

  17. Drosophila IKK-related kinase Ik2 and Katanin p60-like 1 regulate dendrite pruning of sensory neuron during metamorphosis.

    PubMed

    Lee, Hsiu-Hsiang; Jan, Lily Yeh; Jan, Yuh-Nung

    2009-04-14

    Pruning is a widely observed mechanism for developing nervous systems to refine their circuitry. During metamorphosis, certain Drosophila sensory neurons undergo large-scale dendrite pruning to remove their larval branches before regeneration of their adult dendrites. Dendrite pruning involves dendrite severing, followed with debris removal. Little is known about the molecular mechanisms underlying dendrite severing. Here, we show that both the Ik2 kinase and Katanin p60-like 1 (Kat-60L1) of the Katanin family of microtubule severing proteins are required for dendrite severing. Mutant neurons with disrupted Ik2 function have diminished ability in severing their larval dendrites in pupae. Conversely, premature activation of Ik2 triggers precocious dendrite severing in larvae, revealing a critical role of Ik2 in initiating dendrite severing. We found a role for Kat-60L1 in facilitating dendrite severing by breaking microtubule in proximal dendrites, where the dendrites subsequently separate from the soma. Our study thus implicates Ik2 and Kat-60L1 in dendrite severing that involves local microtubule disassembly.

  18. 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.

  19. 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.

  20. Overexpression of Partner of Numb Induces Asymmetric Distribution of the PI4P 5-Kinase Skittles in Mitotic Sensory Organ Precursor Cells in Drosophila

    PubMed Central

    Perdigoto, Carolina N. L. R.; Gervais, Louis; Overstreet, Erin; Fischer, Janice; Guichet, Antoine; Schweisguth, François

    2008-01-01

    Unequal segregation of cell fate determinants at mitosis is a conserved mechanism whereby cell fate diversity can be generated during development. In Drosophila, each sensory organ precursor cell (SOP) divides asymmetrically to produce an anterior pIIb and a posterior pIIa cell. The Par6-aPKC complex localizes at the posterior pole of dividing SOPs and directs the actin-dependent localization of the cell fate determinants Numb, Partner of Numb (Pon) and Neuralized at the opposite pole. The plasma membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the plasma membrane localization and activity of various proteins, including several actin regulators, thereby modulating actin-based processes. Here, we have examined the distribution of PIP2 and of the PIP2-producing kinase Skittles (Sktl) in mitotic SOPs. Our analysis indicates that both Sktl and PIP2 reporters are uniformly distributed in mitotic SOPs. In the course of this study, we have observed that overexpression of full-length Pon or its localization domain (LD) fused to the Red Fluorescent Protein (RFP::PonLD) results in asymmetric distribution of Sktl and PIP2 reporters in dividing SOPs. Our observation that Pon overexpression alters polar protein distribution is relevant because RFP::PonLD is often used as a polarity marker in dividing progenitors. PMID:18728778

  1. Overexpression of partner of numb induces asymmetric distribution of the PI4P 5-Kinase Skittles in mitotic sensory organ precursor cells in Drosophila.

    PubMed

    Perdigoto, Carolina N L R; Gervais, Louis; Overstreet, Erin; Fischer, Janice; Guichet, Antoine; Schweisguth, François

    2008-08-27

    Unequal segregation of cell fate determinants at mitosis is a conserved mechanism whereby cell fate diversity can be generated during development. In Drosophila, each sensory organ precursor cell (SOP) divides asymmetrically to produce an anterior pIIb and a posterior pIIa cell. The Par6-aPKC complex localizes at the posterior pole of dividing SOPs and directs the actin-dependent localization of the cell fate determinants Numb, Partner of Numb (Pon) and Neuralized at the opposite pole. The plasma membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the plasma membrane localization and activity of various proteins, including several actin regulators, thereby modulating actin-based processes. Here, we have examined the distribution of PIP2 and of the PIP2-producing kinase Skittles (Sktl) in mitotic SOPs. Our analysis indicates that both Sktl and PIP2 reporters are uniformly distributed in mitotic SOPs. In the course of this study, we have observed that overexpression of full-length Pon or its localization domain (LD) fused to the Red Fluorescent Protein (RFP::Pon(LD)) results in asymmetric distribution of Sktl and PIP2 reporters in dividing SOPs. Our observation that Pon overexpression alters polar protein distribution is relevant because RFP::Pon(LD) is often used as a polarity marker in dividing progenitors.

  2. Drosophila casein kinase I alpha regulates homolog pairing and genome organization by modulating condensin II subunit Cap-H2 levels.

    PubMed

    Nguyen, Huy Q; Nye, Jonathan; Buster, Daniel W; Klebba, Joseph E; Rogers, Gregory C; Bosco, Giovanni

    2015-01-01

    The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCFSlimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. Here, we identify Casein Kinase I alpha (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein.

  3. Cyclin-dependent Kinase 8 Module Expression Profiling Reveals Requirement of Mediator Subunits 12 and 13 for Transcription of Serpent-dependent Innate Immunity Genes in Drosophila*

    PubMed Central

    Kuuluvainen, Emilia; Hakala, Heini; Havula, Essi; Sahal Estimé, Michelle; Rämet, Mika; Hietakangas, Ville; Mäkelä, Tomi P.

    2014-01-01

    The Cdk8 (cyclin-dependent kinase 8) module of Mediator integrates regulatory cues from transcription factors to RNA polymerase II. It consists of four subunits where Med12 and Med13 link Cdk8 and cyclin C (CycC) to core Mediator. Here we have investigated the contributions of the Cdk8 module subunits to transcriptional regulation using RNA interference in Drosophila cells. Genome-wide expression profiling demonstrated separation of Cdk8-CycC and Med12-Med13 profiles. However, transcriptional regulation by Cdk8-CycC was dependent on Med12-Med13. This observation also revealed that Cdk8-CycC and Med12-Med13 often have opposite transcriptional effects. Interestingly, Med12 and Med13 profiles overlapped significantly with that of the GATA factor Serpent. Accordingly, mutational analyses indicated that GATA sites are required for Med12-Med13 regulation of Serpent-dependent genes. Med12 and Med13 were also found to be required for Serpent-activated innate immunity genes in defense to bacterial infection. The results reveal a novel role for the Cdk8 module in Serpent-dependent transcription and innate immunity. PMID:24778181

  4. Genetic and phenotypic analysis of alleles of the Drosophila chromosomal JIL-1 kinase reveals a functional requirement at multiple developmental stages.

    PubMed Central

    Zhang, Weiguo; Jin, Ye; Ji, Yun; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M

    2003-01-01

    In this study we provide a cytological and genetic characterization of the JIL-1 locus in Drosophila. JIL-1 is an essential chromosomal tandem kinase and in JIL-1 null animals chromatin structure is severely perturbed. Using a range of JIL-1 hypomorphic mutations, we show that they form an allelic series. JIL-1 has a strong maternal effect and JIL-1 activity is required at all stages of development, including embryonic, larval, and pupal stages. Furthermore, we identified a new allele of JIL-1, JIL-1(h9), that encodes a truncated protein missing COOH-terminal sequences. Remarkably, the truncated JIL-1 protein can partially restore viability without rescuing the defects in polytene chromosome organization. This suggests that sequences within this region of JIL-1 play an important role in establishing and/or maintaining normal chromatin structure. By analyzing the effects of JIL-1 mutations we provide evidence that JIL-1 function is necessary for the normal progression of several developmental processes at different developmental stages such as oogenesis and segment specification. We propose that JIL-1 may exert such effects by a general regulation of chromatin structure affecting gene expression. PMID:14668387

  5. 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

  6. Differential localizations of and requirements for the two Drosophila ninaC kinase/myosins in photoreceptor cells.

    PubMed

    Porter, J A; Hicks, J L; Williams, D S; Montell, C

    1992-02-01

    The ninaC gene encodes two retinal specific proteins (p132 and p174) consisting of a protein kinase domain joined to a domain homologous to the head region of the myosin heavy chain. The putative myosin domain of p174 is linked at the COOH-terminus to a tail which has some similarities to myosin-I tails. In the current report, we demonstrate that the ninaC mutation results in light- and age-dependent retinal degeneration. We also show that ninaC flies display an electrophysiological phenotype before any discernible retinal degeneration indicating that the electrophysiological defect is the primary effect of the mutation. This suggests that ninaC has a role in phototransduction and that the retinal degeneration is a secondary effect resulting from the defect in phototransduction. To examine the requirements for the individual ninaC isoforms, mutant alleles were generated which express only p132 or p174. Elimination of p174 resulted in a ninaC phenotype as strong as the null allele; however, elimination of p132 had little if any effect. As a first step in investigating the basis for the difference in requirements for p174 and p132 we performed immuno-localization at the electron microscopic level and found that the two isoforms display different subcellular distributions in the photoreceptor cells. The p132 protein is restricted primarily to the cytoplasm and p174 to the rhabdomeres, the microvillar structure which is the site of action of many of the steps in phototransduction. This suggests that the p174 myosin-I type tail is the domain responsible for association with the rhabdomeres and that the substrate for the p174 putative kinase may be a rhabdomeric protein important in photo-transduction.

  7. Drosophila Casein Kinase I Alpha Regulates Homolog Pairing and Genome Organization by Modulating Condensin II Subunit Cap-H2 Levels

    PubMed Central

    Nguyen, Huy Q.; Nye, Jonathan; Buster, Daniel W.; Klebba, Joseph E.; Rogers, Gregory C.; Bosco, Giovanni

    2015-01-01

    The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCFSlimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. Here, we identify Casein Kinase I alpha (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein. PMID:25723539

  8. 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. Copyright © 2016 the American Physiological Society.

  9. Evidence against a Role for the JIL-1 Kinase in H3S28 Phosphorylation and 14-3-3 Recruitment to Active Genes in Drosophila

    PubMed Central

    Li, Yeran; Cai, Weili; Bao, Xiaomin; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M.

    2013-01-01

    JIL-1 is the major kinase controlling phosphorylation of histone H3S10 and has been demonstrated to function to counteract heterochromatization and gene silencing. However, an alternative model has been proposed in which JIL-1 is required for transcription to occur, additionally phosphorylates H3S28, and recruits 14-3-3 to active genes. Since these findings are incompatible with our previous demonstration that there are robust levels of transcription in the complete absence of JIL-1 and that JIL-1 is not present at developmental or heat shock-induced polytene chromosome puffs, we have reexamined JIL-1’s possible role in H3S28 phosphorylation and 14-3-3 recruitment. Using two different H3S28ph antibodies we show by immunocytochemistry and immunoblotting that in Drosophila the H3S28ph mark is not present at detectable levels above background on polytene chromosomes at interphase but only on chromosomes at pro-, meta-, and anaphase during cell division in S2 cells and third instar larval neuroblasts. Moreover, this mitotic H3S28ph signal is also present in a JIL-1 null mutant background at undiminished levels suggesting that JIL-1 is not the mitotic H3S28ph kinase. We also demonstrate that H3S28ph is not enriched at heat shock puffs. Using two different pan-specific 14-3-3 antibodies as well as an enhancer trap 14-3-3ε-GFP line we show that 14-3-3, while present in salivary gland nuclei, does not localize to chromosomes but only to the nuclear matrix surrounding the chromosomes. In our hands 14-3-3 is not recruited to developmental or heat shock puffs. Furthermore, using a lacO repeat tethering system to target LacI-JIL-1 to ectopic sites on polytene chromosomes we show that only H3S10ph is present and upregulated at such sites, not H3S28ph or 14-3-3. Thus, our results argue strongly against a model where JIL-1 is required for H3S28 phosphorylation and 14-3-3 recruitment at active genes. PMID:23638096

  10. A Single Deoxynucleoside Kinase Variant from Drosophila melanogaster Synthesizes Monophosphates of Nucleosides That Are Components of an Expanded Genetic System.

    PubMed

    Matsuura, Mariko F; Winiger, Christian B; Shaw, Ryan W; Kim, Myong-Jung; Kim, Myong-Sang; Daugherty, Ashley B; Chen, Fei; Moussatche, Patricia; Moses, Jennifer D; Lutz, Stefan; Benner, Steven A

    2017-03-17

    Deoxynucleoside kinase from D. melanogaster (DmdNK) has broad specificity; although it catalyzes the phosphorylation of natural pyrimidine more efficiently than natural purine nucleosides, it accepts all four 2'-deoxynucleosides and many analogues, using ATP as a phosphate donor to give the corresponding deoxynucleoside monophosphates. Here, we show that replacing a single amino acid (glutamine 81 by glutamate) in DmdNK creates a variant that also catalyzes the phosphorylation of nucleosides that form part of an artificially expanded genetic information system (AEGIS). By shuffling hydrogen bonding groups on the nucleobases, AEGIS adds potentially as many as four additional nucleobase pairs to the genetic "alphabet". Specifically, we show that DmdNK Q81E creates the monophosphates from the AEGIS nucleosides dP, dZ, dX, and dK (respectively 2-amino-8-(1'-β-d-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, dP; 6-amino-3-(1'-β-d-2'-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one, dZ; 8-(1'β-d-2'-deoxy-ribofuranosyl)imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione, dX; and 2,4-diamino-5-(1'-β-d-2'-deoxyribofuranosyl)-pyrimidine, dK). Using a coupled enzyme assay, in vitro kinetic parameters were obtained for three of these nucleosides (dP, dX, and dK; the UV absorbance of dZ made it impossible to get its precise kinetic parameters). Thus, DmdNK Q81E appears to be a suitable enzyme to catalyze the first step in the biosynthesis of AEGIS 2'-deoxynucleoside triphosphates in vitro and, perhaps, in vivo, in a cell able to manage plasmids containing AEGIS DNA.

  11. Sample preparation and high-performance liquid chromatographic analysis of deoxyribonucleoside triphosphates in individual rat embryos.

    PubMed

    Mole, M L; Hunter, D L; Gao, P; Lau, C

    1998-06-01

    A rapid, robust, and sensitive method has been developed to measure concentrations of deoxyribonucleoside triphosphates in individual, day 14 rat embryos by modifying and optimizing existing methods for cellular extracts. Significant changes include: (i) oxidative degradation of ribonucleoside triphosphates using methylamine at lower pH (decreased from 6.5 to 4.0) to improve poor HPLC peak shape of early eluting nucleotides; (ii) glass fiber disc solid-phase extraction of the reaction mixture, which dramatically reduces impurities that interfere with nucleotide measurement, eliminates the necessity of column regeneration, and allows mobile phase recycling; and (iii) lower ionic strength (reduced from 0.4 to 0.26 or 0.12 M ammonium phosphate) and higher pH (increased from 3.25 to 5.55 or 6.98, respectively) mobile phase, conditions which are less destructive to the column's bonded phase and silica support, thereby contributing to longer column life. Enhancements include: (i) filtration of the sample prior to HPLC injection and addition of an in-line filter, guard column, and saturating precolumn of silica in the mobile phase flow, which aids substantially in extending column life and improves chromatographic stability, and (ii) inclusion of an internal standard to correct for mechanical losses. Limits of determination at a signal to noise ratio of 6:1 range from 5.5 to 12 pmol on-column or 0.41 to 0.87 pmol/mg of embryonic tissue depending on the specific nucleotide. Recoveries are quantitative for all nucleotides, and interassay variabilities are between 5 and 7% when quantified by peak height. The method has also been applied successfully to analysis of murine erythroleukemic cell cultures and this, when coupled with the embryo results, suggests its general utility.

  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. 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

    Schleede, Justin; Blair, Seth S

    2015-10-01

    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.

  14. The direct interaction between ASH2, a Drosophila trithorax group protein, and SKTL, a nuclear phosphatidylinositol 4-phosphate 5-kinase, implies a role for phosphatidylinositol 4,5-bisphosphate in maintaining transcriptionally active chromatin.

    PubMed Central

    Cheng, Mimi K; Shearn, Allen

    2004-01-01

    The products of trithorax group (trxG) genes maintain active transcription of many important developmental regulatory genes, including homeotic genes. Several trxG proteins have been shown to act in multimeric protein complexes that modify chromatin structure. ASH2, the product of the Drosophila trxG gene absent, small, or homeotic discs 2 (ash2) is a component of a 500-kD complex. In this article, we provide biochemical evidence that ASH2 binds directly to Skittles (SKTL), a predicted phosphatidylinositol 4-phosphate 5-kinase, and genetic evidence that the association of these proteins is functionally significant. We also show that histone H1 hyperphosphorylation is dramatically increased in both ash2 and sktl mutant polytene chromosomes. These results suggest that ASH2 maintains active transcription by binding a producer of nuclear phosphoinositides and downregulating histone H1 hyperphosphorylation. PMID:15280236

  15. The direct interaction between ASH2, a Drosophila trithorax group protein, and SKTL, a nuclear phosphatidylinositol 4-phosphate 5-kinase, implies a role for phosphatidylinositol 4,5-bisphosphate in maintaining transcriptionally active chromatin.

    PubMed

    Cheng, Mimi K; Shearn, Allen

    2004-07-01

    The products of trithorax group (trxG) genes maintain active transcription of many important developmental regulatory genes, including homeotic genes. Several trxG proteins have been shown to act in multimeric protein complexes that modify chromatin structure. ASH2, the product of the Drosophila trxG gene absent, small, or homeotic discs 2 (ash2) is a component of a 500-kD complex. In this article, we provide biochemical evidence that ASH2 binds directly to Skittles (SKTL), a predicted phosphatidylinositol 4-phosphate 5-kinase, and genetic evidence that the association of these proteins is functionally significant. We also show that histone H1 hyperphosphorylation is dramatically increased in both ash2 and sktl mutant polytene chromosomes. These results suggest that ASH2 maintains active transcription by binding a producer of nuclear phosphoinositides and downregulating histone H1 hyperphosphorylation.

  16. 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

    2015-11-27

    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.

  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. Reactions of {4-[bis(2-chloroethyl)amino]phenyl}acetic acid (phenylacetic acid mustard) with 2'-deoxyribonucleosides.

    PubMed

    Florea-Wang, Diana; Ijäs, Inna; Hakala, Kristo; Mattinen, Jorma; Vilpo, Juhani; Hovinen, Jari

    2007-03-01

    Phenylacetic acid mustard (PAM; 2), a major metabolite of the anticancer agent chlorambucil (CLB; 1), was allowed to react with 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG), 2'-deoxycytidine (dC), 2'-deoxy-5-methylcytidine (dMeC), and thymidine (T) at physiological pH (cacodylic acid, 50% base). The reactions were followed by HPLC and analyzed by HPLC/MS and/or (1)H-NMR techniques. Although the predominant reaction observed was hydrolysis of PAM, 2 also reacted with various heteroatoms of the nucleosides to give a series of products: compounds 5-31. PAM (2) was found to be hydrolytically slightly more stable than CLB (1). The principal reaction sites of 2 with dA, dG, and with all pyrimidine nucleosides were N(1), N(7), and N(3), resp. Also, several other adducts were detected and characterized. There was no significant difference in the reactivity of 1 and 2 with dG, dA or T, but the N(3) dC-PAM adduct was deaminated easier than the corresponding CLB derivative. The role of PAM-2'-deoxyribonucleoside adducts on the cytotoxic and mutagenic properties of CLB (1) is discussed.

  19. A genetic screen for novel components of the Ras/Mitogen-activated protein kinase signaling pathway that interact with the yan gene of Drosophila identifies split ends, a new RNA recognition motif-containing protein.

    PubMed Central

    Rebay, I; Chen, F; Hsiao, F; Kolodziej, P A; Kuang, B H; Laverty, T; Suh, C; Voas, M; Williams, A; Rubin, G M

    2000-01-01

    The receptor tyrosine kinase (RTK) signaling pathway is used reiteratively during the development of all multicellular organisms. While the core RTK/Ras/MAPK signaling cassette has been studied extensively, little is known about the nature of the downstream targets of the pathway or how these effectors regulate the specificity of cellular responses. Drosophila yan is one of a few downstream components identified to date, functioning as an antagonist of the RTK/Ras/MAPK pathway. Previously, we have shown that ectopic expression of a constitutively active protein (yan(ACT)) inhibits the differentiation of multiple cell types. In an effort to identify new genes functioning downstream in the Ras/MAPK/yan pathway, we have performed a genetic screen to isolate dominant modifiers of the rough eye phenotype associated with eye-specific expression of yan(ACT). Approximately 190,000 mutagenized flies were screened, and 260 enhancers and 90 suppressors were obtained. Among the previously known genes we recovered are four RTK pathway components, rolled (MAPK), son-of-sevenless, Star, and pointed, and two genes, eyes absent and string, that have not been implicated previously in RTK signaling events. We also isolated mutations in five previously uncharacterized genes, one of which, split ends, we have characterized molecularly and have shown to encode a member of the RRM family of RNA-binding proteins. PMID:10655223

  20. Atg6/UVRAG/Vps34-Containing Lipid Kinase Complex Is Required for Receptor Downregulation through Endolysosomal Degradation and Epithelial Polarity during Drosophila Wing Development

    PubMed Central

    Szatmári, Zsuzsanna; Sass, Miklós

    2014-01-01

    Atg6 (Beclin 1 in mammals) is a core component of the Vps34 PI3K (III) complex, which promotes multiple vesicle trafficking pathways. Atg6 and Vps34 form two distinct PI3K (III) complexes in yeast and mammalian cells, either with Atg14 or with UVRAG. The functions of these two complexes are not entirely clear, as both Atg14 and UVRAG have been suggested to regulate both endocytosis and autophagy. In this study, we performed a microscopic analysis of UVRAG, Atg14, or Atg6 loss-of-function cells in the developing Drosophila wing. Both autophagy and endocytosis are seriously impaired and defective endolysosomes accumulate upon loss of Atg6. We show that Atg6 is required for the downregulation of Notch and Wingless signaling pathways; thus it is essential for normal wing development. Moreover, the loss of Atg6 impairs cell polarity. Atg14 depletion results in autophagy defects with no effect on endocytosis or cell polarity, while the silencing of UVRAG phenocopies all but the autophagy defect of Atg6 depleted cells. Thus, our results indicate that the UVRAG-containing PI3K (III) complex is required for receptor downregulation through endolysosomal degradation and for the establishment of proper cell polarity in the developing wing, while the Atg14-containing complex is involved in autophagosome formation. PMID:25006588

  1. Role of Murine Leukemia Virus Reverse Transcriptase Deoxyribonucleoside Triphosphate-Binding Site in Retroviral Replication and In Vivo Fidelity

    PubMed Central

    Halvas, Elias K.; Svarovskaia, Evguenia S.; Pathak, Vinay K.

    2000-01-01

    Retroviral populations exhibit a high evolutionary potential, giving rise to extensive genetic variation. Error-prone DNA synthesis catalyzed by reverse transcriptase (RT) generates variation in retroviral populations. Structural features within RTs are likely to contribute to the high rate of errors that occur during reverse transcription. We sought to determine whether amino acids within murine leukemia virus (MLV) RT that contact the deoxyribonucleoside triphosphate (dNTP) substrate are important for in vivo fidelity of reverse transcription. We utilized the previously described ANGIE P encapsidating cell line, which expresses the amphotropic MLV envelope and a retroviral vector (pGA-1). pGA-1 expresses the bacterial β-galactosidase gene (lacZ), which serves as a reporter of mutations. Extensive mutagenesis was performed on residues likely to interact with the dNTP substrate, and the effects of these mutations on the fidelity of reverse transcription were determined. As expected, most substitution mutations of amino acids that directly interact with the dNTP substrate significantly reduced viral titers (>10,000-fold), indicating that these residues played a critical role in catalysis and viral replication. However, the D153A and A154S substitutions, which are predicted to affect the interactions with the triphosphate, resulted in statistically significant increases in the mutation rate. In addition, the conservative substitution F155W, which may affect interactions with the base and the ribose, increased the mutation rate 2.8-fold. Substitutions of residues in the vicinity of the dNTP-binding site also resulted in statistically significant decreases in fidelity (1.3- to 2.4-fold). These results suggest that mutations of residues that contact the substrate dNTP can affect viral replication as well as alter the fidelity of reverse transcription. PMID:11044079

  2. Enantioselectivity of human AMP, dTMP and UMP-CMP kinases

    PubMed Central

    Alexandre, Julie A.C.; Roy, Béatrice; Topalis, Dimitri; Pochet, Sylvie; Périgaud, Christian; Deville-Bonne, Dominique

    2007-01-01

    l-Nucleoside analogues such as lamivudine are active for treating viral infections. Like d-nucleosides, the biological activity of the l-enantiomers requires their stepwise phosphorylation by cellular or viral kinases to give the triphosphate. The enantioselectivity of NMP kinases has not been thoroughly studied, unlike that of deoxyribonucleoside kinases. We have therefore investigated the capacity of l-enantiomers of some natural (d)NMP to act as substrates for the recombinant forms of human uridylate-cytidylate kinase, thymidylate kinase and adenylate kinases 1 and 2. Both cytosolic and mitochondrial adenylate kinases were strictly enantioselective, as they phosphorylated only d-(d)AMP. l-dTMP was a substrate for thymidylate kinase, but with an efficiency 150-fold less than d-dTMP. Both l-dUMP and l-(d)CMP were phosphorylated by UMP-CMP kinase although much less efficiently than their natural counterparts. The stereopreference was conserved with the 2′-azido derivatives of dUMP and dUMP while, unexpectedly, the 2′-azido-d-dCMP was a 4-fold better substrate for UMP-CMP kinase than was CMP. Docking simulations showed that the small differences in the binding of d-(d)NMP to their respective kinases could account for the differences in interactions of the l-isomers with the enzymes. This in vitro information was then used to develop the in vivo activation pathway for l-dT. PMID:17626051

  3. 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.

  4. 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

  5. Protective effect of deoxyribonucleosides on UV-irradiated human peripheral blood T-lymphocytes: possibilities for the selective killing of either cycling or non-cycling cells.

    PubMed

    Green, M H; Waugh, A P; Lowe, J E; Harcourt, S A; Clingen, P H; Cole, J; Arlett, C F

    1996-02-19

    Non-cycling human T-lymphocytes from normal subjects show a 10-fold greater sensitivity than fibroblasts to UV-B (280-315 nm) irradiation from a Westinghouse FS20 lamp, but only a 2.7-fold greater sensitivity to UV-C (254 nm) irradiation. Hypersensitivity is associated with a deficiency in the rejoining of excision breaks. Non-cycling T-lymphocytes have extremely low deoxyribonucleotide pools. Addition to the medium of the four deoxyribonucleosides, each at a concentration of 10(-5) M, substantially increases survival and reduces the persistence of excision-related strand breaks following UV-B or UV-C irradiation (Yew and Johnson (1979) Biochim. Biophys. Acta 562, 240-241; Green et al. (1994) Mutation Res., 315, 25-32). UV-resistance of T-lymphocytes is also increased by stimulating the cells into cycle. The addition of deoxyribonucleosides does not further enhance survival of cycling cells and they do not reach the level of resistance achieved by non-cycling cells in the presence of deoxyribonucleosides. We suggest that two opposing effects are in operation. Cells out of cycle can show increased resistance to DNA damage in the absence of division but they also have reduced deoxyribonucleotide pools, which may limit DNA repair. With UV-B irradiation, the exceptionally low dNTP pools in non-cycling T-lymphocytes cause this second effect to predominate. In contrast, with ionising radiation, which forms highly cytotoxic double-strand breaks, non-cycling human T-lymphocytes are slightly more resistant than fibroblasts. Non-cycling cells such as T-lymphocytes should be especially sensitive to agents which produce a high proportion of read excisable damage, but should show normal resistance to agents which highly toxic lesions. It may be possible by choice of DNA damaging agent and manipulation of cellular deoxyribonucleotide pools, to choose regimes which will selectively kill either cycling or non-cycling cells and to improve the efficacy of standard therapeutic

  6. Construction of microbial platform for an energy-requiring bioprocess: practical 2′-deoxyribonucleoside production involving a C−C coupling reaction with high energy substrates

    PubMed Central

    2012-01-01

    Background Reproduction and sustainability are important for future society, and bioprocesses are one technology that can be used to realize these concepts. However, there is still limited variation in bioprocesses and there are several challenges, especially in the operation of energy-requiring bioprocesses. As an example of a microbial platform for an energy-requiring bioprocess, we established a process that efficiently and enzymatically synthesizes 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase. This method consists of the coupling reactions of the reversible nucleoside degradation pathway and energy generation through the yeast glycolytic pathway. Results Using E. coli that co-express deoxyriboaldolase and phosphopentomutase, a high amount of 2′-deoxyribonucleoside was produced with efficient energy transfer under phosphate-limiting reaction conditions. Keeping the nucleobase concentration low and the mixture at a low reaction temperature increased the yield of 2′-deoxyribonucleoside relative to the amount of added nucleobase, indicating that energy was efficiently generated from glucose via the yeast glycolytic pathway under these reaction conditions. Using a one-pot reaction in which small amounts of adenine, adenosine, and acetone-dried yeast were fed into the reaction, 75 mM of 2′-deoxyinosine, the deaminated product of 2′-deoxyadenosine, was produced from glucose (600 mM), acetaldehyde (250 mM), adenine (70 mM), and adenosine (20 mM) with a high yield relative to the total base moiety input (83%). Moreover, a variety of natural dNSs were further synthesized by introducing a base-exchange reaction into the process. Conclusion A critical common issue in energy-requiring bioprocess is fine control of phosphate concentration. We tried to resolve this problem, and provide the convenient recipe for establishment of energy-requiring bioprocesses. It is anticipated that the commercial demand for dNSs, which are primary

  7. Spatial control of the actin cytoskeleton in Drosophila epithelial cells.

    PubMed

    Baum, B; Perrimon, N

    2001-10-01

    The actin cytoskeleton orders cellular space and transduces many of the forces required for morphogenesis. Here we combine genetics and cell biology to identify genes that control the polarized distribution of actin filaments within the Drosophila follicular epithelium. We find that profilin and cofilin regulate actin-filament formation throughout the cell cortex. In contrast, CAP-a Drosophila homologue of Adenylyl Cyclase Associated Proteins-functions specifically to limit actin-filament formation catalysed by Ena at apical cell junctions. The Abl tyrosine kinase also collaborates in this process. We therefore propose that CAP, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila.

  8. CK1 and GSK3 in the Drosophila and mammalian circadian clock.

    PubMed

    Harms, Emily; Young, Michael W; Saez, Lino

    2003-01-01

    Two kinases, DOUBLETIME and SHAGGY, have been shown to play a role in the circadian clock. DOUBLETIME, the Drosophila orthologue of casein kinase 1, can phosphorylate PERIOD in the cytoplasm and in the nucleus. This phosphorylation destabilizes PERIOD in both locations and sets patterns of both cytoplasmic accumulation and nuclear turnover. Cytoplasmic phosphorylation postpones accumulation of PERIOD and affects timing of nuclear accumulation of PERIOD/ TIMELESS complexes. SHAGGY, the Drosophila orthologue of glycogen synthase kinase 3, phosphorylates TIMELESS and promotes nuclear translocation of PERIOD/ TIMELESS complexes. Thus, the opposing effects of these two kinases in the cytoplasm are crucial for establishing the approximately 24 h period of circadian rhythmicity in Drosophila. Casein Kinase 1 has been shown to be a component of the circadian clock in mammals. Recent studies are also pointing to a role for glycogen synthase kinase 3 in the mammalian clock.

  9. 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"…

  10. 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"…

  11. 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

  12. 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

  13. 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.

  14. Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases

    PubMed Central

    Shiu, Shin-Han; Bleecker, Anthony B.

    2001-01-01

    Plant receptor-like kinases (RLKs) are proteins with a predicted signal sequence, single transmembrane region, and cytoplasmic kinase domain. Receptor-like kinases belong to a large gene family with at least 610 members that represent nearly 2.5% of Arabidopsis protein coding genes. We have categorized members of this family into subfamilies based on both the identity of the extracellular domains and the phylogenetic relationships between the kinase domains of subfamily members. Surprisingly, this structurally defined group of genes is monophyletic with respect to kinase domains when compared with the other eukaryotic kinase families. In an extended analysis, animal receptor kinases, Raf kinases, plant RLKs, and animal receptor tyrosine kinases form a well supported group sharing a common origin within the superfamily of serine/threonine/tyrosine kinases. Among animal kinase sequences, Drosophila Pelle and related cytoplasmic kinases fall within the plant RLK clade, which we now define as the RLK/Pelle family. A survey of expressed sequence tag records for land plants reveals that mosses, ferns, conifers, and flowering plants have similar percentages of expressed sequence tags representing RLK/Pelle homologs, suggesting that the size of this gene family may have been close to the present-day level before the diversification of land plant lineages. The distribution pattern of four RLK subfamilies on Arabidopsis chromosomes indicates that the expansion of this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. PMID:11526204

  15. Identification of a Drosophila activin receptor.

    PubMed Central

    Childs, S R; Wrana, J L; Arora, K; Attisano, L; O'Connor, M B; Massagué, J

    1993-01-01

    Activins are cytokines of the transforming growth factor beta superfamily that control various events during vertebrate embryo development and cell differentiation in the adult, and act through transmembrane receptors that contain a cytoplasmic protein-serine/threonine kinase domain. We describe the identification, deduced primary structure, and expression pattern of Atr-II, a receptor serine/threonine kinase found in Drosophila. With the exception of the spacing of 10 cysteine residues, the extracellular domain of Atr-II is very dissimilar from those of vertebrate activin receptors, yet it binds activin with high affinity and specificity. The kinase domain sequence of Atr-II is 60% identical to those of activin receptors from vertebrates, suggesting similarities in their signaling mechanisms. Maternal Atr-II transcript and its product are abundant in the oocyte. During development, the highest levels of Atr-II transcript and protein are observed in the mesoderm and gut. The possible role of an activin signaling system in Drosophila development is discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8415726

  16. 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

  17. Regulation of Drosophila lifespan by JNK signaling

    PubMed Central

    Biteau, Benoit; Karpac, Jason; Hwangbo, DaeSung; Jasper, Heinrich

    2010-01-01

    Cellular responses to extrinsic and intrinsic insults have to be carefully regulated to properly coordinate cytoprotection, repair processes, cell proliferation and apoptosis. Stress signaling pathways, most prominently the Jun-N-terminal Kinase (JNK) pathway, are critical regulators of such cellular responses and have accordingly been implicated in the regulation of lifespan in various organisms. JNK signaling promotes cytoprotective gene expression, but also interacts with the Insulin signaling pathway to influence growth, metabolism, stress tolerance and regeneration. Here, we review recent studies in Drosophila that elucidate the tissue-specific and systemic consequences of JNK activation that ultimately impact lifespan of the organism. PMID:21111799

  18. 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

  19. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  1. Autophosphorylating protein kinase activity in titin-like arthropod projectin.

    PubMed

    Maroto, M; Vinós, J; Marco, R; Cervera, M

    1992-03-20

    The function of the high molecular weight structural proteins from muscle, namely vertebrate titin, arthropod projectin and nematode twitchin, remains to be established. Using a simple method for the purification of projectin from crayfish and Drosophila melanogaster, a polyclonal antibody has been raised against crayfish projectin, and shown to immunocrossreact with Drosophila projectin but not with rat titin. In this study, evidence is presented that projectin and twitchin may share functional protein kinase domains, indicating a possible relationship between them. Projectin has a serine/threonine protein kinase activity. This supports the relationship with twitchin since, in sequence analysis of the latter, a protein-kinase-like domain has been found. Moreover, projectin is capable of autophosphorylation in vitro. These kinase activities imply regulatory functions for this group of proteins, extending its previously assumed structural role in the sarcomere. We also show here that projectin is phosphorylated in vivo at serine residues, as described for titin.

  2. Polo-like kinase-activating kinases: Aurora A, Aurora B and what else?

    PubMed

    Archambault, Vincent; Carmena, Mar

    2012-04-15

    The events of cell division are regulated by a complex interplay between kinases and phosphatases. Cyclin-dependent kinases (Cdks), polo-like kinases (Plks) and Aurora kinases play central roles in this process. Polo kinase (Plk1 in humans) regulates a wide range of events in mitosis and cytokinesis. To ensure the accuracy of these processes, polo activity itself is subject to complex regulation. Phosphorylation of polo in its T loop (or activation loop) increases its kinase activity several-fold. It has been shown that Aurora A kinase, with its co-factor Bora, activates Plk1 in G(2), and that this is essential for recovery from cell cycle arrest induced by DNA damage. In a recent article published in PLoS Biology, we report that Drosophila polo is activated by Aurora B kinase at centromeres, and that this is crucial for polo function in regulating chromosome dynamics in prometaphase. Our results suggest that this regulatory pathway is conserved in humans. Here, we propose a model for the collaboration between Aurora B and polo in the regulation of kinetochore attachment to microtubules in early mitosis. Moreover, we suggest that Aurora B could also function to activate Polo/Plk1 in cytokinesis. Finally, we discuss recent findings and open questions regarding the activation of polo and polo-like kinases by different kinases in mitosis, cytokinesis and other processes.

  3. 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

  4. 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.

  5. New insights into Drosophila vision.

    PubMed

    Dolph, Patrick

    2008-01-10

    Studies of the Drosophila visual system have provided valuable insights into the function and regulation of phototransduction signaling pathways. Much of this work has stemmed from or relied upon the genetic tools offered by the Drosophila system. In this issue of Neuron, Wang and colleagues and Acharya and colleagues have further exploited the Drosophila genetic system to characterize two new phototransduction players.

  6. Characterization of PDZ-binding kinase, a mitotic kinase

    PubMed Central

    Gaudet, Suzanne; Branton, Daniel; Lue, Robert A.

    2000-01-01

    hDlg, the human homologue of the Drosophila Discs-large (Dlg) tumor suppressor protein, is known to interact with the tumor suppressor protein APC and the human papillomavirus E6 transforming protein. In a two-hybrid screen, we identified a 322-aa serine/threonine kinase that binds to the PDZ2 domain of hDlg. The mRNA for this PDZ-binding kinase, or PBK, is most abundant in placenta and absent from adult brain tissue. The protein sequence of PBK has all the characteristic protein kinase subdomains and a C-terminal PDZ-binding T/SXV motif. In vitro, PBK binds specifically to PDZ2 of hDlg through its C-terminal T/SXV motif. PBK and hDlg are phosphorylated at mitosis in HeLa cells, and the mitotic phosphorylation of PBK is required for its kinase activity. In vitro, cdc2/cyclin B phosphorylates PBK. This evidence shows how PBK could link hDlg or other PDZ-containing proteins to signal transduction pathways regulating the cell cycle or cellular proliferation. PMID:10779557

  7. Protected deoxyribonucleoside-3' aryl phosphodiesters as key intermediates in polynucleotide synthesis. Construction of an icosanucleotide analogous to the sequence at the ends of Rous sarcoma virus 35S RNA.

    PubMed Central

    Gough, G R; Singleton, C K; Weith, H L; Gilham, P T

    1979-01-01

    Several modifications have been incorporated into the phosphotriester strategy for chemical synthesis of oligodeoxyribonucleotides. These include high-yield methods of preparation and isolation of O5', N-protected deoxyribonucleoside-3' p-chlorophenyl phosphates which serve as key intermediates, and the elimination of some superfluous manipulation and purification steps commonly used in the process of synthesizing oligonucleotide blocks. In addition, two new arylsulfonyl nitroimidazole derivatives have been prepared and found to be highly effective agents for internucleotide bond formation. These techniques have been applied in construction of the iconsamer d(G-C-C-A-T-T-T-T-A-C-C-A-T-T-C-A-C-C-A)-rC, equivalent to a ribonucleotide sequence located at both the 5' and 3' ends of Rous sarcoma virus 35S RNA. Images PMID:221888

  8. 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.

  9. Hearing regulates Drosophila aggression

    PubMed Central

    Versteven, Marijke; Vanden Broeck, Lies; Geurten, Bart; Zwarts, Liesbeth; Decraecker, Lisse; Beelen, Melissa; Göpfert, Martin C.; Heinrich, Ralf; Callaerts, Patrick

    2017-01-01

    Aggression is a universal social behavior important for the acquisition of food, mates, territory, and social status. Aggression in Drosophila is context-dependent and can thus be expected to involve inputs from multiple sensory modalities. Here, we use mechanical disruption and genetic approaches in Drosophila melanogaster to identify hearing as an important sensory modality in the context of intermale aggressive behavior. We demonstrate that neuronal silencing and targeted knockdown of hearing genes in the fly’s auditory organ elicit abnormal aggression. Further, we show that exposure to courtship or aggression song has opposite effects on aggression. Our data define the importance of hearing in the control of Drosophila intermale aggression and open perspectives to decipher how hearing and other sensory modalities are integrated at the neural circuit level. PMID:28115690

  10. Sterile Inflammation in Drosophila

    PubMed Central

    Shaukat, Zeeshan; Liu, Dawei; Gregory, Stephen

    2015-01-01

    The study of immune responses in Drosophila has already yielded significant results with impacts on our understanding of vertebrate immunity, such as the characterization of the Toll receptor. Several recent papers have focused on the humoral response to damage signals rather than pathogens, particularly damage signals from tumour-like tissues generated by loss of cell polarity or chromosomal instability. Both the triggers that generate this sterile inflammation and the systemic and local effects of it are only just beginning to be characterized in Drosophila. Here we review the molecular mechanisms that are known that give rise to the recruitment of Drosophila phagocytes, called hemocytes, as well as the signals, such as TNFα, that stimulated hemocytes emit at sites of perceived damage. The signalling consequences of inflammation, such as the activation of JNK, and the potential for modifying this response are also discussed. PMID:25948885

  11. Hearing regulates Drosophila aggression.

    PubMed

    Versteven, Marijke; Vanden Broeck, Lies; Geurten, Bart; Zwarts, Liesbeth; Decraecker, Lisse; Beelen, Melissa; Göpfert, Martin C; Heinrich, Ralf; Callaerts, Patrick

    2017-02-21

    Aggression is a universal social behavior important for the acquisition of food, mates, territory, and social status. Aggression in Drosophila is context-dependent and can thus be expected to involve inputs from multiple sensory modalities. Here, we use mechanical disruption and genetic approaches in Drosophila melanogaster to identify hearing as an important sensory modality in the context of intermale aggressive behavior. We demonstrate that neuronal silencing and targeted knockdown of hearing genes in the fly's auditory organ elicit abnormal aggression. Further, we show that exposure to courtship or aggression song has opposite effects on aggression. Our data define the importance of hearing in the control of Drosophila intermale aggression and open perspectives to decipher how hearing and other sensory modalities are integrated at the neural circuit level.

  12. Studying aging in Drosophila.

    PubMed

    He, Ying; Jasper, Heinrich

    2014-06-15

    Drosophila melanogaster represents one of the most important genetically accessible model organisms for aging research. Studies in flies have identified single gene mutations that influence lifespan and have characterized endocrine signaling interactions that control homeostasis systemically. Recent studies have focused on the effects of aging on specific tissues and physiological processes, providing a comprehensive picture of age-related tissue dysfunction and the loss of systemic homeostasis. Here we review methodological aspects of this work and highlight technical considerations when using Drosophila to study aging and age-related diseases.

  13. Chemical sensing in Drosophila.

    PubMed

    Benton, Richard

    2008-08-01

    Chemical sensing begins when peripheral receptor proteins recognise specific environmental stimuli and translate them into spatial and temporal patterns of sensory neuron activity. The chemosensory system of the fruit fly, Drosophila melanogaster, has become a dominant model to understand this process, through its accessibility to a powerful combination of molecular, genetic and electrophysiological analysis. Recent results have revealed many surprises in the biology of peripheral chemosensation in Drosophila, including novel structural and signalling properties of the insect odorant receptors (ORs), combinatorial mechanisms of chemical recognition by the gustatory receptors (GRs), and the implication of Transient Receptor Potential (TRP) ion channels as a novel class of chemosensory receptors.

  14. 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

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

    PubMed

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

    2014-05-01

    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.

  16. 3'-Phosphorylated nucleotides are tight binding inhibitors of nucleoside diphosphate kinase activity.

    PubMed

    Schneider, B; Xu, Y W; Janin, J; Véron, M; Deville-Bonne, D

    1998-10-30

    Nucleoside diphosphate (NDP) kinase catalyzes the phosphorylation of ribo- and deoxyribonucleosides diphosphates into triphosphates. NDP kinase is also involved in malignant tumors and was shown to activate in vitro transcription of the c-myc oncogene by binding to its NHE sequence. The structure of the complex of NDP kinase with bound ADP shows that the nucleotide adopts a different conformation from that observed in other phosphokinases with an internal H bond between the 3'-OH and the beta-O made free by the phosphate transfer. We use intrinsic protein fluorescence to investigate the inhibitory and binding potential of nucleotide analogues phosphorylated in 3'-OH position of the ribose to both wild type and F64W mutant NDP kinase from Dictyostelium discoideum. Due to their 3'-phosphate, 5'-phosphoadenosine 3'-phosphate (PAP) and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) can be regarded as structural analogues of enzyme-bound ADP. The KD of PAPS (10 microM) is three times lower than the KD of ADP. PAPS also acts as a competitive inhibitor toward natural substrates during catalysis, with a KI in agreement with binding data. The crystal structure of the binary complex between Dictyostelium NDP kinase and PAPS was solved at 2.8-A resolution. It shows a new mode of nucleotide binding at the active site with the 3'-phosphate of PAPS located near the catalytic histidine, at the same position as the gamma-phosphate in the transition state. The sulfate group is directed toward the protein surface. PAPS will be useful for the design of high affinity drugs targeted to NDP kinases.

  17. 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

  18. The Dictyostelium Kinome—Analysis of the Protein Kinases from a Simple Model Organism

    PubMed Central

    Liu, Allen; Fey, Petra; Pilcher, Karen E; Xu, Yanji; Smith, Janet L

    2006-01-01

    Dictyostelium discoideum is a widely studied model organism with both unicellular and multicellular forms in its developmental cycle. The Dictyostelium genome encodes 285 predicted protein kinases, similar to the count of the much more advanced Drosophila. It contains members of most kinase classes shared by fungi and metazoans, as well as many previously thought to be metazoan specific, indicating that they have been secondarily lost from the fungal lineage. This includes the entire tyrosine kinase–like (TKL) group, which is expanded in Dictyostelium and includes several novel receptor kinases. Dictyostelium lacks tyrosine kinase group kinases, and most tyrosine phosphorylation appears to be mediated by TKL kinases. About half of Dictyostelium kinases occur in subfamilies not present in yeast or metazoa, suggesting that protein kinases have played key roles in the adaptation of Dictyostelium to its habitat. This study offers insights into kinase evolution and provides a focus for signaling analysis in this system. PMID:16596165

  19. 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

  20. 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.

  1. Modeling Human Cancers in Drosophila.

    PubMed

    Sonoshita, M; Cagan, R L

    2017-01-01

    Cancer is a complex disease that affects multiple organs. Whole-body animal models provide important insights into oncology that can lead to clinical impact. Here, we review novel concepts that Drosophila studies have established for cancer biology, drug discovery, and patient therapy. Genetic studies using Drosophila have explored the roles of oncogenes and tumor-suppressor genes that when dysregulated promote cancer formation, making Drosophila a useful model to study multiple aspects of transformation. Not limited to mechanism analyses, Drosophila has recently been showing its value in facilitating drug development. Flies offer rapid, efficient platforms by which novel classes of drugs can be identified as candidate anticancer leads. Further, we discuss the use of Drosophila as a platform to develop therapies for individual patients by modeling the tumor's genetic complexity. Drosophila provides both a classical and a novel tool to identify new therapeutics, complementing other more traditional cancer tools. © 2017 Elsevier Inc. All rights reserved.

  2. Aging studies in Drosophila melanogaster.

    PubMed

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

    2013-01-01

    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.

  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. Octopamine Regulates Sleep in Drosophila through PKA Dependent Mechanisms

    PubMed Central

    Crocker, Amanda; Sehgal, Amita

    2008-01-01

    Sleep is a fundamental process, but its regulation and function are still not well understood. The Drosophila model for sleep provides a powerful system to address the genetic and molecular mechanisms underlying sleep and wakefulness. Here we show that a Drosophila biogenic amine, octopamine, is a potent wake-promoting signal. Mutations in the octopamine biosynthesis pathway produced a phenotype of increased sleep, which was restored to wild type levels by pharmacological treatment with octopamine. Moreover, electrical silencing of octopamine-producing cells decreased wakefulness while excitation of these neurons promoted wakefulness. Since protein kinase A (PKA) is a putative target of octopamine signaling and is also implicated in Drosophila sleep, we investigated its role in the effects of octopamine on sleep. We found that decreased PKA activity in neurons rendered flies insensitive to the wake-promoting effects of octopamine. However, this effect of PKA was not exerted in the mushroom bodies (MB), a site previously associated with PKA action on sleep. These studies identify a novel pathway that regulates sleep in Drosophila. PMID:18799671

  5. CASK and CaMKII function in Drosophila memory

    PubMed Central

    Malik, Bilal R.; Hodge, James J. L.

    2014-01-01

    Calcium (Ca2+) and Calmodulin (CaM)-dependent serine/threonine kinase II (CaMKII) plays a central role in synaptic plasticity and memory due to its ability to phosphorylate itself and regulate its own kinase activity. Autophosphorylation at threonine 287 (T287) switches CaMKII to a Ca2+ independent and constitutively active state replicated by overexpression of a phosphomimetic CaMKII-T287D transgene or blocked by expression of a T287A transgene. A second pair of sites, T306 T307 in the CaM binding region once autophosphorylated, prevents CaM binding and inactivates the kinase during synaptic plasticity and memory, and can be blocked by a TT306/7AA transgene. Recently the synaptic scaffolding molecule called CASK (Ca2+/CaM-associated serine kinase) has been shown to control both sets of CaMKII autophosphorylation events during neuronal growth, Ca2+ signaling and memory in Drosophila. Deletion of either full length CASK or just its CaMK-like and L27 domains removed middle-term memory (MTM) and long-term memory (LTM), with CASK function in the α′/ß′ mushroom body neurons being required for memory. In a similar manner directly changing the levels of CaMKII autophosphorylation (T287D, T287A, or TT306/7AA) in the α′/ß′ neurons also removed MTM and LTM. In the CASK null mutant expression of either the Drosophila or human CASK transgene in the α′/ß′ neurons was found to completely rescue memory, confirming that CASK signaling in α′/β′ neurons is necessary and sufficient for Drosophila memory formation and that the neuronal function of CASK is conserved between Drosophila and human. Expression of human CASK in Drosophila also rescued the effect of CASK deletion on the activity state of CaMKII, suggesting that human CASK may also regulate CaMKII autophosphorylation. Mutations in human CASK have recently been shown to result in intellectual disability and neurological defects suggesting a role in plasticity and learning possibly via regulation of Ca

  6. A Drosophila ABC Transporter Regulates Lifespan

    PubMed Central

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

    2014-01-01

    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

  7. 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.

  8. Temperature sensation in Drosophila.

    PubMed

    Barbagallo, Belinda; Garrity, Paul A

    2015-10-01

    Animals use thermosensory systems to achieve optimal temperatures for growth and reproduction and to avoid damaging extremes. Thermoregulation is particularly challenging for small animals like the fruit fly Drosophila melanogaster, whose body temperature rapidly changes in response to environmental temperature fluctuation. Recent work has uncovered some of the key molecules mediating fly thermosensation, including the Transient Receptor Potential (TRP) channels TRPA1 and Painless, and the Gustatory Receptor Gr28b, an unanticipated thermosensory regulator normally associated with a different sensory modality. There is also evidence the Drosophila phototransduction cascade may have some role in thermosensory responses. Together, the fly's diverse thermosensory molecules act in an array of functionally distinct thermosensory neurons to drive a suite of complex, and often exceptionally thermosensitive, behaviors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. Transcriptional feedback and definition of the circadian pacemaker in Drosophila and animals.

    PubMed

    Rosbash, M; Bradley, S; Kadener, S; Li, Y; Luo, W; Menet, J S; Nagoshi, E; Palm, K; Schoer, R; Shang, Y; Tang, C-H A

    2007-01-01

    The modern era of Drosophila circadian rhythms began with the landmark Benzer and Konopka paper and its definition of the period gene. The recombinant DNA revolution then led to the cloning and sequencing of this gene. This work did not result in a coherent view of circadian rhythm biochemistry, but experiments eventually gave rise to a transcription-centric view of circadian rhythm generation. Although these circadian transcription-translation feedback loops are still important, their contribution to core timekeeping is under challenge. Indeed, kinases and posttranslational regulation may be more important, based in part on recent in vitro work from cyanobacteria. In addition, kinase mutants or suspected kinase substrate mutants have unusually large period effects in Drosophila. This chapter discusses our recent experiments, which indicate that circadian transcription does indeed contribute to period determination in this system. We propose that cyanobacteria and animal clocks reflect two independent origins of circadian rhythms.

  15. Glial dysfunction causes age-related memory impairment in Drosophila.

    PubMed

    Yamazaki, Daisuke; Horiuchi, Junjiro; Ueno, Kohei; Ueno, Taro; Saeki, Shinjiro; Matsuno, Motomi; Naganos, Shintaro; Miyashita, Tomoyuki; Hirano, Yukinori; Nishikawa, Hiroyuki; Taoka, Masato; Yamauchi, Yoshio; Isobe, Toshiaki; Honda, Yoshiko; Kodama, Tohru; Masuda, Tomoko; Saitoe, Minoru

    2014-11-19

    Several aging phenotypes, including age-related memory impairment (AMI), are thought to be caused by cumulative oxidative damage. In Drosophila, age-related impairments in 1 hr memory can be suppressed by reducing activity of protein kinase A (PKA). However, the mechanism for this effect has been unclear. Here we show that decreasing PKA suppresses AMI by reducing activity of pyruvate carboxylase (PC), a glial metabolic enzyme whose amounts increase upon aging. Increased PC activity causes AMI through a mechanism independent of oxidative damage. Instead, increased PC activity is associated with decreases in D-serine, a glia-derived neuromodulator that regulates NMDA receptor activity. D-serine feeding suppresses both AMI and memory impairment caused by glial overexpression of dPC, indicating that an oxidative stress-independent dysregulation of glial modulation of neuronal activity contributes to AMI in Drosophila.

  16. Circadian Rhythms and Sleep in Drosophila melanogaster.

    PubMed

    Dubowy, Christine; Sehgal, Amita

    2017-04-01

    The advantages of the model organism Drosophila melanogaster, including low genetic redundancy, functional simplicity, and the ability to conduct large-scale genetic screens, have been essential for understanding the molecular nature of circadian (∼24 hr) rhythms, and continue to be valuable in discovering novel regulators of circadian rhythms and sleep. In this review, we discuss the current understanding of these interrelated biological processes in Drosophila and the wider implications of this research. Clock genes period and timeless were first discovered in large-scale Drosophila genetic screens developed in the 1970s. Feedback of period and timeless on their own transcription forms the core of the molecular clock, and accurately timed expression, localization, post-transcriptional modification, and function of these genes is thought to be critical for maintaining the circadian cycle. Regulators, including several phosphatases and kinases, act on different steps of this feedback loop to ensure strong and accurately timed rhythms. Approximately 150 neurons in the fly brain that contain the core components of the molecular clock act together to translate this intracellular cycling into rhythmic behavior. We discuss how different groups of clock neurons serve different functions in allowing clocks to entrain to environmental cues, driving behavioral outputs at different times of day, and allowing flexible behavioral responses in different environmental conditions. The neuropeptide PDF provides an important signal thought to synchronize clock neurons, although the details of how PDF accomplishes this function are still being explored. Secreted signals from clock neurons also influence rhythms in other tissues. SLEEP is, in part, regulated by the circadian clock, which ensures appropriate timing of sleep, but the amount and quality of sleep are also determined by other mechanisms that ensure a homeostatic balance between sleep and wake. Flies have been useful

  17. The Drosophila melanogaster host model

    PubMed Central

    Igboin, Christina O.; Griffen, Ann L.; Leys, Eugene J.

    2012-01-01

    The deleterious and sometimes fatal outcomes of bacterial infectious diseases are the net result of the interactions between the pathogen and the host, and the genetically tractable fruit fly, Drosophila melanogaster, has emerged as a valuable tool for modeling the pathogen–host interactions of a wide variety of bacteria. These studies have revealed that there is a remarkable conservation of bacterial pathogenesis and host defence mechanisms between higher host organisms and Drosophila. This review presents an in-depth discussion of the Drosophila immune response, the Drosophila killing model, and the use of the model to examine bacterial–host interactions. The recent introduction of the Drosophila model into the oral microbiology field is discussed, specifically the use of the model to examine Porphyromonas gingivalis–host interactions, and finally the potential uses of this powerful model system to further elucidate oral bacterial-host interactions are addressed. PMID:22368770

  18. Feeding regulation in Drosophila

    PubMed Central

    Pool, Allan-Hermann; Scott, Kristin

    2014-01-01

    Neuromodulators play a key role in adjusting animal behavior based on environmental cues and internal needs. Here, we review the regulation of Drosophila feeding behavior to illustrate how neuromodulators achieve behavioral plasticity. Recent studies have made rapid progress in determining molecular and cellular mechanisms that translate the metabolic needs of the fly into changes in neuroendocrine and neuromodulatory states. These neuromodulators in turn promote or inhibit discrete feeding behavioral subprograms. This review highlights the links between physiological needs, neuromodulatory states, and feeding decisions. PMID:24937262

  19. Myc Function in Drosophila

    PubMed Central

    Gallant, Peter

    2013-01-01

    Drosophila contains a single MYC gene. Like its vertebrate homologs, it encodes a transcription factor that activates many targets, including prominently genes involved in ribosome biogenesis and translation. This activity makes Myc a central regulator of growth and/or proliferation of many cell types, such as imaginal disc cells, polyploid cells, stem cells, and blood cells. Importantly, not only does Myc act cell autonomously but it also affects the fate of adjacent cells and tissues. This potential of Myc is harnessed by many different signaling pathways, involving, among others, Wg, Dpp, Hpo, ecdysone, insulin, and mTOR. PMID:24086064

  20. 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.

  1. Deconstructing Memory in Drosophila

    PubMed Central

    Margulies, Carla; Tully, Tim; Dubnau, Josh

    2011-01-01

    Unlike most organ systems, which have evolved to maintain homeostasis, the brain has been selected to sense and adapt to environmental stimuli by constantly altering interactions in a gene network that functions within a larger neural network. This unique feature of the central nervous system provides a remarkable plasticity of behavior, but also makes experimental investigations challenging. Each experimental intervention ramifies through both gene and neural networks, resulting in unpredicted and sometimes confusing phenotypic adaptations. Experimental dissection of mechanisms underlying behavioral plasticity ultimately must accomplish an integration across many levels of biological organization, including genetic pathways acting within individual neurons, neural network interactions which feed back to gene function, and phenotypic observations at the behavioral level. This dissection will be more easily accomplished for model systems such as Drosophila, which, compared with mammals, have relatively simple and manipulable nervous systems and genomes. The evolutionary conservation of behavioral phenotype and the underlying gene function ensures that much of what we learn in such model systems will be relevant to human cognition. In this essay, we have not attempted to review the entire Drosophila memory field. Instead, we have tried to discuss particular findings that provide some level of intellectual synthesis across three levels of biological organization: behavior, neural circuitry and biochemical pathways. We have attempted to use this integrative approach to evaluate distinct mechanistic hypotheses, and to propose critical experiments that will advance this field. PMID:16139203

  2. 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.

  3. Drosophila pupal abdomen immunohistochemistry.

    PubMed

    Wang, Wei; Yoder, John H

    2011-10-02

    The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies. During pupation, which spans approximately 96 hours (at 25 °C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests). Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

  4. Epigenetic regulation in Drosophila.

    PubMed

    Lyko, F; Beisel, C; Marhold, J; Paro, R

    2006-01-01

    Epigenetic regulation of gene transcription relies on molecular marks like DNA methylation or histone modifications. Here we review recent advances in our understanding of epigenetic regulation in the fruit fly Drosophila melanogaster. In the past, DNA methylation research has primarily utilized mammalian model systems. However, several recent landmark discoveries have been made in other organisms. For example, the interaction between DNA methylation and histone methylation was first described in the filamentous fungus Neurospora crassa. Another example is provided by the interaction between epigenetic modifications and the RNA interference (RNAi) machinery that was first reported in the fission yeast Schizosaccharomyces pombe. Another organism with great experimental power is the fruit fly Drosophila. Epigenetic regulation by chromatin has been extensively analyzed in the fly and several of the key components have been discovered in this organism. In this chapter, we will focus on three aspects that represent the complexity of epigenetic gene regulation. (1) We will discuss the available data about the DNA methylation system, (2) we will illuminate the interaction between DNA methylation and chromatin regulation, and (3) we will provide an overview over the Polycomb system of epigenetic chromatin modifiers that has proved to be an important paradigm for a chromatin system regulating epigenetic programming.

  5. Null EPAC Mutants Reveal a Sequential Order of Versatile cAMP Effects during "Drosophila" Aversive Odor Learning

    ERIC Educational Resources Information Center

    Richlitzki, Antje; Latour, Philipp; Schwärzel, Martin

    2017-01-01

    Here, we define a role of the cAMP intermediate EPAC in "Drosophila" aversive odor learning by means of null epac mutants. Complementation analysis revealed that EPAC acts downstream from the "rutabaga" adenylyl cyclase and in parallel to protein kinase A. By means of targeted knockdown and genetic rescue we identified mushroom…

  6. Transcriptional regulation during Drosophila spermatogenesis

    PubMed Central

    Lim, Cindy; Tarayrah, Lama; Chen, Xin

    2012-01-01

    Drosophila spermatogenesis has become a paradigmatic system for the study of mechanisms that regulate adult stem cell maintenance, proliferation and differentiation. The dramatic cellular differentiation process from germline stem cell (GSC) to mature sperm is accompanied by dynamic changes in gene expression, which are regulated at transcriptional, post-transcriptional (including translational) and post-translational levels. Post-transcriptional regulation has been proposed as a unique feature of germ cells. However, recent studies have provided new insights into transcriptional regulation during Drosophila spermatogenesis. Both signaling pathways and epigenetic mechanisms act to orchestrate the transcriptional regulation of distinct genes at different germ cell differentiation stages. Many of the regulatory pathways that control male gamete differentiation in Drosophila are conserved in mammals. Therefore, studies using Drosophila spermatogenesis will provide insight into the molecular mechanisms that regulate mammalian germ cell differentiation pathways. PMID:23087835

  7. Safeguarding genetic information in Drosophila.

    PubMed

    Su, Tin Tin

    2011-12-01

    Eukaryotic cells employ a plethora of conserved proteins and mechanisms to ensure genome integrity. In metazoa, these mechanisms must operate in the context of organism development. This mini-review highlights two emerging features of DNA damage responses in Drosophila: a crosstalk between DNA damage responses and components of the spindle assembly checkpoint, and increasing evidence for the effect of DNA damage on the developmental program at multiple points during the Drosophila life cycle.

  8. 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

  9. Cytoplasmic myosin from Drosophila melanogaster

    PubMed Central

    1986-01-01

    Myosin is identified and purified from three different established Drosophila melanogaster cell lines (Schneider's lines 2 and 3 and Kc). Purification entails lysis in a low salt, sucrose buffer that contains ATP, chromatography on DEAE-cellulose, precipitation with actin in the absence of ATP, gel filtration in a discontinuous KI-KCl buffer system, and hydroxylapatite chromatography. Yield of pure cytoplasmic myosin is 5-10%. This protein is identified as myosin by its cross-reactivity with two monoclonal antibodies against human platelet myosin, the molecular weight of its heavy chain, its two light chains, its behavior on gel filtration, its ATP-dependent affinity for actin, its characteristic ATPase activity, its molecular morphology as demonstrated by platinum shadowing, and its ability to form bipolar filaments. The molecular weight of the cytoplasmic myosin's light chains and peptide mapping and immunochemical analysis of its heavy chains demonstrate that this myosin, purified from Drosophila cell lines, is distinct from Drosophila muscle myosin. Two-dimensional thin layer maps of complete proteolytic digests of iodinated muscle and cytoplasmic myosin heavy chains demonstrate that, while the two myosins have some tryptic and alpha-chymotryptic peptides in common, most peptides migrate with unique mobility. One-dimensional peptide maps of SDS PAGE purified myosin heavy chain confirm these structural data. Polyclonal antiserum raised and reacted against Drosophila myosin isolated from cell lines cross-reacts only weakly with Drosophila muscle myosin isolated from the thoraces of adult Drosophila. Polyclonal antiserum raised against Drosophila muscle myosin behaves in a reciprocal fashion. Taken together our data suggest that the myosin purified from Drosophila cell lines is a bona fide cytoplasmic myosin and is very likely the product of a different myosin gene than the muscle myosin heavy chain gene that has been previously identified and characterized. PMID

  10. Gilgamesh is required for the maintenance of germline stem cells in Drosophila testis.

    PubMed

    Chen, Dongsheng; Zhu, Xiangxiang; Zhou, Lijuan; Wang, Jian; Tao, Xiaoqian; Wang, Shuang; Sun, Fuling; Kan, Xianzhao; Han, Zhengqi; Gu, Yuelin

    2017-07-18

    Emerging evidence supports that stem cells are regulated by both intrinsic and extrinsic mechanisms. However, factors that determine the fate of stem cells remain incompletely understood. The Drosophila testis provides an exclusive powerful model in searching for potential important regulatory factors and their underlying mechanisms for controlling the fate of germline stem cells (GSCs). In this study, we have found that Drosophila gilgamesh (gish), which encodes a homologue of human CK1-γ (casein kinase 1-gamma), is required intrinsically for GSC maintenance. Our genetic analyses indicate gish is not required for Dpp/Gbb signaling silencing of bam and is dispensable for Dpp/Gbb signaling-dependent Dad expression. Finally, we show that overexpression of gish fail to dramatically increase the number of GSCs. These findings demonstrate that gish controls the fate of GSCs in Drosophila testis by a novel Dpp/Gbb signaling-independent pathway.

  11. The eye of Drosophila as a model system for studying intracellular signaling in ontogenesis and pathogenesis.

    PubMed

    Katanaev, V L; Kryuchkov, M V

    2011-12-01

    Many human diseases are caused by malfunction of basic types of cellular activity such as proliferation, differentiation, apoptosis, cell polarization, and migration. In turn, these processes are associated with different routes of intracellular signal transduction. A number of model systems have been designed to study normal and abnormal cellular and molecular processes associated with pathogenesis. The developing eye of the fruit fly Drosophila melanogaster is one of these systems. The sequential development of compound eyes of this insect makes it possible to model human neurodegenerative diseases and mechanisms of carcinogenesis. In this paper we overview the program of the eye development in Drosophila, with emphasis on intracellular signaling pathways that regulate this complex process. We discuss in detail the roles of the Notch, Hedgehog, TGFβ, Wnt, and receptor tyrosine kinase signaling pathways in Drosophila eye development and human pathology. We also briefly describe the modern methods of experimentation with this model organism to analyze the function of human pathogenic proteins.

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

    PubMed Central

    Cinalli, Ryan M.; Lehmann, Ruth

    2013-01-01

    The primordial germ cells (PGCs) are the first cells to form during Drosophila melanogaster embryogenesis. While the process of somatic cell formation has been studied in detail, the mechanics of PGC formation are poorly understood. Here, using 4D multi-photon imaging combined with genetic and pharmacological manipulations, we find that PGC formation requires an anaphase spindle-independent cleavage pathway. In addition to utilizing core regulators of cleavage, including the small GTPase RhoA (Drosophila Rho) and the Rho associated kinase, ROCK (Drosophila Rok), we show that this pathway requires Germ cell-less (Gcl), a conserved BTB-domain protein not previously implicated in cleavage mechanics. This alternate form of cell formation suggests that organisms have evolved multiple molecular strategies for regulating the cytoskeleton during cleavage. PMID:23728423

  13. 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.

  14. Peroxiredoxin 5 modulates immune response in Drosophila

    PubMed Central

    Radyuk, Svetlana N.; Michalak, Katarzyna; Klichko, Vladimir I.; Benes, Judith; Orr, William C.

    2010-01-01

    Background Peroxiredoxins are redox-sensing enzymes with multiple cellular functions. Previously, we reported on the potent antioxidant function of Drosophila peroxiredoxin 5 (dPrx5). Studies with mammalian and human cells suggest that peroxiredoxins can modulate immune-related signaling. Methods Survivorship studies and bacteriological analysis were used to determine resistance of flies to fungal and bacterial infections. RT-PCR and immunoblot analyses determined expression of dPrx5 and immunity factors in response to bacterial challenge. Double mutants for dprx5 gene and genes comprising the Imd/Relish and dTak1/Basket branches of the immune signaling pathways were used in epistatic analysis. Results The dprx5 mutant flies were more resistant to bacterial infection than controls, while flies overexpressing dPrx5 were more susceptible. The enhanced resistance to bacteria was accompanied by rapid induction of the Imd-dependent antimicrobial peptides, phosphorylation of the JNK kinase Basket and altered transcriptional profiling of the transient response genes, puckered, ets21C and relish, while the opposite effects were observed in flies over-expressing dPrx5. Epistatic analysis of double mutants, using attacin D and Puckered as read outs of activation of the Imd and JNK pathways, implicated dPrx5 function in the control of the dTak1-JNK arm of immune signaling. Conclusions Differential effects on fly survivorship suggested a trade-off between the antioxidant and immune functions of dPrx5. Molecular and epistatic analyses identified dPrx5 as a negative regulator in the dTak1-JNK arm of immune signaling. General significance Our findings suggest that peroxiredoxins play an important modulatory role in the Drosophila immune response. PMID:20600624

  15. 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.

  16. Cytosolic Ras Supports Eye Development in Drosophila

    PubMed Central

    Sung, Pamela J.; Rodrigues, Aloma B.; Kleinberger, Andrew; Quatela, Steven; Bach, Erika A.; Philips, Mark R.

    2010-01-01

    Ras proteins associate with cellular membranes as a consequence of a series of posttranslational modifications of a C-terminal CAAX sequence that include prenylation and are thought to be required for biological activity. In Drosophila melanogaster, Ras1 is required for eye development. We found that Drosophila Ras1 is inefficiently prenylated as a consequence of a lysine in the A1 position of its CAAX sequence such that a significant pool remains soluble in the cytosol. We used mosaic analysis with a repressible cell marker (MARCM) to assess if various Ras1 transgenes could restore photoreceptor fate to eye disc cells that are null for Ras1. Surprisingly, we found that whereas Ras1 with an enhanced efficiency of membrane targeting could not rescue the Ras1 null phenotype, Ras1 that was not at all membrane targeted by virtue of a mutation of the CAAX cysteine was able to fully rescue eye development. In addition, constitutively active Ras112V,C186S not targeted to membranes produced a hypermorphic phenotype and stimulated mitogen-activated protein kinase (MAPK) signaling in S2 cells. We conclude that the membrane association of Drosophila Ras1 is not required for eye development. PMID:20937772

  17. Cytokines in Drosophila immunity.

    PubMed

    Vanha-Aho, Leena-Maija; Valanne, Susanna; Rämet, Mika

    2016-02-01

    Cytokines are a large and diverse group of small proteins that can affect many biological processes, but most commonly cytokines are known as mediators of the immune response. In the event of an infection, cytokines are produced in response to an immune stimulus, and they function as key regulators of the immune response. Cytokines come in many shapes and sizes, and although they vary greatly in structure, their functions have been well conserved in evolution. The immune signaling pathways that respond to cytokines are remarkably conserved from fly to man. Therefore, Drosophila melanogaster, provides an excellent platform for studying the biology and function of cytokines. In this review, we will describe the cytokines and cytokine-like molecules found in the fly and discuss their roles in host immunity. Copyright © 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  18. Transposable elements in Drosophila

    PubMed Central

    McCullers, Tabitha J.; Steiniger, Mindy

    2017-01-01

    ABSTRACT Transposable elements (TEs) are mobile genetic elements that can mobilize within host genomes. As TEs comprise more than 40% of the human genome and are linked to numerous diseases, understanding their mechanisms of mobilization and regulation is important. Drosophila melanogaster is an ideal model organism for the study of eukaryotic TEs as its genome contains a diverse array of active TEs. TEs universally impact host genome size via transposition and deletion events, but may also adopt unique functional roles in host organisms. There are 2 main classes of TEs: DNA transposons and retrotransposons. These classes are further divided into subgroups of TEs with unique structural and functional characteristics, demonstrating the significant variability among these elements. Despite this variability, D. melanogaster and other eukaryotic organisms utilize conserved mechanisms to regulate TEs. This review focuses on the transposition mechanisms and regulatory pathways of TEs, and their functional roles in D. melanogaster. PMID:28580197

  19. 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.

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

    PubMed Central

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

    2015-01-01

    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

  1. 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

  2. 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.

  3. Wallenda regulates JNK-mediated cell death in Drosophila

    PubMed Central

    Ma, X; Xu, W; Zhang, D; Yang, Y; Li, W; Xue, L

    2015-01-01

    The c-Jun N-terminal kinase (JNK) pathway plays essential roles in regulating a variety of cellular processes including proliferation, migration and survival. Previous genetic studies in Drosophila have identified numerous cell death regulating genes, providing new insights into the mechanisms for related diseases. Despite the known role of the small GTPase Rac1 in regulating cell death, the downstream components and underlying mechanism remain largely elusive. Here, we show that Rac1 promotes JNK-dependent cell death through Wallenda (Wnd). In addition, we find that Wnd triggers JNK activation and cell death via its kinase domain. Moreover, we show that both MKK4 and Hep are critical for Wnd-induced cell death. Furthermore, Wnd is essential for ectopic Egr- or Rho1-induced JNK activation and cell death. Finally, Wnd is physiologically required for loss of scribble-induced JNK-dependent cell death. Thus, our data suggest that wnd encodes a novel essential cell death regulator in Drosophila. PMID:25950467

  4. Transcriptional regulation of Profilin during wound closure in Drosophila larvae

    PubMed Central

    Brock, Amanda R.; Wang, Yan; Berger, Susanne; Renkawitz-Pohl, Renate; Han, Violet C.; Wu, Yujane; Galko, Michael J.

    2012-01-01

    Summary Injury is an inevitable part of life, making wound healing essential for survival. In postembryonic skin, wound closure requires that epidermal cells recognize the presence of a gap and change their behavior to migrate across it. In Drosophila larvae, wound closure requires two signaling pathways [the Jun N-terminal kinase (JNK) pathway and the Pvr receptor tyrosine kinase signaling pathway] and regulation of the actin cytoskeleton. In this and other systems, it remains unclear how the signaling pathways that initiate wound closure connect to the actin regulators that help execute wound-induced cell migrations. Here, we show that chickadee, which encodes the Drosophila Profilin, a protein important for actin filament recycling and cell migration during development, is required for the physiological process of larval epidermal wound closure. After injury, chickadee is transcriptionally upregulated in cells proximal to the wound. We found that JNK, but not Pvr, mediates the increase in chic transcription through the Jun and Fos transcription factors. Finally, we show that chic-deficient larvae fail to form a robust actin cable along the wound edge and also fail to form normal filopodial and lamellipodial extensions into the wound gap. Our results thus connect a factor that regulates actin monomer recycling to the JNK signaling pathway during wound closure. They also reveal a physiological function for an important developmental regulator of actin and begin to tease out the logic of how the wound repair response is organized. PMID:22976306

  5. Pak functions downstream of Dock to regulate photoreceptor axon guidance in Drosophila.

    PubMed

    Hing, H; Xiao, J; Harden, N; Lim, L; Zipursky, S L

    1999-06-25

    The SH2/SH3 adaptor protein Dock has been proposed to transduce signals from guidance receptors to the actin cytoskeleton in Drosophila photoreceptor (R cell) growth cones. Here, we demonstrate that Drosophila p21-activated kinase (Pak) is required in a Dock pathway regulating R cell axon guidance and targeting. Dock and Pak colocalize to R cell axons and growth cones, physically interact, and their loss-of-function phenotypes are indistinguishable. Normal patterns of R cell connectivity require Pak's kinase activity and binding sites for both Dock and Cdc42/Rac. A membrane-tethered form of Pak (Pak(myr) acts as a dominant gain-of-function protein. Retinal expression of Pak(myr) rescues the R cell connectivity phenotype in dock mutants. These data establish Pak as a critical regulator of axon guidance and a downstream effector of Dock in vivo.

  6. Strong dietary restrictions protect Drosophila against anoxia/reoxygenation injuries.

    PubMed

    Vigne, Paul; Tauc, Michel; Frelin, Christian

    2009-01-01

    Reoxygenation of ischemic tissues is a major factor that determines the severity of cardiovascular diseases. This paper describes the consequences of anoxia/reoxygenation (A/R) stresses on Drosophila, a useful, anoxia tolerant, model organism. Newly emerged adult male flies were exposed to anoxic conditions (<1% O2) for 1 to 6 hours, reoxygenated and their survival was monitored. A/R stresses induced a transient increase in mortality which peaked at the time of reoxygenation. Then flies recovered low mortality rates similar to those of control flies. A/R induced mortality was strongly dependent on dietary conditions during the 48 h that preceded anoxia. Well fed flies were anoxia sensitive. Strong dietary restrictions and starvation conditions protected flies against A/R injuries. The tolerance to anoxia was associated to large decreases in glycogen, protein, and ATP contents. During anoxia, anoxia tolerant flies produced more lactate, less phosphate and they maintained more stable ATP levels than anoxia sensitive flies. Moderate dietary restrictions, which increased the longevity of normoxic flies, did not promote resistance to A/R stresses. Diet dependent A/R injuries were still observed in sigma loss of function mutants and they were insensitive to dietary rapamycin or resveratrol. AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribose-furanoside), an activator AMP kinase decreased A/R injuries. Mutants in the insulin signalling pathway were more anoxia tolerant in a fed state. Long A/R stresses induce a transient increase in mortality in Drosophila. This mortality is highly dependent on dietary conditions prior to the stress. Strong dietary restrictions and starvation conditions protect flies against A/R injuries, probably by inducing a major remodelling of energy metabolism. The results also indicate that mechanistically different responses develop in response to dietary restrictions of different strengths. AMP kinase and the insulin signalling pathway are

  7. Strong Dietary Restrictions Protect Drosophila against Anoxia/Reoxygenation Injuries

    PubMed Central

    Vigne, Paul; Tauc, Michel; Frelin, Christian

    2009-01-01

    Background Reoxygenation of ischemic tissues is a major factor that determines the severity of cardiovascular diseases. This paper describes the consequences of anoxia/reoxygenation (A/R) stresses on Drosophila, a useful, anoxia tolerant, model organism. Methodology/Principal Findings Newly emerged adult male flies were exposed to anoxic conditions (<1% O2) for 1 to 6 hours, reoxygenated and their survival was monitored. Results A/R stresses induced a transient increase in mortality which peaked at the time of reoxygenation. Then flies recovered low mortality rates similar to those of control flies. A/R induced mortality was strongly dependent on dietary conditions during the 48 h that preceded anoxia. Well fed flies were anoxia sensitive. Strong dietary restrictions and starvation conditions protected flies against A/R injuries. The tolerance to anoxia was associated to large decreases in glycogen, protein, and ATP contents. During anoxia, anoxia tolerant flies produced more lactate, less phosphate and they maintained more stable ATP levels than anoxia sensitive flies. Moderate dietary restrictions, which increased the longevity of normoxic flies, did not promote resistance to A/R stresses. Diet dependent A/R injuries were still observed in sima loss of function mutants and they were insensitive to dietary rapamycin or resveratrol. AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribosefuranoside), an activator AMP kinase decreased A/R injuries. Mutants in the insulin signalling pathway were more anoxia tolerant in a fed state. Conclusion/Significance Long A/R stresses induce a transient increase in mortality in Drosophila. This mortality is highly dependent on dietary conditions prior to the stress. Strong dietary restrictions and starvation conditions protect flies against A/R injuries, probably by inducing a major remodelling of energy metabolism. The results also indicate that mechanistically different responses develop in response to dietary restrictions of

  8. The PIKE homolog Centaurin gamma regulates developmental timing in Drosophila.

    PubMed

    Gündner, Anna Lisa; Hahn, Ines; 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

    2008-08-07

    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.

  10. An epidermal barrier wound repair pathway in Drosophila is mediated by grainy head.

    PubMed

    Mace, Kimberly A; Pearson, Joseph C; McGinnis, William

    2005-04-15

    We used wounded Drosophila embryos to define an evolutionarily conserved pathway for repairing the epidermal surface barrier. This pathway includes a wound response enhancer from the Ddc gene that requires grainy head (grh) function and binding sites for the Grh transcription factor. At the signaling level, tyrosine kinase and extracellular signal-regulated kinase (ERK) activities are induced in epidermal cells near wounds, and activated ERK is required for a robust wound response. The conservation of this Grh-dependent pathway suggests that the repair of insect cuticle and mammal skin is controlled by an ancient, shared control system for constructing and healing the animal body surface barrier.

  11. Rho1-Wnd signaling regulates loss-of-cell polarity-induced cell invasion in Drosophila.

    PubMed

    Ma, X; Chen, Y; Zhang, S; Xu, W; Shao, Y; Yang, Y; Li, W; Li, M; Xue, L

    2016-02-18

    Both cell polarity and c-Jun N-terminal kinase (JNK) activity are essential to the maintenance of tissue homeostasis, and disruption of either is commonly seen in cancer progression. Despite the established connection between loss-of-cell polarity and JNK activation, much less is known about the molecular mechanism by which aberrant cell polarity induces JNK-mediated cell migration and tumor invasion. Here we show results from a genetic screen using an in vivo invasion model via knocking down cell polarity gene in Drosophila wing discs, and identify Rho1-Wnd signaling as an important molecular link that mediates loss-of-cell polarity-triggered JNK activation and cell invasion. We show that Wallenda (Wnd), a protein kinase of the mitogen-activated protein kinase kinase kinase family, by forming a complex with the GTPase Rho1, is both necessary and sufficient for Rho1-induced JNK-dependent cell invasion, MMP1 activation and epithelial-mesenchymal transition. Furthermore, Wnd promotes cell proliferation and tissue growth through wingless production when apoptosis is inhibited by p35. Finally, Wnd shows oncogenic cooperation with Ras(V12) to trigger tumor growth in eye discs and causes invasion into the ventral nerve cord. Together, our data not only provides a novel mechanistic insight on how cell polarity loss contributes to cell invasion, but also highlights the value of the Drosophila model system to explore human cancer biology.

  12. Genome of Drosophila suzukii, the Spotted Wing Drosophila

    PubMed Central

    Chiu, Joanna C.; Jiang, Xuanting; Zhao, Li; Hamm, Christopher A.; Cridland, Julie M.; Saelao, Perot; Hamby, Kelly A.; Lee, Ernest K.; Kwok, Rosanna S.; Zhang, Guojie; Zalom, Frank G.; Walton, Vaughn M.; Begun, David J.

    2013-01-01

    Drosophila suzukii Matsumura (spotted wing drosophila) has recently become a serious pest of a wide variety of fruit crops in the United States as well as in Europe, leading to substantial yearly crop losses. To enable basic and applied research of this important pest, we sequenced the D. suzukii genome to obtain a high-quality reference sequence. Here, we discuss the basic properties of the genome and transcriptome and describe patterns of genome evolution in D. suzukii and its close relatives. Our analyses and genome annotations are presented in a web portal, SpottedWingFlyBase, to facilitate public access. PMID:24142924

  13. Cytokinesis in Drosophila male meiosis

    PubMed Central

    Giansanti, Maria Grazia; Sechi, Stefano; Frappaolo, Anna; Belloni, Giorgio; Piergentili, Roberto

    2012-01-01

    Cytokinesis separates the cytoplasm and the duplicated genome into two daughter cells at the end of cell division. This process must be finely regulated to maintain ploidy and prevent tumor formation. Drosophila male meiosis provides an excellent cell system for investigating cytokinesis. Mutants affecting this process can be easily identified and spermatocytes are large cells particularly suitable for cytological analysis of cytokinetic structures. Over the past decade, the powerful tools of Drosophila genetics and the unique characteristics of this cell system have led researchers to identify molecular players of the cell cleavage machinery and to address important open questions. Although spermatocyte cytokinesis is incomplete, resulting in formation of stable intercellular bridges, the molecular mechanisms are largely conserved in somatic cells. Thus, studies of Drosophila male meiosis will shed new light on the complex cell circuits regulating furrow ingression and substantially further our knowledge of cancer and other human diseases. PMID:23094234

  14. 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

  15. 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.

  16. Modelling the Drosophila embryo.

    PubMed

    Jaeger, Johannes

    2009-12-01

    I provide a historical overview on the use of mathematical models to gain insight into pattern formation during early development of the fruit fly Drosophila melanogaster. It is my intention to illustrate how the aims and methodology of modelling have changed from the early beginnings of a theoretical developmental biology in the 1960s to modern-day systems biology. I show that even early modelling attempts addressed interesting and relevant questions, which were not tractable by experimental approaches. Unfortunately, their validation was severely hampered by a lack of specificity and appropriate experimental evidence. There is a simple lesson to be learned from this: we cannot deduce general rules for pattern formation from first principles or spurious reproduction of developmental phenomena. Instead, we must infer such rules (if any) from detailed and accurate studies of specific developmental systems. To achieve this, mathematical modelling must be closely integrated with experimental approaches. I report on progress that has been made in this direction in the past few years and illustrate the kind of novel insights that can be gained from such combined approaches. These insights demonstrate the great potential (and some pitfalls) of an integrative, systems-level investigation of pattern formation.

  17. Protein Kinases of the Hippo Pathway: Regulation and Substrates

    PubMed Central

    Avruch, Joseph; Zhou, Dawang; Fitamant, Julien; Bardeesy, Nabeel; Mou, Fan; Barrufet, Laura Regué

    2012-01-01

    The “Hippo” signaling pathway has emerged as a major regulator of cell proliferation and survival in metazoans. The pathway, as delineated by genetic and biochemical studies in Drosophila, consists of a kinase cascade regulated by cell-cell contact and cell polarity that inhibits the transcriptional coactivator Yorkie and its proliferative, anti-differentiation, antiapoptotic transcriptional program. The core pathway components are the GC kinase Hippo, which phosphorylates the noncatalytic polypeptide Mats/Mob1 and, with the assistance of the scaffold protein Salvador, phosphorylates the ndr-family kinase Lats. In turn phospho-Lats, after binding to phospho-Mats, autoactivates and phosphorylates Yorkie, resulting in its nuclear exit. Hippo also uses the scaffold protein Furry and a different Mob protein to control another ndr-like kinase, the morphogenetic regulator Tricornered. Architecturally homologous kinase cascades consisting of a GC kinase, a Mob protein, a scaffolding polypeptide and an ndr-like kinase are well described in yeast; in S. cerevisiae e.g., the MEN pathway promotes mitotic exit whereas the RAM network, using a different GC kinase, Mob protein, scaffold and ndr-like kinase, regulates cell polarity and morphogenesis. In mammals, the Hippo orthologues Mst1 and Mst2 utilize the Salvador ortholog WW45/Sav1 and other scaffolds to regulate the kinases Lats1/Lats2 and ndr1/ndr2. As in Drosophila, murine Mst1/Mst2, in a redundant manner, negatively regulate the Yorkie ortholog YAP in the epithelial cells of the liver and gut; loss of both Mst1 and Mst2 results in hyperproliferation and tumorigenesis that can be largely negated by reduction or elimination of YAP. Despite this conservation, considerable diversification in pathway composition and regulation is already evident; in skin e.g., YAP phosphorylation is independent of Mst1Mst2 and Lats1Lats2. Moreover, in lymphoid cells, Mst1/Mst2, under the control of the Rap1 GTPase and independent of YAP

  18. 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.

  19. A humoral stress response in Drosophila.

    PubMed

    Ekengren, S; Tryselius, Y; Dushay, M S; Liu, G; Steiner, H; Hultmark, D

    2001-05-01

    The ability to react to unfavorable environmental changes is crucial for survival and reproduction, and several adaptive responses to stress have been conserved during evolution [1-3]. Specific immune and heat shock responses mediate the elimination of invading pathogens and of damaged proteins or cells [4-6]. Furthermore, MAP kinases and other signaling factors mediate cellular responses to a very broad range of environmental insults [7-9]. Here we describe a novel systemic response to stress in Drosophila. The Turandot A (TotA) gene encodes a humoral factor, which is secreted from the fat body and accumulates in the body fluids. TotA is strongly induced upon bacterial challenge, as well as by other types of stress such as high temperature, mechanical pressure, dehydration, UV irradiation, and oxidative agents. It is also upregulated during metamorphosis and at high age. Strikingly, flies that overexpress TotA show prolonged survival and retain normal activity at otherwise lethal temperatures. Although TotA is only induced by severe stress, it responds to a much wider range of stimuli than heat shock genes such as hsp70 or immune genes such as Cecropin A1.

  20. Active forgetting of olfactory memories in Drosophila.

    PubMed

    Berry, Jacob A; Davis, Ronald L

    2014-01-01

    Failure to remember, or forgetting, is a phenomenon familiar to everyone and despite more than a century of scientific inquiry, why we forget what we once knew remains unclear. If the brain marshals significant resources to form and store memories, why is it that these memories become lost? In the last century, psychological studies have divided forgetting into decay theory, in which memory simply dissipates with time, and interference theory, in which additional learning or mental activity hinders memory by reducing its stability or retrieval (for review, Dewar et al., 2007; Wixted, 2004). Importantly, these psychological models of forgetting posit that forgetting is a passive property of the brain and thus a failure of the brain to retain memories. However, recent neuroscience research on olfactory memory in Drosophila has offered evidence for an alternative conclusion that forgetting is an "active" process, with specific, biologically regulated mechanisms that remove existing memories (Berry et al., 2012; Shuai et al., 2010). Similar to the bidirectional regulation of cell number by mitosis and apoptosis, protein concentration by translation and lysosomal or proteomal degradation, and protein phosphate modification by kinases and phosphatases, biologically regulated memory formation and removal would be yet another example in biological systems where distinct and separate pathways regulate the creation and destruction of biological substrates.

  1. 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.

  2. The Role of Deoxycytidine Kinase (dCK) in Radiation-Induced Cell Death

    PubMed Central

    Zhong, Rui; Xin, Rui; Chen, Zongyan; Liang, Nan; Liu, Yang; Ma, Shumei; Liu, Xiaodong

    2016-01-01

    Deoxycytidine kinase (dCK) is a key enzyme in deoxyribonucleoside salvage and the anti-tumor activity for many nucleoside analogs. dCK is activated in response to ionizing radiation (IR)-induced DNA damage and it is phosphorylated on Serine 74 by the Ataxia-Telangiectasia Mutated (ATM) kinase in order to activate the cell cycle G2/M checkpoint. However, whether dCK plays a role in radiation-induced cell death is less clear. In this study, we genetically modified dCK expression by knocking down or expressing a WT (wild-type), S74A (abrogates phosphorylation) and S74E (mimics phosphorylation) of dCK. We found that dCK could decrease IR-induced total cell death and apoptosis. Moreover, dCK increased IR-induced autophagy and dCK-S74 is required for it. Western blotting showed that the ratio of phospho-Akt/Akt, phospho-mTOR/mTOR, phospho-P70S6K/P70S6K significantly decreased in dCK-WT and dCK-S74E cells than that in dCK-S74A cells following IR treatment. Reciprocal experiment by co-immunoprecipitation showed that mTOR can interact with wild-type dCK. IR increased polyploidy and decreased G2/M arrest in dCK knock-down cells as compared with control cells. Taken together, phosphorylated and activated dCK can inhibit IR-induced cell death including apoptosis and mitotic catastrophe, and promote IR-induced autophagy through PI3K/Akt/mTOR pathway. PMID:27879648

  3. The Role of Deoxycytidine Kinase (dCK) in Radiation-Induced Cell Death.

    PubMed

    Zhong, Rui; Xin, Rui; Chen, Zongyan; Liang, Nan; Liu, Yang; Ma, Shumei; Liu, Xiaodong

    2016-11-21

    Deoxycytidine kinase (dCK) is a key enzyme in deoxyribonucleoside salvage and the anti-tumor activity for many nucleoside analogs. dCK is activated in response to ionizing radiation (IR)-induced DNA damage and it is phosphorylated on Serine 74 by the Ataxia-Telangiectasia Mutated (ATM) kinase in order to activate the cell cycle G2/M checkpoint. However, whether dCK plays a role in radiation-induced cell death is less clear. In this study, we genetically modified dCK expression by knocking down or expressing a WT (wild-type), S74A (abrogates phosphorylation) and S74E (mimics phosphorylation) of dCK. We found that dCK could decrease IR-induced total cell death and apoptosis. Moreover, dCK increased IR-induced autophagy and dCK-S74 is required for it. Western blotting showed that the ratio of phospho-Akt/Akt, phospho-mTOR/mTOR, phospho-P70S6K/P70S6K significantly decreased in dCK-WT and dCK-S74E cells than that in dCK-S74A cells following IR treatment. Reciprocal experiment by co-immunoprecipitation showed that mTOR can interact with wild-type dCK. IR increased polyploidy and decreased G2/M arrest in dCK knock-down cells as compared with control cells. Taken together, phosphorylated and activated dCK can inhibit IR-induced cell death including apoptosis and mitotic catastrophe, and promote IR-induced autophagy through PI3K/Akt/mTOR pathway.

  4. Teaching resources. Protein kinases.

    PubMed

    Caplan, Avrom

    2005-02-22

    This Teaching Resource provides lecture notes and slides for a class covering the structure and function of protein kinases and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the genomics and evolutionary relationships among kinases and then proceeds to describe the structure-function relationships of specific kinases, the molecular mechanisms underlying substrate specificity, and selected issues in regulation of kinase activity.

  5. Two Kinase Family Dramas

    PubMed Central

    Leonard, Thomas A.; Hurley, James H.

    2007-01-01

    In this issue, Lietha and colleagues (2007) report the structure of focal adhesion kinase (FAK) and reveal how FAK maintains an autoinhibited state. Together with the structure of another tyrosine kinase, ZAP-70 (Deindl et al., 2007), this work highlights the diversity of mechanisms that nature has evolved within the kinase superfamily to regulate their activity through autoinhibition. PMID:17574014

  6. 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

  7. Iron absorption in Drosophila melanogaster.

    PubMed

    Mandilaras, Konstantinos; Pathmanathan, Tharse; Missirlis, Fanis

    2013-05-17

    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.

  8. Actin is an evolutionarily-conserved damage-associated molecular pattern that signals tissue injury in Drosophila melanogaster

    PubMed Central

    Srinivasan, Naren; Gordon, Oliver; Ahrens, Susan; Franz, Anna; Deddouche, Safia; Chakravarty, Probir; Phillips, David; Yunus, Ali A; Rosen, Michael K; Valente, Rita S; Teixeira, Luis; Thompson, Barry; Dionne, Marc S; Wood, Will; Reis e Sousa, Caetano

    2016-01-01

    Damage-associated molecular patterns (DAMPs) are molecules released by dead cells that trigger sterile inflammation and, in vertebrates, adaptive immunity. Actin is a DAMP detected in mammals by the receptor, DNGR-1, expressed by dendritic cells (DCs). DNGR-1 is phosphorylated by Src-family kinases and recruits the tyrosine kinase Syk to promote DC cross-presentation of dead cell-associated antigens. Here we report that actin is also a DAMP in invertebrates that lack DCs and adaptive immunity. Administration of actin to Drosophila melanogaster triggers a response characterised by selective induction of STAT target genes in the fat body through the cytokine Upd3 and its JAK/STAT-coupled receptor, Domeless. Notably, this response requires signalling via Shark, the Drosophila orthologue of Syk, and Src42A, a Drosophila Src-family kinase, and is dependent on Nox activity. Thus, extracellular actin detection via a Src-family kinase-dependent cascade is an ancient means of detecting cell injury that precedes the evolution of adaptive immunity. DOI: http://dx.doi.org/10.7554/eLife.19662.001 PMID:27871362

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

    PubMed Central

    Schulz, Adriana; Preiss, Anette; Nagel, Anja C.

    2015-01-01

    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

  10. 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

  11. Methods to assay Drosophila behavior.

    PubMed

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

    2012-03-07

    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

  12. Tissue localization of Drosophila melanogaster insulin receptor transcripts during development.

    PubMed Central

    Garofalo, R S; Rosen, O M

    1988-01-01

    The Drosophila melanogaster insulin receptor (Drosophila insulin receptor homolog [dIRH]) is similar to its mammalian counterpart in deduced amino acid sequence, subunit structure, and ligand-stimulated protein tyrosine kinase activity. The function of this receptor in D. melanogaster is not yet known. However, a role in development is suggested by the observations that levels of insulin-stimulated kinase activity and expression of dIRH mRNA are maximal during Drosophila midembryogenesis. In this study, a 2.9-kilobase (kb) cDNA clone corresponding to both the dIRH tyrosine kinase domain and some of the 3' untranslated sequence was used to determine the tissue distribution of dIRH mRNA during development. Two principal mRNAs of 11 and 8.6 kb hybridized with the dIRH cDNA in Northern (RNA) blot analysis. The abundance of the 8.6-kb mRNA increased transiently in early embryos, whereas the 11-kb species was most abundant during midembryogenesis. A similar pattern of expression was previously determined by Northern analysis, using a dIRH genomic clone (L. Petruzzelli, R. Herrera, R. Arenas-Garcia, R. Fernandez, M. J. Birnbaum, and O. M. Rosen, Proc. Natl. Acad. Sci. USA 83:4710-4714, 1986). In situ hybridization revealed dIRH transcripts in the ovaries of adult flies, in which the transcripts appeared to be synthesized by nurse cells for eventual storage as maternal RNA in the mature oocyte. Throughout embryogenesis, dIRH transcripts were ubiquitously expressed, although after midembryogenesis, higher levels were detected in the developing nervous system. Nervous system expression remained elevated throughout the larval stages and persisted in the adult, in which the cortex of the brain and ganglion cells were among the most prominently labeled tissues. In larvae, the imaginal disk cells exhibited comparatively high levels of dIRH mRNA expression. The broad distribution of dIRH mRNA in embryos and imaginal disks is compatible with a role for dIRH in anabolic processes

  13. The chromosomal passenger complex activates Polo kinase at centromeres.

    PubMed

    Carmena, Mar; Pinson, Xavier; Platani, Melpi; Salloum, Zeina; Xu, Zhenjie; Clark, Anthony; Macisaac, Fiona; Ogawa, Hiromi; Eggert, Ulrike; Glover, David M; Archambault, Vincent; Earnshaw, William C

    2012-01-01

    The coordinated activities at centromeres of two key cell cycle kinases, Polo and Aurora B, are critical for ensuring that the two sister kinetochores of each chromosome are attached to microtubules from opposite spindle poles prior to chromosome segregation at anaphase. Initial attachments of chromosomes to the spindle involve random interactions between kinetochores and dynamic microtubules, and errors occur frequently during early stages of the process. The balance between microtubule binding and error correction (e.g., release of bound microtubules) requires the activities of Polo and Aurora B kinases, with Polo promoting stable attachments and Aurora B promoting detachment. Our study concerns the coordination of the activities of these two kinases in vivo. We show that INCENP, a key scaffolding subunit of the chromosomal passenger complex (CPC), which consists of Aurora B kinase, INCENP, Survivin, and Borealin/Dasra B, also interacts with Polo kinase in Drosophila cells. It was known that Aurora A/Bora activates Polo at centrosomes during late G2. However, the kinase that activates Polo on chromosomes for its critical functions at kinetochores was not known. We show here that Aurora B kinase phosphorylates Polo on its activation loop at the centromere in early mitosis. This phosphorylation requires both INCENP and Aurora B activity (but not Aurora A activity) and is critical for Polo function at kinetochores. Our results demonstrate clearly that Polo kinase is regulated differently at centrosomes and centromeres and suggest that INCENP acts as a platform for kinase crosstalk at the centromere. This crosstalk may enable Polo and Aurora B to achieve a balance wherein microtubule mis-attachments are corrected, but proper attachments are stabilized allowing proper chromosome segregation.

  14. Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis

    PubMed Central

    2013-01-01

    Background In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. Results Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof1/+; mnkp6/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. Conclusion mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry

  15. Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis.

    PubMed

    Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Ramaiah, M Janaki; Chowdhury, Debabani Roy; Bhadra, Utpal; Pal-Bhadra, Manika

    2013-01-24

    In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof¹/+; mnkp⁶/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry out the interaction of MOF

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

    PubMed Central

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

    2014-01-01

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

  17. A Drosophila model for developmental nicotine exposure.

    PubMed

    Velazquez-Ulloa, Norma Andrea

    2017-01-01

    Despite the known health risks of tobacco smoking, many people including pregnant women continue smoking. The effects of developmental nicotine exposure are known, but the underlying mechanisms are not well understood. Drosophila melanogaster is a model organism that can be used for uncovering genetic and molecular mechanisms for drugs of abuse. Here I show that Drosophila can be a model to elucidate the mechanisms for nicotine's effects on a developing organism. Drosophila reared on nicotine food display developmental and behavioral effects similar to those in mammals including decreased survival and weight, increased developmental time, and decreased sensitivity to acute nicotine and ethanol. The Drosophila nicotinic acetylcholine receptor subunit alpha 7 (Dα7) mediates some of these effects. A novel role for Dα7 on ethanol sedation in Drosophila is also shown. Future research taking advantage of the genetic and molecular tools for Drosophila will allow additional discovery of the mechanisms behind the effects of nicotine during development.

  18. SlgA, the homologue of the human schizophrenia associated PRODH gene, acts in clock neurons to regulate Drosophila aggression.

    PubMed

    Zwarts, Liesbeth; Vulsteke, Veerle; Buhl, Edgar; Hodge, James J L; Callaerts, Patrick

    2017-03-22

    Mutations in proline dehydrogenase (PRODH) are linked to behavioral alterations in schizophrenia and as part of DiGeorge and velo-cardio-facial syndromes, but the role of PRODH in their etiology remains unclear. We here establish a Drosophila model to study the role of PRODH in behavioral disorders. We determine the distribution of the Drosophila PRODH homolog slgA in the brain and show that knock-down and overexpression of human PRODH and slgA in the lateral neurons ventral (LNv) lead to altered aggressive behavior. SlgA acts in an isoform-specific manner and is regulated by casein kinase II (CkII). Our data suggest that these effects are, at least partially, due to effects on mitochondrial function. We thus show that precise regulation of proline metabolism is essential to drive normal behavior and we identify Drosophila aggression as a model behavior relevant for the study of mechanisms impaired in neuropsychiatric disorders.

  19. Ion and solute transport by Prestin in Drosophila and Anopheles.

    PubMed

    Hirata, Taku; Czapar, Anna; Brin, Lauren; Haritonova, Alyona; Bondeson, Daniel P; Linser, Paul; Cabrero, Pablo; Thompson, James; Dow, Julian A T; Romero, Michael F

    2012-04-01

    The gut and Malpighian tubules of insects are the primary sites of active solute and water transport for controlling hemolymph and urine composition, pH, and osmolarity. These processes depend on ATPase (pumps), channels and solute carriers (Slc proteins). Maturation of genomic databases enables us to identify the putative molecular players for these processes. Anion transporters of the Slc4 family, AE1 and NDAE1, have been reported as HCO(3)(-) transporters, but are only part of the story. Here we report Dipteran (Drosophila melanogaster (d) and Anopheles gambiae (Ag)) anion exchangers, belonging to the Slc26 family, which are multi-functional anion exchangers. One Drosophila and two Ag homologues of mammalian Slc26a5 (Prestin) and Slc26a6 (aka, PAT1, CFEX) were identified and designated dPrestin, AgPrestinA and AgPrestinB. dPrestin and AgPrestinB show electrogenic anion exchange (Cl(-)/nHCO(3)(-), Cl(-)/SO(4)(2-) and Cl(-)/oxalate(2-)) in an oocyte expression system. Since these transporters are the only Dipteran Slc26 proteins whose transport is similar to mammalian Slc26a6, we submit that Dipteran Prestin are functional and even molecular orthologues of mammalian Slc26a6. OSR1 kinase increases dPrestin ion transport, implying another set of physiological processes controlled by WNK/SPAK signaling in epithelia. All of these mRNAs are highly expressed in the gut and Malpighian tubules. Dipteran Prestin proteins appear suited for central roles in bicarbonate, sulfate and oxalate metabolism including generating the high pH conditions measured in the Dipteran midgut lumen. Finally, we present and discuss Drosophila genetic models that integrate these processes.

  20. Ion and solute transport by prestin in Drosophila and Anopheles

    PubMed Central

    Hirata, Taku; Czapar, Anna; Brin, Lauren R.; Haritonova, Alyona; Bondeson, Daniel P.; Linser, Paul J.; Cabrero, Pablo; Dow, Julian A. T.; Romero, Michael F.

    2012-01-01

    The gut and Malpighian tubules of insects are the primary sites of active solute and water transport for controlling hemolymph and urine composition, pH, and osmolarity. These processes depend on ATPase (pumps), channels and solute carriers (Slc proteins). Maturation of genomic databases enables us to identify the putative molecular players for these processes. Anion transporters of the Slc4 family, AE1 and NDAE1, have been reported as HCO3− transporters, but are only part of the story. Here we report Dipteran (Drosophila melanogaster (d) and Anopheles gambiae (Ag)) anion exchangers, belonging to the Slc26 family, which are multi-functional anion exchangers. One Drosophila and two Ag homologues of mammalian Slc26a5 (prestin) and Slc26a6 (aka, PAT1, CFEX) were identified and designated dPrestin, AgPrestinA and AgPrestinB. dPrestin and AgPrestinB show electrogenic anion exchange (Cl−/nHCO3−, Cl−/SO42− and Cl−/oxalate2−) in an oocyte expression system. Since these transporters are the only Dipteran Slc26 proteins whose transport is similar to mammalian Slc26a6, we submit that Dipteran Prestin are functional and even molecular orthologues of mammalian Slc26a6. OSR1 kinase increases dPrestin ion transport, implying another set of physiological processes controlled by WNK/SPAK signaling in epithelia. All of these mRNAs are highly expressed in the gut and Malpighian tubules. Dipteran Prestin proteins appear suited for central roles in bicarbonate, sulfate and oxalate metabolism including generating the high pH conditions measured in the Dipteran midgut lumen. Finally, we present and discuss Drosophila genetic models that integrate these processes. PMID:22321763

  1. Identification of autosomal regions involved in Drosophila Raf function.

    PubMed Central

    Li, W; Noll, E; Perrimon, N

    2000-01-01

    Raf is an essential downstream effector of activated p21(Ras) (Ras) in transducing proliferation or differentiation signals. Following binding to Ras, Raf is translocated to the plasma membrane, where it is activated by a yet unidentified "Raf activator." In an attempt to identify the Raf activator or additional molecules involved in the Raf signaling pathway, we conducted a genetic screen to identify genomic regions that are required for the biological function of Drosophila Raf (Draf). We tested a collection of chromosomal deficiencies representing approximately 70% of the autosomal euchromatic genomic regions for their abilities to enhance the lethality associated with a hypomorphic viable allele of Draf, Draf(Su2). Of the 148 autosomal deficiencies tested, 23 behaved as dominant enhancers of Draf(Su2), causing lethality in Draf(Su2) hemizygous males. Four of these deficiencies identified genes known to be involved in the Drosophila Ras/Raf (Ras1/Draf) pathway: Ras1, rolled (rl, encoding a MAPK), 14-3-3epsilon, and bowel (bowl). Two additional deficiencies removed the Drosophila Tec and Src homologs, Tec29A and Src64B. We demonstrate that Src64B interacts genetically with Draf and that an activated form of Src64B, when overexpressed in early embryos, causes ectopic expression of the Torso (Tor) receptor tyrosine kinase-target gene tailless. In addition, we show that a mutation in Tec29A partially suppresses a gain-of-function mutation in tor. These results suggest that Tec29A and Src64B are involved in Tor signaling, raising the possibility that they function to activate Draf. Finally, we discovered a genetic interaction between Draf(Su2) and Df(3L)vin5 that revealed a novel role of Draf in limb development. We find that loss of Draf activity causes limb defects, including pattern duplications, consistent with a role for Draf in regulation of engrailed (en) expression in imaginal discs. PMID:11014822

  2. 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

  3. Loss of putzig Activity Results in Apoptosis during Wing Imaginal Development in Drosophila

    PubMed Central

    Zimmermann, Mirjam; Kugler, Sabrina J.; Schulz, Adriana; Nagel, Anja C.

    2015-01-01

    The Drosophila gene putzig (pzg) encodes a nuclear protein that is an integral component of the Trf2/Dref complex involved in the transcription of proliferation-related genes. Moreover, Pzg is found in a complex together with the nucleosome remodeling factor NURF, where it promotes Notch target gene activation. Here we show that downregulation of pzg activity in the developing wing imaginal discs induces an apoptotic response, accompanied by the induction of the pro-apoptotic gene reaper, repression of Drosophila inhibitor of apoptosis protein accumulation and the activation of the caspases Drice, Caspase3 and Dcp1. As a further consequence ‘Apoptosis induced Proliferation’ (AiP) and ‘Apoptosis induced Apoptosis’ (AiA) are triggered. As expected, the activity of the stress kinase Jun N-terminal kinase (JNK), proposed to mediate both processes, is ectopically induced in response to pzg loss. In addition, the expression of the mitogen wingless (wg) but not of decapentaplegic (dpp) is observed. We present evidence that downregulation of Notch activates Dcp1 caspase and JNK signaling, however, neither induces ectopic wg nor dpp expression. In contrast, the consequences of Dref-RNAi were largely indistinguishable from pzg-RNAi with regard to apoptosis induction. Moreover, overexpression of Dref ameliorated the downregulation of pzg compatible with the notion that the two are required together to maintain cell and tissue homeostasis in Drosophila. PMID:25894556

  4. Dissociation of rugose-dependent short-term memory component from memory consolidation in Drosophila.

    PubMed

    Zhao, J; Lu, Y; Zhao, X; Yao, X; Shuai, Y; Huang, C; Wang, L; Jeong, S H; Zhong, Y

    2013-08-01

    Extensive investigations show several molecular and neuroanatomical mechanisms underlying short-lived and long-lasting memory in Drosophila. At the molecular level, the genetic pathway of memory formation, which was obtained through mutant research, seems to occur sequentially. So far, studies of Drosophila mutants appear to support the idea that mutants defective in short-term memory (STM) are always associated with long-term memory (LTM) impairment. At the neuroanatomical level, distinct memory traces are partially independently distributed. However, whether memory phase dissociation also exists at the molecular level remains unclear. Here, we report on molecular separation of STM and consolidated memory through genetic dissection of rugose mutants. Mutants in the rugose gene, which encodes an evolutionarily conserved A-kinase anchor protein, show immediate memory defects as assayed through aversive olfactory conditioning. Intriguingly, two well-defined consolidated memory components, anesthesia-resistant memory and protein synthesis-dependent LTM, are both normal in spite of the defective immediate memory after 10-session massed and spaced training. Moreover, rugose genetically interacts with cyclic AMP-protein kinase A signaling during STM formation. Considering our previous study that AKAP Yu specifically participates in LTM formation, these results suggest that there exists a molecular level of memory phase dissociation with distinct AKAPs in Drosophila.

  5. DaPKC-dependent phosphorylation of Crumbs is required for epithelial cell polarity in Drosophila

    PubMed Central

    Sotillos, Sol; Díaz-Meco, María Teresa; Caminero, Eva; Moscat, Jorge; Campuzano, Sonsoles

    2004-01-01

    Both in Drosophila and vertebrate epithelial cells, the establishment of apicobasal polarity requires the apically localized, membrane-associated Par-3–Par-6–aPKC protein complex. In Drosophila, this complex colocalizes with the Crumbs–Stardust (Sdt)–Pals1-associated TJ protein (Patj) complex. Genetic and molecular analyses suggest a functional relationship between them. We show, by overexpression of a kinase-dead Drosophila atypical PKC (DaPKC), the requirement for the kinase activity of DaPKC to maintain the position of apical determinants and to restrict the localization of basolateral ones. We demonstrate a novel physical interaction between the apical complexes, via direct binding of DaPKC to both Crb and Patj, and identify Crumbs as a phosphorylation target of DaPKC. This phosphorylation of Crumbs is functionally significant. Thus, a nonphosphorylatable Crumbs protein behaves in vivo as a dominant negative. Moreover, the phenotypic effect of overexpressing wild-type Crumbs is suppressed by reducing DaPKC activity. These results provide a mechanistic framework for the functional interaction between the Par-3–Par-6–aPKC and Crumbs–Sdt–Patj complexes based in the posttranslational modification of Crb by DaPKC. PMID:15302858

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

    PubMed

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

    2013-01-01

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

  7. The Drosophila MAPK p38c Regulates Oxidative Stress and Lipid Homeostasis in the Intestine

    PubMed Central

    Chakrabarti, Sveta; Poidevin, Mickaël; Lemaitre, Bruno

    2014-01-01

    The p38 mitogen-activated protein (MAP) kinase signaling cassette has been implicated in stress and immunity in evolutionarily diverse species. In response to a wide variety of physical, chemical and biological stresses p38 kinases phosphorylate various substrates, transcription factors of the ATF family and other protein kinases, regulating cellular adaptation to stress. The Drosophila genome encodes three p38 kinases named p38a, p38b and p38c. In this study, we have analyzed the role of p38c in the Drosophila intestine. The p38c gene is expressed in the midgut and upregulated upon intestinal infection. We showed that p38c mutant flies are more resistant to infection with the lethal pathogen Pseudomonas entomophila but are more susceptible to the non-pathogenic bacterium Erwinia carotovora 15. This phenotype was linked to a lower production of Reactive Oxygen Species (ROS) in the gut of p38c mutants, whereby the transcription of the ROS-producing enzyme Duox is reduced in p38c mutant flies. Our genetic analysis shows that p38c functions in a pathway with Mekk1 and Mkk3 to induce the phosphorylation of Atf-2, a transcription factor that controls Duox expression. Interestingly, p38c deficient flies accumulate lipids in the intestine while expressing higher levels of antimicrobial peptide and metabolic genes. The role of p38c in lipid metabolism is mediated by the Atf3 transcription factor. This observation suggests that p38c and Atf3 function in a common pathway in the intestine to regulate lipid metabolism and immune homeostasis. Collectively, our study demonstrates that p38c plays a central role in the intestine of Drosophila. It also reveals that many roles initially attributed to p38a are in fact mediated by p38c. PMID:25254641

  8. Sculpting epithelia with planar polarized actomyosin networks: Principles from Drosophila.

    PubMed

    Harris, Tony J C

    2017-07-29

    Drosophila research has revealed how planar polarized actomyosin networks affect various types of tissue morphogenesis. The networks are positioned by both tissue-wide patterning factors (including Even-skipped, Runt, Engrailed, Invected, Hedgehog, Notch, Wingless, Epidermal Growth Factor, Jun N-terminal kinase, Sex combs reduced and Fork head) and local receptor complexes (including Echinoid, Crumbs and Toll receptors). Networks with differing super-structure and contractile output have been discovered. Their contractility can affect individual cells or can be coordinated across groups of cells, and such contractility can drive or resist physical change. For what seem to be simple tissue changes, multiple types of actomyosin networks can contribute, acting together as contractile elements or braces within the developing structure. This review discusses the positioning and effects of planar polarized actomyosin networks for a number of developmental events in Drosophila, including germband extension, dorsal closure, head involution, tracheal pit formation, salivary gland development, imaginal disc boundary formation, and tissue rotation of the male genitalia and the egg chamber. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. The development of germline stem cells in Drosophila.

    PubMed

    Dansereau, David A; Lasko, Paul

    2008-01-01

    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.

  10. 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.

  11. Rescue of easily shocked mutant seizure sensitivity in Drosophila adults.

    PubMed

    Kroll, Jason R; Tanouye, Mark A

    2013-10-15

    Genetic factors that influence seizure susceptibility can act transiently during the development of neural circuits or might be necessary for the proper functioning of existing circuits. We provide evidence that the Drosophila seizure-sensitive mutant easily shocked (eas) represents a neurological disorder in which abnormal functioning of existing neural circuits leads to seizure sensitivity. The eas(+) gene encodes for the protein Ethanolamine Kinase, involved in phospholipid biosynthesis. We show that induction of eas(+) in adult mutant flies rescues them from seizure sensitivity despite previously known developmental defects in brain morphology. Additionally, through cell-type-specific rescue, our results suggest a specific role for eas(+) in excitatory rather than inhibitory neural transmission. Overall, our findings emphasize an important role for proper phospholipid metabolism in normal brain function and suggest that certain classes of epilepsy syndromes could have the potential to be treated with gene therapy techniques. Copyright © 2013 Wiley Periodicals, Inc.

  12. Quantitative Evaluation of Signaling Events in Drosophila S2 Cells

    PubMed Central

    2008-01-01

    Drosophila activates a robust defense response to gram-negative bacteria through the Immune deficiency (Imd) pathway. Imd signaling proceeds through c-Jun N-terminal Kinase (JNK), NF-kB and caspase modules. The individual signaling modules act in a highly coordinated manner to yield a stereotypical response to infection. While considerable attention has focused on NF-kB-mediated antimicrobial activities, more recent studies have highlighted the involvement of JNK signaling in the Imd pathway response. JNK signaling occurs in a transitory burst and drives the expression of a number of gene products through the AP-1 transcription factor. In this report, we describe a simple method for the quantification of JNK activation by Western blot analysis or directly in tissue culture plates. PMID:18385808

  13. Misexpression screen for genes altering the olfactory map in Drosophila.

    PubMed

    Zhang, Dongsheng; Zhou, Weiguang; Yin, Chong; Chen, Weitao; Ozawa, Rie; Ang, Lay-Hong; Anandan, Lavanya; Aigaki, Toshiro; Hing, Huey

    2006-04-01

    Despite the identification of a number of guidance molecules, a comprehensive picture has yet to emerge to explain the precise anatomy of the olfactory map. From a misexpression screen of 1,515 P{GS} lines, we identified 23 genes that, when forcibly expressed in the olfactory receptor neurons, disrupted the stereotyped anatomy of the Drosophila antennal lobes. These genes, which have not been shown previously to control olfactory map development, encode novel proteins as well as proteins with known roles in axonal outgrowth and cytoskeletal remodeling. We analyzed Akap200, which encodes a Protein Kinase A-binding protein. Overexpression of Akap200 resulted in fusion of the glomeruli, while its loss resulted in misshapen and ectopic glomeruli. The requirement of Akap200 validates our screen as an effective approach for recovering genes controlling glomerular map patterning. Our finding of diverse classes of genes reveals the complexity of the mechanisms that underlie olfactory map development. Published 2006 Wiley-Liss, Inc.

  14. Genes regulating dendritic outgrowth, branching, and routing in Drosophila

    PubMed Central

    Gao, Fen-Biao; Brenman, Jay E.; Jan, Lily Yeh; Jan, Yuh Nung

    1999-01-01

    Signaling between neurons requires highly specialized subcellular structures, including dendrites and axons. Dendrites exhibit diverse morphologies yet little is known about the mechanisms controlling dendrite formation in vivo. We have developed methods to visualize the stereotyped dendritic morphogenesis in living Drosophila embryos. Dendrite development is altered in prospero mutants and in transgenic embryos expressing a constitutively active form of the small GTPase cdc42. From a genetic screen, we have identified several genes that control different aspects of dendrite development including dendritic outgrowth, branching, and routing. These genes include kakapo, a large cytoskeletal protein related to plectin and dystrophin; flamingo, a seven-transmembrane protein containing cadherin-like repeats; enabled, a substrate of the tyrosine kinase Abl; and nine potentially novel loci. These findings begin to reveal the molecular mechanisms controlling dendritic morphogenesis. PMID:10521399

  15. Genes regulating dendritic outgrowth, branching, and routing in Drosophila.

    PubMed

    Gao, F B; Brenman, J E; Jan, L Y; Jan, Y N

    1999-10-01

    Signaling between neurons requires highly specialized subcellular structures, including dendrites and axons. Dendrites exhibit diverse morphologies yet little is known about the mechanisms controlling dendrite formation in vivo. We have developed methods to visualize the stereotyped dendritic morphogenesis in living Drosophila embryos. Dendrite development is altered in prospero mutants and in transgenic embryos expressing a constitutively active form of the small GTPase cdc42. From a genetic screen, we have identified several genes that control different aspects of dendrite development including dendritic outgrowth, branching, and routing. These genes include kakapo, a large cytoskeletal protein related to plectin and dystrophin; flamingo, a seven-transmembrane protein containing cadherin-like repeats; enabled, a substrate of the tyrosine kinase Abl; and nine potentially novel loci. These findings begin to reveal the molecular mechanisms controlling dendritic morphogenesis.

  16. Limited taste discrimination in Drosophila.

    PubMed

    Masek, Pavel; Scott, Kristin

    2010-08-17

    In the gustatory systems of mammals and flies, different populations of sensory cells recognize different taste modalities, such that there are cells that respond selectively to sugars and others to bitter compounds. This organization readily allows animals to distinguish compounds of different modalities but may limit the ability to distinguish compounds within one taste modality. Here, we developed a behavioral paradigm in Drosophila melanogaster to evaluate directly the tastes that a fly distinguishes. These studies reveal that flies do not discriminate among different sugars, or among different bitter compounds, based on chemical identity. Instead, flies show a limited ability to distinguish compounds within a modality based on intensity or palatability. Taste associative learning, similar to olfactory learning, requires the mushroom bodies, suggesting fundamental similarities in brain mechanisms underlying behavioral plasticity. Overall, these studies provide insight into the discriminative capacity of the Drosophila gustatory system and the modulation of taste behavior.

  17. 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.

  18. Drosophila 14-3-3/PAR-5 is an essential mediator of PAR-1 function in axis formation.

    PubMed

    Benton, Richard; Palacios, Isabel M; St Johnston, Daniel

    2002-11-01

    PAR-1 kinases are required to determine the anterior-posterior (A-P) axis in C. elegans and Drosophila, but little is known about their molecular function. We identified 14-3-3 proteins as Drosophila PAR-1 interactors and show that PAR-1 binds a domain of 14-3-3 distinct from the phosphoserine binding pocket. PAR-1 kinases phosphorylate proteins to generate 14-3-3 binding sites and may therefore directly deliver 14-3-3 to these targets. 14-3-3 mutants display identical phenotypes to par-1 mutants in oocyte determination and the polarization of the A-P axis. Together, these results indicate that PAR-1's function is mediated by the binding of 14-3-3 to its substrates. The C. elegans 14-3-3 protein, PAR-5, is also required for A-P polarization, suggesting that this is a conserved mechanism by which PAR-1 establishes cellular asymmetries.

  19. 'Peer pressure' in larval Drosophila?

    PubMed

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

    2014-06-06

    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. © 2014. Published by The Company of Biologists Ltd.

  20. Drosophila's view on insect vision.

    PubMed

    Borst, Alexander

    2009-01-13

    Within the last 400 million years, insects have radiated into at least a million species, accounting for more than half of all known living organisms: they are the most successful group in the animal kingdom, found in almost all environments of the planet, ranging in body size from a mere 0.1 mm up to half a meter. Their eyes, together with the respective parts of the nervous system dedicated to the processing of visual information, have long been the subject of intense investigation but, with the exception of some very basic reflexes, it is still not possible to link an insect's visual input to its behavioral output. Fortunately for the field, the fruit fly Drosophila is an insect, too. This genetic workhorse holds great promise for the insect vision field, offering the possibility of recording, suppressing or stimulating any single neuron in its nervous system. Here, I shall give a brief synopsis of what we currently know about insect vision, describe the genetic toolset available in Drosophila and give some recent examples of how the application of these tools have furthered our understanding of color and motion vision in Drosophila.

  1. 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

  2. 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

  3. 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

  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. Using Drosophila for Studies of Intermediate Filaments.

    PubMed

    Bohnekamp, Jens; Cryderman, Diane E; Thiemann, Dylan A; Magin, Thomas M; Wallrath, Lori L

    2016-01-01

    Drosophila melanogaster is a useful organism for determining protein function and modeling human disease. Drosophila offers a rapid generation time and an abundance of genomic resources and genetic tools. Conservation in protein structure, signaling pathways, and developmental processes make studies performed in Drosophila relevant to other species, including humans. Drosophila models have been generated for neurodegenerative diseases, muscular dystrophy, cancer, and many other disorders. Recently, intermediate filament protein diseases have been modeled in Drosophila. These models have revealed novel mechanisms of pathology, illuminated potential new routes of therapy, and make whole organism compound screens feasible. The goal of this chapter is to outline steps to study intermediate filament function and model intermediate filament-associated diseases in Drosophila. The steps are general and can be applied to study the function of almost any protein. The protocols outlined here are for both the novice and experienced Drosophila researcher, allowing the rich developmental and cell biology that Drosophila offers to be applied to studies of intermediate filaments. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. 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.

  7. Bällchen is required for self-renewal of germline stem cells in Drosophila melanogaster.

    PubMed

    Herzig, Bettina; Yakulov, Toma A; Klinge, Kathrin; Günesdogan, Ufuk; Jäckle, Herbert; Herzig, Alf

    2014-05-29

    Self-renewing stem cells are pools of undifferentiated cells, which are maintained in cellular niche environments by distinct tissue-specific signalling pathways. In Drosophila melanogaster, female germline stem cells (GSCs) are maintained in a somatic niche of the gonads by BMP signalling. Here we report a novel function of the Drosophila kinase Bällchen (BALL), showing that its cell autonomous role is to maintain the self-renewing capacity of female GSCs independent of BMP signalling. ball mutant GSCs are eliminated from the niche and subsequently differentiate into mature eggs, indicating that BALL is largely dispensable for differentiation. Similar to female GSCs, BALL is required to maintain self-renewal of male GSCs, suggesting a tissue independent requirement of BALL for self-renewal of germline stem cells.

  8. Loss of Drosophila Vps16A enhances autophagosome formation through reduced Tor activity

    PubMed Central

    Takáts, Szabolcs; Varga, Ágnes; Pircs, Karolina; Juhász, Gábor

    2015-01-01

    The HOPS tethering complex facilitates autophagosome-lysosome fusion by binding to Syx17 (Syntaxin 17), the autophagosomal SNARE. Here we show that loss of the core HOPS complex subunit Vps16A enhances autophagosome formation and slows down Drosophila development. Mechanistically, Tor kinase is less active in Vps16A mutants likely due to impaired endocytic and biosynthetic transport to the lysosome, a site of its activation. Tor reactivation by overexpression of Rheb suppresses autophagosome formation and restores growth and developmental timing in these animals. Thus, Vps16A reduces autophagosome numbers both by indirectly restricting their formation rate and by directly promoting their clearance. In contrast, the loss of Syx17 blocks autophagic flux without affecting the induction step in Drosophila. PMID:26061715

  9. Loss of PI3K blocks cell-cycle progression in a Drosophila tumor model.

    PubMed

    Willecke, M; Toggweiler, J; Basler, K

    2011-09-29

    Tumorigenesis is a complex process, which requires alterations in several tumor suppressor or oncogenes. Here, we use a Drosophila tumor model to identify genes, which are specifically required for tumor growth. We found that reduction of phosphoinositide 3-kinase (PI3K) activity resulted in very small tumors while only slightly affecting growth of wild-type tissue. The observed inhibition on tumor growth occurred at the level of cell-cycle progression. We conclude that tumor cells become dependent on PI3K function and that reduction of PI3K activity synthetically interferes with tumor growth. The results presented here broaden our insights into the intricate mechanisms underling tumorigenesis and illustrate the power of Drosophila genetics in revealing weak points of tumor progression.

  10. 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

  11. Organogenesis and tumorigenesis: Insight from the JAK/STAT pathway in the Drosophila eye

    PubMed Central

    Wang, Ying-Hsuan; Huang, Min-Lang

    2010-01-01

    The Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway is one of the main signaling pathways in eukaryotic cells. This pathway is used during diverse growth and developmental processes in multiple tissues to control cell proliferation, differentiation, survival, and apoptosis. In addition to its role during development, the JAK/STAT pathway has also been implicated in tumorigenesis. Drosophila melanogaster is a powerful genetic tool, and its eyes have been used extensively as a platform to study signaling pathways. Many reports have demonstrated that the JAK/STAT pathway plays pleiotropic roles in Drosophila eye development. Its functions and activation are decided by its interplay with other signal pathways and the epigenetic status. In this review, we focus on the functions and regulation of the JAK/STAT pathway during eye development and provide some insights into the study of this pathway in tumorigenesis. Developmental Dynamics 239:2522–2533, 2010. © 2010 Wiley-Liss, Inc. PMID:20737505

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

    PubMed

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

    2016-01-29

    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.

  13. The deubiquitinating enzyme Usp5 regulates Notch and RTK signaling during Drosophila eye development.

    PubMed

    Ling, Xuemei; Huang, Qinzhu; Xu, Yanqin; Jin, Yuxiao; Feng, Ying; Shi, Weijie; Ye, Xiaolei; Lin, Yi; Hou, Ling; Lin, Xinhua

    2017-03-01

    Usp5 belongs to the USP family of deubiquitinating enzymes (DUBs), which comprises the largest class of DUBs. We previously reported that loss of Usp5 impairs development of photoreceptors in Drosophila eyes, although the detailed mechanism remained unclear. In the present study, we demonstrate that Usp5 regulates both Notch and receptor tyrosine kinase (RTK) signaling. Loss of Usp5 results in upregulation of Notch signaling and downregulation of RTK signaling, leading to impaired photoreceptor development. Moreover, genetic rescue experiments with the DNA binding protein Suppressor of Hairless or Notch RNAi indicate that they mediate the regulation of RTK signaling by Usp5. The present study provides mechanistic insight into how Usp5 regulates photoreceptor differentiation by Notch and RTK signaling in the Drosophila eye. © 2017 Federation of European Biochemical Societies.

  14. Organogenesis and tumorigenesis: insight from the JAK/STAT pathway in the Drosophila eye.

    PubMed

    Wang, Ying-Hsuan; Huang, Min-Lang

    2010-10-01

    The Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway is one of the main signaling pathways in eukaryotic cells. This pathway is used during diverse growth and developmental processes in multiple tissues to control cell proliferation, differentiation, survival, and apoptosis. In addition to its role during development, the JAK/STAT pathway has also been implicated in tumorigenesis. Drosophila melanogaster is a powerful genetic tool, and its eyes have been used extensively as a platform to study signaling pathways. Many reports have demonstrated that the JAK/STAT pathway plays pleiotropic roles in Drosophila eye development. Its functions and activation are decided by its interplay with other signal pathways and the epigenetic status. In this review, we focus on the functions and regulation of the JAK/STAT pathway during eye development and provide some insights into the study of this pathway in tumorigenesis.

  15. Activity-dependent facilitation of Synaptojanin and synaptic vesicle recycling by the Minibrain kinase

    PubMed Central

    Chen, Chun-Kan; Bregere, Catherine; Paluch, Jeremy; Lu, Jason; Dickman, Dion K.; Chang, Karen T.

    2014-01-01

    Phosphorylation has emerged as a crucial regulatory mechanism in the nervous system to integrate the dynamic signaling required for proper synaptic development, function, and plasticity, particularly during changes in neuronal activity. Here we present evidence that Minibrain (Mnb; also known as Dyrk1A), a serine/threonine kinase implicated in autism spectrum disorder and Down syndrome, is required presynaptically for normal synaptic growth and rapid synaptic vesicle endocytosis at the Drosophila neuromuscular junction (NMJ). We find that Mnb-dependent phosphorylation of synaptojanin (Synj) is required, in vivo, for complex endocytic protein interactions and to enhance Synj activity. Neuronal stimulation drives Mnb mobilization to endocytic zones and triggers Mnb-dependent phosphorylation of Synj. Our data identify Mnb as a synaptic kinase that promotes efficient synaptic vesicle recycling by dynamically calibrating Synj function at the Drosophila NMJ, and in turn endocytic capacity, to adapt to conditions of high synaptic activity. PMID:24977345

  16. Activity-dependent facilitation of Synaptojanin and synaptic vesicle recycling by the Minibrain kinase.

    PubMed

    Chen, Chun-Kan; Bregere, Catherine; Paluch, Jeremy; Lu, Jason F; Dickman, Dion K; Chang, Karen T

    2014-06-30

    Phosphorylation has emerged as a crucial regulatory mechanism in the nervous system to integrate the dynamic signalling required for proper synaptic development, function and plasticity, particularly during changes in neuronal activity. Here we present evidence that Minibrain (Mnb; also known as Dyrk1A), a serine/threonine kinase implicated in autism spectrum disorder and Down syndrome, is required presynaptically for normal synaptic growth and rapid synaptic vesicle endocytosis at the Drosophila neuromuscular junction (NMJ). We find that Mnb-dependent phosphorylation of Synaptojanin (Synj) is required, in vivo, for complex endocytic protein interactions and to enhance Synj activity. Neuronal stimulation drives Mnb mobilization to endocytic zones and triggers Mnb-dependent phosphorylation of Synj. Our data identify Mnb as a synaptic kinase that promotes efficient synaptic vesicle recycling by dynamically calibrating Synj function at the Drosophila NMJ, and in turn endocytic capacity, to adapt to conditions of high synaptic activity.

  17. Mobile phone electromagnetic radiation activates MAPK signaling and regulates viability in Drosophila.

    PubMed

    Lee, Kyu-Sun; Choi, Jong-Soon; Hong, Sae-Yong; Son, Tae-Ho; Yu, Kweon

    2008-07-01

    Mobile phones are widely used in the modern world. However, biological effects of electromagnetic radiation produced by mobile phones are largely unknown. In this report, we show biological effects of the mobile phone 835 MHz electromagnetic field (EMF) in the Drosophila model system. When flies were exposed to the specific absorption rate (SAR) 1.6 W/kg, which is the proposed exposure limit by the American National Standards Institute (ANSI), more than 90% of the flies were viable even after the 30 h exposure. However, in the SAR 4.0 W/kg strong EMF exposure, viability dropped from the 12 h exposure. These EMF exposures triggered stress response and increased the production of reactive oxygen species. The EMF exposures also activated extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling, but not p38 kinase signaling. Interestingly, SAR 1.6 W/kg activated mainly ERK signaling and expression of an anti-apoptotic gene, whereas SAR 4.0 W/kg strongly activated JNK signaling and expression of apoptotic genes. In addition, SAR 4.0 W/kg amplified the number of apoptotic cells in the fly brain. These findings demonstrate that the exposure limit on electromagnetic radiation proposed by ANSI triggered ERK-survival signaling but the strong electromagnetic radiation activated JNK-apoptotic signaling in Drosophila.

  18. Genome-wide identification of phospho-regulators of Wnt signaling in Drosophila.

    PubMed

    Swarup, Sharan; Pradhan-Sundd, Tirthadipa; Verheyen, Esther M

    2015-04-15

    Evolutionarily conserved intercellular signaling pathways regulate embryonic development and adult tissue homeostasis in metazoans. The precise control of the state and amplitude of signaling pathways is achieved in part through the kinase- and phosphatase-mediated reversible phosphorylation of proteins. In this study, we performed a genome-wide in vivo RNAi screen for kinases and phosphatases that regulate the Wnt pathway under physiological conditions in the Drosophila wing disc. Our analyses have identified 54 high-confidence kinases and phosphatases capable of modulating the Wnt pathway, including 22 novel regulators. These candidates were also assayed for a role in the Notch pathway, and numerous phospho-regulators were identified. Additionally, each regulator of the Wnt pathway was evaluated in the wing disc for its ability to affect the mechanistically similar Hedgehog pathway. We identified 29 dual regulators that have the same effect on the Wnt and Hedgehog pathways. As proof of principle, we established that Cdc37 and Gilgamesh/CK1γ inhibit and promote signaling, respectively, by functioning at analogous levels of these pathways in both Drosophila and mammalian cells. The Wnt and Hedgehog pathways function in tandem in multiple developmental contexts, and the identification of several shared phospho-regulators serve as potential nodes of control under conditions of aberrant signaling and disease. © 2015. Published by The Company of Biologists Ltd.

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

    PubMed

    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.

  20. 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

  1. Ectopic histone H3S10 phosphorylation causes chromatin structure remodeling in Drosophila.

    PubMed

    Deng, Huai; Bao, Xiaomin; Cai, Weili; Blacketer, Melissa J; Belmont, Andrew S; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M

    2008-02-01

    Histones are subject to numerous post-translational modifications that correlate with the state of higher-order chromatin structure and gene expression. However, it is not clear whether changes in these epigenetic marks are causative regulatory factors in chromatin structure changes or whether they play a mainly reinforcing or maintenance role. In Drosophila phosphorylation of histone H3S10 in euchromatic chromatin regions by the JIL-1 tandem kinase has been implicated in counteracting heterochromatization and gene silencing. Here we show, using a LacI-tethering system, that JIL-1 mediated ectopic histone H3S10 phosphorylation is sufficient to induce a change in higher-order chromatin structure from a condensed heterochromatin-like state to a more open euchromatic state. This effect was absent when a ;kinase dead' LacI-JIL-1 construct without histone H3S10 phosphorylation activity was expressed. Instead, the 'kinase dead' construct had a dominant-negative effect, leading to a disruption of chromatin structure that was associated with a global repression of histone H3S10 phosphorylation levels. These findings provide direct evidence that the epigenetic histone tail modification of H3S10 phosphorylation at interphase can function as a causative regulator of higher-order chromatin structure in Drosophila in vivo.

  2. 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

  3. 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.

  4. 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.

  5. 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

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

    PubMed

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

    2015-12-01

    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.

  7. A phospholipid kinase regulates actin organization and intercellular bridge formation during germline cytokinesis.

    PubMed

    Brill, J A; Hime, G R; Scharer-Schuksz, M; Fuller, M T

    2000-09-01

    The endgame of cytokinesis can follow one of two pathways depending on developmental context: resolution into separate cells or formation of a stable intercellular bridge. Here we show that the four wheel drive (fwd) gene of Drosophila melanogaster is required for intercellular bridge formation during cytokinesis in male meiosis. In fwd mutant males, contractile rings form and constrict in dividing spermatocytes, but cleavage furrows are unstable and daughter cells fuse together, producing multinucleate spermatids. fwd is shown to encode a phosphatidylinositol 4-kinase (PI 4-kinase), a member of a family of proteins that perform the first step in the synthesis of the key regulatory membrane phospholipid PIP2. Wild-type activity of the fwd PI 4-kinase is required for tyrosine phosphorylation in the cleavage furrow and for normal organization of actin filaments in the constricting contractile ring. Our results suggest a critical role for PI 4-kinases and phosphatidylinositol derivatives during the final stages of cytokinesis.

  8. SKK4, a novel activator of stress-activated protein kinase-1 (SAPK1/JNK).

    PubMed

    Lawler, S; Cuenda, A; Goedert, M; Cohen, P

    1997-09-01

    A cDNA was cloned and expressed that encodes human stress-activated protein kinase kinase-4 (SKK4), a novel MAP kinase kinase family member whose mRNA is widely expressed in human tissues. SKK4 activated SAPK1/JNK in vitro, but not SAPK2a/p38, SAPK2b/p38beta, SAPK3/ERK6 or SAPK4. It appears to be the mammalian homologue of HEP, an activator of SAPK1/JNK in Drosophila. In human epithelial KB cells SKK4 and SKK1/MKK4 (another activator of SAPK1/JNK) were both activated by stressful stimuli, but only SKK4 was activated by proinflammatory cytokines. The identification of SKK4 explains why the major SAPK1/JNK activator detected in many mammalian cell extracts is chromatographically separable from SKK1/MKK4.

  9. Evolutionary Conserved Role of c-Jun-N-Terminal Kinase in CO2-Induced Epithelial Dysfunction

    PubMed Central

    Vadász, István; Dada, Laura A.; Briva, Arturo; Helenius, Iiro Taneli; Sharabi, Kfir; Welch, Lynn C.; Kelly, Aileen M.; Grzesik, Benno A.; Budinger, G. R. Scott; Liu, Jing; Seeger, Werner; Beitel, Greg J.; Gruenbaum, Yosef; Sznajder, Jacob I.

    2012-01-01

    Elevated CO2 levels (hypercapnia) occur in patients with respiratory diseases and impair alveolar epithelial integrity, in part, by inhibiting Na,K-ATPase function. Here, we examined the role of c-Jun N-terminal kinase (JNK) in CO2 signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO2 levels rapidly induced activation of JNK leading to downregulation of Na,K-ATPase and alveolar epithelial dysfunction. Hypercapnia-induced activation of JNK required AMP-activated protein kinase (AMPK) and protein kinase C-ζ leading to subsequent phosphorylation of JNK at Ser-129. Importantly, elevated CO2 levels also caused a rapid and prominent activation of JNK in Drosophila S2 cells and in C. elegans. Paralleling the results with mammalian epithelial cells, RNAi against Drosophila JNK fully prevented CO2-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO2 signaling was additionally demonstrated by the ability of mutations in the C. elegans JNK homologs, jnk-1 and kgb-2 to partially rescue the hypercapnia-induced fertility defects but not the pharyngeal pumping defects. Together, these data provide evidence that deleterious effects of hypercapnia are mediated by JNK which plays an evolutionary conserved, specific role in CO2 signaling in mammals, diptera and nematodes. PMID:23056407

  10. Chromosome Conformation Capture in Drosophila.

    PubMed

    Li, Hua-Bing

    2016-01-01

    Linear chromatin fiber is packed inside the nuclei as a complex three-dimensional structure, and the organization of the chromatin has important roles in the appropriate spatial and temporal regulation of gene expression. To understand how chromatin organizes inside nuclei, and how regulatory proteins physically interact with genes, chromosome conformation capture (3C) technique provides a powerful and sensitive tool to detect both short- and long-range DNA-DNA interaction. Here I describe the 3C technique to detect the DNA-DNA interactions mediated by insulator proteins that are closely related to PcG in Drosophila, which is also broadly applicable to other systems.

  11. 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.

  12. 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.

  13. In Focus: Spotted wing drosophila, Drosophila suzukii, across perspectives.

    PubMed

    Lee, Jana C; Bruck, Denny J; Dreves, Amy J; Ioriatti, Claudio; Vogt, Heidrun; Baufeld, Peter

    2011-11-01

    In August 2008, the first detection of the spotted wing drosophila, Drosophila suzukii, to the North America mainland in California caused great concern, as the fly was found infesting a variety of commercial fruits. Subsequent detections followed in Oregon, Washington, Florida and British Columbia in 2009; in Utah, North Carolina, South Carolina, Michigan, and Louisiana in 2010; and in Virginia, Montana, Wisconsin, Pennsylvania, New Jersey, Maryland and Mexico in 2011. In Europe, it has been detected in Italy and Spain in 2009 and in France in 2010. Economic costs to the grower from D. suzukii include the increased cost of production (increased labor and materials for chemical inputs, monitoring and other management tools) and crop loss. An effective response to the invasion of D. suzukii requires proper taxonomic identification at the initial phase, understanding basic biology and phenology, developing management tools, transferring information and technology quickly to user groups, and evaluating the impact of the research and extension program on an economic, social, and environmental level. As D. suzukii continues to expand its range, steps must be initiated in each new region to educate and inform the public as well as formulate management tactics suitable for the crops and growing conditions in each. Copyright © 2011 Society of Chemical Industry.

  14. Drosophila as an unconventional substrate for microfabrication

    NASA Astrophysics Data System (ADS)

    Shum, Angela J.; Parviz, Babak A.

    2007-02-01

    We present the application of Drosophila fruit flies as an unconventional substrate for microfabrication. Drosophila by itself represents a complex system capable of many functions not attainable with current microfabrication technology. By using Drosophila as a substrate, we are able to capitalize on these natural functions while incorporating additional functionality into a superior hybrid system. In the following, development of microfabrication processes for Drosophila substrates is discussed. In particular, results of a study on Drosophila tolerance to vacuum pressure during multiple stages of development are given. A remarkable finding that adult Drosophila may withstand up to 3 hours of exposure to vacuum with measurable survival is noted. This finding opens a number of new opportunities for performing fabrication processes, similar to the ones performed on a silicon wafer, on a fruit fly as a live substrate. As a model microfabrication process, it is shown how a collection of Drosophila can be made to self-assemble into an array of microfabricated recesses on a silicon wafer and how a shadow mask can be used to thermally evaporate 100 nm of indium on flies. The procedure resulted in the production of a number of live flies with a pre-designed metal micropattern on their wings. This demonstration of vacuum microfabrication on a live organism provides the first step towards the development of a hybrid biological/solid-state manufacturing process for complex microsystems.

  15. [Tyrosine kinase inhibitors].

    PubMed

    Robert, Jacques

    2011-11-01

    Membrane receptors with tyrosine kinase activity and cytoplasmic tyrosine kinases have emerged as important potential targets in oncology. Starting from basic structures such as anilino-quinazoline, numerous compounds have been synthesised, with the help of tyrosine kinase crystallography, which has allowed to optimise protein-ligand interactions. The catalytic domains of all kinases present similar three-dimensional structures, which explains that it may be difficult to identify molecules having a high specificity for a given tyrosine kinase. Some tyrosine kinase inhibitors are relatively specific for epidermal growth factor receptor (EGFR) such as géfitinib and erlotinib; other are mainly active against platelet-derived growth factor receptor (PDGFR) and the receptor KIT, such as imatinib or nilotinib, and other against vascular endothelial growth factor (VEGF) receptors involved in angiogenesis, such as sunitinib and sorafenib. The oral formulation of tyrosine kinase inhibitors is well accepted by the patients but may generate sometimes compliance problems requiring pharmacokinetic monitoring. This chemical family is in full expansion and several dozens of compounds have entered clinical trials.

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

    PubMed

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

    1996-05-31

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

  17. Drosophila Genetics in the Classroom

    PubMed Central

    Sofer, W.; Tompkins, L.

    1994-01-01

    Drosophila has long been useful for demonstrating the principles of classical Mendelian genetics in the classroom. In recent years, the organism has also helped students understand biochemical and behavioral genetics. In this connection, this article describes the development of a set of integrated laboratory exercises and descriptive materials--a laborotory module--in biochemical genetics for use by high-school students. The module focuses on the Adh gene and its product, the alcohol dehydrogenase enzyme. Among other activities, students using the module get to measure alcohol tolerance and to assay alcohol dehydrogenase activity in Adh-negative and -postive flies. To effectively present the module in the classroom, teachers attend a month-long Dissemination Institute in the summer. During this period, they learn about other research activities that can be adapted for classroom use. One such activity that has proved popular with teachers and students utilizes Drosophila to introduce some of the concepts of behavioral genetics to the high-school student. By establishing closer interactions between high-school educators and research scientists, the gulf between the two communities can begin to be bridged. It is anticipated that the result of a closer relationship will be that the excitement and creativity of science will be more effectively conveyed to students. PMID:8138175

  18. The genetics of Drosophila transgenics.

    PubMed

    Roman, Gregg

    2004-11-01

    In Drosophila, the genetic approach is still the method of choice for answering fundamental questions on cell biology, signal transduction, development, physiology and behavior. In this approach, a gene's function is ascertained by altering either the amount or quality of the gene product, and then observing the consequences. The genetic approach is itself polymorphous, encompassing new and more complex techniques that typically employ the growing collections of transgenes. The keystone of these modern Drosophila transgenic techniques has been the Gal4 binary system. Recently, several new techniques have modified this binary system to offer greater control over the timing, tissue specificity and magnitude of gene expression. Additionally, the advances in post-transcriptional gene silencing, or RNAi, have greatly expanded the ability to knockdown almost any gene's function. Regardless of the growing experimental intricacy, the application of these advances to modify gene activity still obeys the fundamental principles of genetic analysis. Several of these transgenic techniques, which offer more precise control over a gene's activity, will be reviewed here with a discussion on how they may be used for determining a gene's function.

  19. 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

  20. Haploidy and androgenesis in Drosophila.

    PubMed Central

    Komma, D J; Endow, S A

    1995-01-01

    Adrogenesis, development from paternal but not maternal chromosomes, can be induced to occur in some organisms, including vertebrates, but has only been reported to occur naturally in interspecific hybrids of the Sicilian stick insect. Androgenesis has not been described previously in Drosophila. We now report the recovery of androgenetic offspring from Drosophila melanogaster females mutant for a gene that affects an oocyte- and embryo-specific alpha-tubulin. The androgenetic exceptions are X,X diploid females that develop from haploid embryos and express paternal markers on all 4 chromosomes. The exceptional females arise by fusion of haploid cleavage nuclei or failure of newly replicated haploid chromosomes to segregate, rather than fusion of two inseminating sperm. The frequency of androgenetic offspring is greatly enhanced by a partial loss-of-function mutant of the NCD (nonclaret disjunctional) microtubule motor protein, suggesting that wild-type NCD functions is pronuclear fusion. Diploidization of haploid paternal chromosome complements results in complete genetic homozygosity, which could facilitate studies of gene variation and mutational load in populations. Images Fig. 2 Fig. 3 PMID:8524868

  1. MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase.

    PubMed Central

    Stokoe, D; Campbell, D G; Nakielny, S; Hidaka, H; Leevers, S J; Marshall, C; Cohen, P

    1992-01-01

    A novel protein kinase, which was only active when phosphorylated by the mitogen-activated protein kinase (MAP kinase), has been purified 85,000-fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase-2, was distinguished from S6 kinase-II (MAPKAP kinase-1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase-2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase-2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase-2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase-2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor-stimulated protein kinase cascade bifurcates. Images PMID:1327754

  2. Gaining insights into diabetic cardiomyopathy from Drosophila

    PubMed Central

    Diop, Soda Balla; Bodmer, Rolf

    2015-01-01

    The high degree of genetic conservation between Drosophila melanogaster and mammals has helped to translate many important findings into new knowledge, and has led to better understanding of many biological processes in vertebrates. For over a century, the Drosophila model has been used in studies aimed at understanding molecular mechanisms implicated in heredity, development, disease progression, and aging. The current epidemic of obesity and associated diabetic cardiomyopathy and heart failure has led to a shift in Drosophila research towards understanding the basic mechanisms leading to metabolic syndrome and associated cardiac risk factors. Here, we discuss recent findings in Drosophila that highlight the importance of this organism as an excellent model to study the effects of metabolic imbalance on cardiac function. PMID:26482877

  3. 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

  4. 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.

  5. Ecdysteroid receptors in Drosophila melanogaster adult females

    USDA-ARS?s Scientific Manuscript database

    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...

  6. F-actin staining of Drosophila testes.

    PubMed

    Bonaccorsi, Silvia; Giansanti, Maria G; Cenci, Giovanni; Gatti, Maurizio

    2012-01-01

    Preparations of Drosophila testes fixed with paraformaldehyde can be stained for F-actin according to the protocol described here. This staining procedure is particularly suitable for staining the male fusome and the cytokinetic contractile ring.

  7. The Drosophila cyst stem cell lineage

    PubMed Central

    Zoller, Richard; Schulz, Cordula

    2012-01-01

    In all animals, germline cells differentiate in intimate contact with somatic cells and interactions between germline and soma are particularly important for germline development and function. In the male gonad of Drosophila melanogaster, the developing germline cells are enclosed by somatic cyst cells. The cyst cells are derived from cyst stem cells (CySCs) of somatic origin and codifferentiate with the germline cells. The fast generation cycle and the genetic tractability of Drosophila has made the Drosophila testis an excellent model for studying both the roles of somatic cells in guiding germline development and the interdependence of two separate stem cell lineages. This review focuses on our current understanding of CySC specification, CySC self-renewing divisions, cyst cell differentiation, and soma-germline interactions. Many of the mechanisms guiding these processes in Drosophila testes are similarly essential for the development and function of tissues in other organisms, most importantly for gametogenesis in mammals. PMID:23087834

  8. A Kinome RNAi Screen in Drosophila Identifies Novel Genes Interacting with Lgl, aPKC, and Crb Cell Polarity Genes in Epithelial Tissues

    PubMed Central

    Parsons, Linda M.; Grzeschik, Nicola A.; Amaratunga, Kasun; Burke, Peter; Quinn, Leonie M.; Richardson, Helena E.

    2017-01-01

    In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein networks. To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, we screened a boutique collection of RNAi stocks targeting the kinome for their capacity to modify Drosophila “cell polarity” eye and wing phenotypes. Initially, we identified kinase or phosphatase genes whose depletion modified adult eye phenotypes associated with the manipulation of cell polarity complexes (via overexpression of Crb or aPKC). We next conducted a secondary screen to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, Jun kinase (JNK), and Notch signaling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras, and lipid/Phospho-inositol-3-kinase (PI3K) signaling pathways. Additionally, we demonstrated that the “nutrient sensing” kinases Salt Inducible Kinase 2 and 3 (SIK2 and 3) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. Overall, our screen has revealed novel cell polarity-interacting kinases and phosphatases that affect tissue growth, providing a platform for investigating molecular mechanisms coordinating cell polarity and tissue growth during development. PMID:28611255

  9. A Kinome RNAi Screen in Drosophila Identifies Novel Genes Interacting with Lgl, aPKC, and Crb Cell Polarity Genes in Epithelial Tissues.

    PubMed

    Parsons, Linda M; Grzeschik, Nicola A; Amaratunga, Kasun; Burke, Peter; Quinn, Leonie M; Richardson, Helena E

    2017-08-07

    In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein networks. To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, we screened a boutique collection of RNAi stocks targeting the kinome for their capacity to modify Drosophila "cell polarity" eye and wing phenotypes. Initially, we identified kinase or phosphatase genes whose depletion modified adult eye phenotypes associated with the manipulation of cell polarity complexes (via overexpression of Crb or aPKC). We next conducted a secondary screen to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, Jun kinase (JNK), and Notch signaling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras, and lipid/Phospho-inositol-3-kinase (PI3K) signaling pathways. Additionally, we demonstrated that the "nutrient sensing" kinases Salt Inducible Kinase 2 and 3 (SIK2 and 3) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. Overall, our screen has revealed novel cell polarity-interacting kinases and phosphatases that affect tissue growth, providing a platform for investigating molecular mechanisms coordinating cell polarity and tissue growth during development. Copyright © 2017 Parsons et al.

  10. Progress in understanding the Drosophila dnc locus.

    PubMed

    Nighorn, A; Qiu, Y; Davis, R L

    1994-05-01

    The genetic dissection of learning and memory in Drosophila is two decades old. Recently, a great deal of progress has been made towards isolating new mutants as well as towards a better understanding of the originally isolated ones. This paper reviews the recent developments in the understanding of the structure and function of the gene identified by the first and best-characterized of these mutants, the Drosophila dunce mutant.

  11. [Development of Drosophila melanogaster in space flight].

    PubMed

    Ogneva, I V; Larina, I M; Sarantseva, S V

    2014-01-01

    The review deals with the available literary data on different aspects of Drosophila melanogaster vital functions in the conditions of real and modeled microgravity. The developmental stages, embryogenesis and aging, specifically, and behavioral reactions are discussed. The presented results of morphological as well as molecular genetic analyses are indicative of structural changes in early Drosophila embryos and their compensation during subsequent development, and formation of an adaptive gene-expression pattern in microgravity.

  12. The c-Jun kinase signaling cascade promotes glial engulfment activity through activation of draper and phagocytic function.

    PubMed

    Macdonald, J M; Doherty, J; Hackett, R; Freeman, M R

    2013-09-01

    After neuronal injury or death glial cells become reactive, exhibiting dramatic changes in morphology and patterns of gene expression and ultimately engulfing neuronal debris. Rapid clearance of degenerating neuronal material is thought to be crucial for suppression of inflammation and promotion of functional recovery. Here we demonstrate that Drosophila c-Jun N-terminal kinase (dJNK) signaling is a critical in vivo mediator of glial engulfment activity. In response to axotomy, we find glial dJNK signals through a cascade involving the upstream mitogen-activated protein kinase kinase kinases Slipper and Tak1, the mitogen-activated protein kinase kinase MKK4, and ultimately the Drosophila activator protein 1 (AP-1) transcriptional complex composed of Jra and Kayak to initiate glial phagocytosis of degenerating axons. Interestingly, loss of dJNK also blocked injury-induced upregulation of Draper levels in glia, and glial-specific overexpression of Draper was sufficient to rescue engulfment defects associated with loss of dJNK signaling. This work identifies that the dJNK pathway is a novel mediator of glial engulfment activity and a primary role for the glial Slipper/Tak1 →MKK4 →dJNK →dAP-1 signaling cascade appears to be activation of draper expression after axon injury.

  13. The c-Jun kinase signaling cascade promotes glial engulfment activity through activation of draper and phagocytic function

    PubMed Central

    MacDonald, J M; Doherty, J; Hackett, R; Freeman, M R

    2013-01-01

    After neuronal injury or death glial cells become reactive, exhibiting dramatic changes in morphology and patterns of gene expression and ultimately engulfing neuronal debris. Rapid clearance of degenerating neuronal material is thought to be crucial for suppression of inflammation and promotion of functional recovery. Here we demonstrate that Drosophila c-Jun N-terminal kinase (dJNK) signaling is a critical in vivo mediator of glial engulfment activity. In response to axotomy, we find glial dJNK signals through a cascade involving the upstream mitogen-activated protein kinase kinase kinases Slipper and Tak1, the mitogen-activated protein kinase kinase MKK4, and ultimately the Drosophila activator protein 1 (AP-1) transcriptional complex composed of Jra and Kayak to initiate glial phagocytosis of degenerating axons. Interestingly, loss of dJNK also blocked injury-induced upregulation of Draper levels in glia, and glial-specific overexpression of Draper was sufficient to rescue engulfment defects associated with loss of dJNK signaling. This work identifies that the dJNK pathway is a novel mediator of glial engulfment activity and a primary role for the glial Slipper/Tak1→MKK4→dJNK→dAP-1 signaling cascade appears to be activation of draper expression after axon injury. PMID:23618811

  14. mom identifies a receptor for the Drosophila JAK/STAT signal transduction pathway and encodes a protein distantly related to the mammalian cytokine receptor family

    PubMed Central

    Chen, Hua-Wei; Chen, Xiu; Oh, Su-Wan; Marinissen, Maria J.; Gutkind, J. Silvio; Hou, Steven X.

    2002-01-01

    The JAK/STAT signal transduction pathway controls numerous events in Drosophila melanogaster development. Receptors for the pathway have yet to be identified. Here we have identified a Drosophila gene that shows embryonic mutant phenotypes identical to those in the hopscotch (hop)/JAK kinase and marelle (mrl)/Stat92e mutations. We named this gene master of marelle (mom). Genetic analyses place mom's function between upd (the ligand) and hop. We further show that cultured cells transfected with the mom gene bind UPD and activate the HOP/STAT92E signal transduction pathway. mom encodes a protein distantly related to the mammalian cytokine receptor family. These data show that mom functions as a receptor of the Drosophila JAK/STAT signal transduction pathway. PMID:11825879

  15. Ectoparasitic mites and their Drosophila hosts.

    PubMed

    Perez-Leanos, Alejandra; Loustalot-Laclette, Mariana Ramirez; Nazario-Yepiz, Nestor; Markow, Therese Ann

    2017-01-02

    Only two parasite interactions are known for Drosophila to date: Allantonematid nematodes associated with mycophagous Drosophilids and the ectoparasitic mite Macrocheles subbadius with the Sonoran Desert endemic Drosophila nigrospiracula. Unlike the nematode-Drosophila association, breadth of mite parasitism on Drosophila species is unknown. As M. subbadius is a generalist, parasitism of additional Drosophilids is expected. We determined the extent and distribution of mite parasitism in nature Drosophilids collected in Mexico and southern California. Thirteen additional species of Drosophilids were infested. Interestingly, 10 belong to the repleta species group of the subgenus Drosophila, despite the fact that the majority of flies collected were of the subgenus Sophophora. In all cases but 2, the associated mites were M. subbadius. Drosophila hexastigma was found to have not only M. subbadius, but another Mesostigmatid mite, Paragarmania bakeri, as well. One D. hydei was also found to have a mite from genus Lasioseius attached. In both choice and no-choice experiments, mites were more attracted to repleta group species than to Sophophoran. The extent of mite parasitism clearly is much broader than previously reported and suggests a host bias mediated either by mite preference and/or some mechanism of resistance in particular Drosophilid lineages.

  16. Characterization of Autophagic Responses in Drosophila melanogaster.

    PubMed

    Xu, T; Kumar, S; Denton, D

    2017-01-01

    Drosophila is an excellent model system for studying autophagy during animal development due to the availability of genetic reagents and opportunity for in vivo cell biological analysis. The regulation and mechanism of autophagy are highly evolutionarily conserved and the role of autophagy has been characterized during various stages of Drosophila development as well as following starvation. Studies in Drosophila have revealed novel insights into the role of distinct components of the autophagy machinery. This chapter describes protocols for examining autophagy during Drosophila development. A crucial step in the induction of autophagy is the incorporation of Atg8a into the autophagosome. This can be measured as autophagic puncta using live fluorescent imaging, immunostaining, or immunoblot analysis of LC3/Atg8a processing. The level of autophagy can also be examined using other specific components of the autophagy pathway as markers detected by immunofluorescent imaging. Based on the distinct morphology of autophagy, it can also be examined by transmission electron microscopy. In addition, one of the advantages of using Drosophila as a model is the ability to undertake genetic analysis of individual components of the autophagy machinery. Current approaches that can be used to monitor autophagy, including the overall flux and individual steps in Drosophila melanogaster, will be discussed. © 2017 Elsevier Inc. All rights reserved.

  17. The Role of AMPK in Drosophila melanogaster.

    PubMed

    Sinnett, Sarah E; Brenman, Jay E

    2016-01-01

    In the fruit fly, Drosophila melanogaster, mono-allelic expression of AMPK-α, -β, and -γ yields a single heterotrimeric energy sensor that regulates cellular and whole-body energetic homeostasis. The genetic simplicity of Drosophila, with only a single gene for each subunit, makes the fruit fly an appealing organism for elucidating the effects of AMPK mutations on signaling pathways and phenotypes. In addition, Drosophila presents researchers with an opportunity to use straightforward genetic approaches to elucidate metabolic signaling pathways that contain a level of complexity similar to that observed in mammalian pathways. Just as in mammals, however, the regulatory realm of AMPK function extends beyond metabolic rates and lipid metabolism. Indeed, experiments using Drosophila have shown that AMPK may exert protective effects with regard to life span and neurodegeneration. This chapter addresses a few of the research areas in which Drosophila has been used to elucidate the physiological functions of AMPK. In doing so, this chapter provides a primer for basic Drosophila nomenclature, thereby eliminating a communication barrier that persists for AMPK researchers trained in mammalian genetics.

  18. Saccadic body turns in walking Drosophila

    PubMed Central

    Geurten, Bart R. H.; Jähde, Philipp; Corthals, Kristina; Göpfert, Martin C.

    2014-01-01

    Drosophila melanogaster structures its optic flow during flight by interspersing translational movements with abrupt body rotations. Whether these “body saccades” are accompanied by steering movements of the head is a matter of debate. By tracking single flies moving freely in an arena, we now discovered that walking Drosophila also perform saccades. Movement analysis revealed that the flies separate rotational from translational movements by quickly turning their bodies by 15 degrees within a tenth of a second. Although walking flies moved their heads by up to 20 degrees about their bodies, their heads moved with the bodies during saccadic turns. This saccadic strategy contrasts with the head saccades reported for e.g., blowflies and honeybees, presumably reflecting optical constraints: modeling revealed that head saccades as described for these latter insects would hardly affect the retinal input in Drosophila because of the lower acuity of its compound eye. The absence of head saccades in Drosophila was associated with the absence of haltere oscillations, which seem to guide head movements in other flies. In addition to adding new twists to Drosophila walking behavior, our analysis shows that Drosophila does not turn its head relative to its body when turning during walking. PMID:25386124

  19. Ectoparasitic mites and their Drosophila hosts

    PubMed Central

    Perez-Leanos, Alejandra; Loustalot-Laclette, Mariana Ramirez; Nazario-Yepiz, Nestor; Markow, Therese Ann

    2017-01-01

    ABSTRACT Only two parasite interactions are known for Drosophila to date: Allantonematid nematodes associated with mycophagous Drosophilids and the ectoparasitic mite Macrocheles subbadius with the Sonoran Desert endemic Drosophila nigrospiracula. Unlike the nematode-Drosophila association, breadth of mite parasitism on Drosophila species is unknown. As M. subbadius is a generalist, parasitism of additional Drosophilids is expected. We determined the extent and distribution of mite parasitism in nature Drosophilids collected in Mexico and southern California. Thirteen additional species of Drosophilids were infested. Interestingly, 10 belong to the repleta species group of the subgenus Drosophila, despite the fact that the majority of flies collected were of the subgenus Sophophora. In all cases but 2, the associated mites were M. subbadius. Drosophila hexastigma was found to have not only M. subbadius, but another Mesostigmatid mite, Paragarmania bakeri, as well. One D. hydei was also found to have a mite from genus Lasioseius attached. In both choice and no-choice experiments, mites were more attracted to repleta group species than to Sophophoran. The extent of mite parasitism clearly is much broader than previously reported and suggests a host bias mediated either by mite preference and/or some mechanism of resistance in particular Drosophilid lineages. PMID:27540774

  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. A role for the segment polarity gene shaggy/GSK-3 in the Drosophila circadian clock.

    PubMed

    Martinek, S; Inonog, S; Manoukian, A S; Young, M W

    2001-06-15

    Tissue-specific overexpression of the glycogen synthase kinase-3 (GSK-3) ortholog shaggy (sgg) shortens the period of the Drosophila circadian locomotor activity cycle. The short period phenotype was attributed to premature nuclear translocation of the PERIOD/TIMELESS heterodimer. Reducing SGG/GSK-3 activity lengthens period, demonstrating an intrinsic role for the kinase in circadian rhythmicity. Lowered sgg activity decreased TIMELESS phosphorylation, and it was found that GSK-3 beta specifically phosphorylates TIMELESS in vitro. Overexpression of sgg in vivo converts hypophosphorylated TIMELESS to a hyperphosphorylated protein whose electrophoretic mobility, and light and phosphatase sensitivity, are indistinguishable from the rhythmically produced hyperphosphorylated TIMELESS of wild-type flies. Our results indicate a role for SGG/GSK-3 in TIMELESS phosphorylation and in the regulated nuclear translocation of the PERIOD/TIMELESS heterodimer.

  2. Gilgamesh is required for rutabaga-independent olfactory learning in Drosophila.

    PubMed

    Tan, Ying; Yu, Dinghui; Pletting, Jennifer; Davis, Ronald L

    2010-09-09

    Cyclic AMP signaling in Drosophila mushroom body neurons, anchored by the adenylyl cyclase encoded by the rutabaga gene, is indispensable for olfactory memory formation. From a screen for new memory mutants, we identified alleles of the gilgamesh (gish) gene, which encodes a casein kinase Iγ homolog that is preferentially expressed in the mushroom body neurons. The gish-encoded kinase participates in the physiology of these neurons underlying memory formation since the mutant memory deficit was rescued with expression of a gish cDNA in these neurons only during adulthood. A cellular memory trace, detected as increased calcium influx into the α'/β' neuron processes in response to the odor used for conditioning, was disrupted in gish mutants. Epistasis experiments indicated a lack of genetic interactions between gish and rutabaga. Therefore, gish participates in a rutabaga-independent pathway for memory formation and accounts for some of the residual learning that occurs in rutabaga mutants. 2010 Elsevier Inc. All rights reserved.

  3. Gilgamesh is required for rutabaga-independent olfactory learning in Drosophila

    PubMed Central

    Tan, Ying; Yu, Dinghui; Pletting, Jennifer; Davis, Ronald L.

    2010-01-01

    Summary Cyclic AMP signaling in Drosophila mushroom body neurons, anchored by the adenylyl cyclase encoded by the rutabaga gene, is indispensable for olfactory memory formation. From a novel screen for new memory mutants, we identified alleles of the gilgamesh (gish) gene, which encodes a casein kinase Iγ homologue that is preferentially expressed in the mushroom body neurons. The gish-encoded kinase participates in the physiology of these neurons underlying memory formation since the mutant memory deficit was rescued with expression of a gish cDNA in these neurons only during adulthood. A cellular memory trace, detected as increased calcium influx into the α′/β′ neuron processes in response to the odor used for conditioning, was disrupted in gish mutants. Epistasis experiments indicated a lack of genetic interactions between gish and rutabaga. Therefore, gish participates in a rutabaga-independent pathway for memory formation and accounts for some of the residual learning that occurs in rutabaga mutants. PMID:20826312

  4. Development of a Drosophila cell-based error correction assay.

    PubMed

    Salemi, Jeffrey D; McGilvray, Philip T; Maresca, Thomas J

    2013-01-01

    Accurate transmission of the genome through cell division requires microtubules from opposing spindle poles to interact with protein super-structures called kinetochores that assemble on each sister chromatid. Most kinetochores establish erroneous attachments that are destabilized through a process called error correction. Failure to correct improper kinetochore-microtubule (kt-MT) interactions before anaphase onset results in chromosomal instability (CIN), which has been implicated in tumorigenesis and tumor adaptation. Thus, it is important to characterize the molecular basis of error correction to better comprehend how CIN occurs and how it can be modulated. An error correction assay has been previously developed in cultured mammalian cells in which incorrect kt-MT attachments are created through the induction of monopolar spindle assembly via chemical inhibition of kinesin-5. Error correction is then monitored following inhibitor wash out. Implementing the error correction assay in Drosophila melanogaster S2 cells would be valuable because kt-MT attachments are easily visualized and the cells are highly amenable to RNAi and high-throughput screening. However, Drosophila kinesin-5 (Klp61F) is unaffected by available small molecule inhibitors. To overcome this limitation, we have rendered S2 cells susceptible to kinesin-5 inhibitors by functionally replacing Klp61F with human kinesin-5 (Eg5). Eg5 expression rescued the assembly of monopolar spindles typically caused by Klp61F depletion. Eg5-mediated bipoles collapsed into monopoles due, in part, to kinesin-14 (Ncd) activity when treated with the kinesin-5 inhibitor S-trityl-L-cysteine (STLC). Furthermore, bipolar spindles reassembled and error correction was observed after STLC wash out. Importantly, error correction in Eg5-expressing S2 cells was dependent on the well-established error correction kinase Aurora B. This system provides a powerful new cell-based platform for studying error correction and CIN.

  5. JAK/Stat signaling regulates heart precursor diversification in Drosophila.

    PubMed

    Johnson, Aaron N; Mokalled, Mayssa H; Haden, Tom N; Olson, Eric N

    2011-11-01

    Intercellular signal transduction pathways regulate the NK-2 family of transcription factors in a conserved gene regulatory network that directs cardiogenesis in both flies and mammals. The Drosophila NK-2 protein Tinman (Tin) was recently shown to regulate Stat92E, the Janus kinase (JAK) and Signal transducer and activator of transcription (Stat) pathway effector, in the developing mesoderm. To understand whether the JAK/Stat pathway also regulates cardiogenesis, we performed a systematic characterization of JAK/Stat signaling during mesoderm development. Drosophila embryos with mutations in the JAK/Stat ligand upd or in Stat92E have non-functional hearts with luminal defects and inappropriate cell aggregations. Using strong Stat92E loss-of-function alleles, we show that the JAK/Stat pathway regulates tin expression prior to heart precursor cell diversification. tin expression can be subdivided into four phases and, in Stat92E mutant embryos, the broad phase 2 expression pattern in the dorsal mesoderm does not restrict to the constrained phase 3 pattern. These embryos also have an expanded pericardial cell domain. We show the E(spl)-C gene HLHm5 is expressed in a pattern complementary to tin during phase 3 and that this expression is JAK/Stat dependent. In addition, E(spl)-C mutant embryos phenocopy the cardiac defects of Stat92E embryos. Mechanistically, JAK/Stat signals activate E(spl)-C genes to restrict Tin expression and the subsequent expression of the T-box transcription factor H15 to direct heart precursor diversification. This study is the first to characterize a role for the JAK/Stat pathway during cardiogenesis and identifies an autoregulatory circuit in which tin limits its own expression domain.

  6. Life span extension and neuronal cell protection by Drosophila nicotinamidase.

    PubMed

    Balan, Vitaly; Miller, Gregory S; Kaplun, Ludmila; Balan, Karina; Chong, Zhao-Zhong; Li, Faqi; Kaplun, Alexander; VanBerkum, Mark F A; Arking, Robert; Freeman, D Carl; Maiese, Kenneth; Tzivion, Guri

    2008-10-10

    The life span of model organisms can be modulated by environmental conditions that influence cellular metabolism, oxidation, or DNA integrity. The yeast nicotinamidase gene pnc1 was identified as a key transcriptional target and mediator of calorie restriction and stress-induced life span extension. PNC1 is thought to exert its effect on yeast life span by modulating cellular nicotinamide and NAD levels, resulting in increased activity of Sir2 family class III histone deacetylases. In Caenorhabditis elegans, knockdown of a pnc1 homolog was shown recently to shorten the worm life span, whereas its overexpression increased survival under conditions of oxidative stress. The function and regulation of nicotinamidases in higher organisms has not been determined. Here, we report the identification and biochemical characterization of the Drosophila nicotinamidase, D-NAAM, and demonstrate that its overexpression significantly increases median and maximal fly life span. The life span extension was reversed in Sir2 mutant flies, suggesting Sir2 dependence. Testing for physiological effectors of D-NAAM in Drosophila S2 cells, we identified oxidative stress as a primary regulator, both at the transcription level and protein activity. In contrast to the yeast model, stress factors such as high osmolarity and heat shock, calorie restriction, or inhibitors of TOR and phosphatidylinositol 3-kinase pathways do not appear to regulate D-NAAM in S2 cells. Interestingly, the expression of D-NAAM in human neuronal cells conferred protection from oxidative stress-induced cell death in a sirtuin-dependent manner. Together, our findings establish a life span extending the ability of nicotinamidase in flies and offer a role for nicotinamide-modulating genes in oxidative stress regulated pathways influencing longevity and neuronal cell survival.

  7. 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

  8. New record for the invasive Spotted Wing Drosophila, Drosophila suzukii Matsumura (Diptera: Drosophilidae) in Anillaco, Argentina

    USDA-ARS?s Scientific Manuscript database

    The invasive Spotted Wing Drosophila (SWD), Drosophila suzukii Matsumura, is reported for the first time in La Rioja, Argentina. This represents a major range expansion for this species. The natural enemies of SWD, Leptopilina clavipes and Ganaspis hookeri were also collected with the SWD at the s...

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  12. Effect of non-nutritive sugars to decrease the survivorship of spotted wing drosophila, Drosophila suzukii

    USDA-ARS?s Scientific Manuscript database

    In this study, we investigated the effects of non-nutritive sugars and sugar alcohols on the survivorship of spotted wing drosophila, Drosophila suzukii, and found erythritol and erythrose as potentially toxic to the fly. In a dose-dependent study, erythritol and erythrose significantly reduced fly ...

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  16. The warts gene as a novel target of the Drosophila DRE/DREF transcription pathway.

    PubMed

    Fujiwara, Shunsuke; Ida, Hiroyuki; Yoshioka, Yasuhide; Yoshida, Hideki; Yamaguchi, Masamitsu

    2012-01-01

    The Hippo tumor suppressor pathway in Drosophila represses expression of DIAP1 and Cyclin E via inactivation of the transcription co-activator Yorkie, resulting in cell cycle arrest and induction of apoptosis. The warts (wts) gene is well known as a core kinase in this pathway, but its transcriptional regulation has yet to be clarified. In Drosophila, DREF binds to a target sequence named DRE (5'-TATCGATA) and regulates transcription of cell proliferation-related genes containing the DRE sequence in their promoter regions. Here we found half reduction of the wts gene dose to enhance the DREF-induced rough eye phenotype, suggesting a DREF genetic interaction with the Hippo pathway in vivo. Three DREs indentified in the wts gene promoter region exhibited strong promoter activity with a luciferase transient expression assay in Drosophila S2 cells, this decreasing under DREF-RNAi conditions. In addition, knockdown of DREF in S2 cells reduced the level of endogenous wts mRNA. Chromatin immunoprecipitation assays with anti-DREF antibody revealed that DREF binds specifically to the wts gene promoter region containing DREs in vivo. These results indicate that the DRE/DREF pathway is required for transcriptional regulation of the wts gene, indicating a novel link between the DRE/DREF and the Hippo pathways.

  17. Genetic Dissection of Aversive Associative Olfactory Learning and Memory in Drosophila Larvae

    PubMed Central

    Widmann, Annekathrin; Artinger, Marc; Biesinger, Lukas; Boepple, Kathrin; Schlechter, Jana; Selcho, Mareike; Thum, Andreas S.

    2016-01-01

    Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes—besides other forms—a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. Here we deconstruct the larval mnemonic organization after aversive olfactory conditioning. We show that after odor-high salt conditioning larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3’5’-monophosphate (cAMP) signaling and synapsin gene function. In addition, we show for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution. PMID:27768692

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

    PubMed

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

    2015-09-01

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

  19. Polo-mediated phosphorylation of Maelstrom regulates oocyte determination during oogenesis in Drosophila.

    PubMed

    Pek, Jun Wei; Ng, Bing Fu; Kai, Toshie

    2012-12-01

    In Drosophila, Maelstrom is a conserved component of the perinuclear nuage, a germline-unique structure that appears to serve as a site for Piwi-interacting RNA (piRNA) production to repress deleterious transposons. Maelstrom also functions in the nucleus as a transcriptional regulator to repress the expression of microRNA-7, a process that is essential for the proper differentiation of germline stem cells. In this paper, we report another function of Maelstrom in regulating oocyte determination independently of its transposon silencing and germline stem cell differentiation activities. In Drosophila, the conserved serine 138 residue in Maelstrom is required for its phosphorylation, an event that promotes oocyte determination. Phosphorylation of Maelstrom is required for the repression of the pachytene checkpoint protein Sir2, but not for transposon silencing or for germline stem cell differentiation. We identify Polo as a kinase that mediates the phosphorylation of Maelstrom. Our results suggest that the Polo-mediated phosphorylation of Maelstrom may be a mechanism that controls oocyte determination by inactivating the pachytene checkpoint via the repression of Sir2 in Drosophila ovaries.

  20. PI4KIIIα is required for cortical integrity and cell polarity during Drosophila oogenesis.

    PubMed

    Tan, Julie; Oh, Karen; Burgess, Jason; Hipfner, David R; Brill, Julie A

    2014-03-01

    Phosphoinositides regulate myriad cellular processes, acting as potent signaling molecules in conserved signaling pathways and as organelle gatekeepers that recruit effector proteins to membranes. Phosphoinositide-generating enzymes have been studied extensively in yeast and cultured cells, yet their roles in animal development are not well understood. Here, we analyze Drosophila melanogaster phosphatidylinositol 4-kinase IIIα (PI4KIIIα) during oogenesis. We demonstrate that PI4KIIIα is required for production of plasma membrane PtdIns4P and PtdIns(4,5)P2 and is crucial for actin organization, membrane trafficking and cell polarity. Female germ cells mutant for PI4KIIIα exhibit defects in cortical integrity associated with failure to recruit the cytoskeletal-membrane crosslinker Moesin and the exocyst subunit Sec5. These effects reflect a unique requirement for PI4KIIIα, as egg chambers from flies mutant for either of the other Drosophila PI4Ks, fwd or PI4KII, show Golgi but not plasma membrane phenotypes. Thus, PI4KIIIα is a vital regulator of a functionally distinct pool of PtdIns4P that is essential for PtdIns(4,5)P2-dependent processes in Drosophila development.

  1. 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

  2. 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.

  3. 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

  4. Studying Polyglutamine Diseases in Drosophila

    PubMed Central

    Xu, Zhen; Tito, Antonio; Rui, Yan-Ning; Zhang, Sheng

    2015-01-01

    Polyglutamine (polyQ) diseases are a family of dominantly transmitted neurodegenerative disorders caused by an abnormal expansion of CAG trinucleotide repeats in the protein-coding regions of the respective disease-causing genes. Despite their simple genetic basis, the etiology of these diseases is far from clear. Over the past two decades, Drosophila has proven to be successful in modeling this family of neurodegenerative disorders, including the faithful recapitulation of pathological features such as polyQ length-dependent formation of protein aggregates and progressive neuronal degeneration. Additionally, it has been valuable in probing the pathogenic mechanisms, in identifying and evaluating disease modifiers, and in helping elucidate the normal functions of disease-causing genes. Knowledge learned from this simple invertebrate organism has had a large impact on our understanding of these devastating brain diseases. PMID:26257024

  5. A Drosophila mechanosensory transduction channel.

    PubMed

    Walker, R G; Willingham, A T; Zuker, C S

    2000-03-24

    Mechanosensory transduction underlies a wide range of senses, including proprioception, touch, balance, and hearing. The pivotal element of these senses is a mechanically gated ion channel that transduces sound, pressure, or movement into changes in excitability of specialized sensory cells. Despite the prevalence of mechanosensory systems, little is known about the molecular nature of the transduction channels. To identify such a channel, we analyzed Drosophila melanogaster mechanoreceptive mutants for defects in mechanosensory physiology. Loss-of-function mutations in the no mechanoreceptor potential C (nompC) gene virtually abolished mechanosensory signaling. nompC encodes a new ion channel that is essential for mechanosensory transduction. As expected for a transduction channel, D. melanogaster NOMPC and a Caenorhabditis elegans homolog were selectively expressed in mechanosensory organs.

  6. The organization of Drosophila genes.

    PubMed

    Maroni, G

    1994-01-01

    This study was designed to examine the range of size variations in the major functional elements of Drosophila genes and to test whether those size variations occur independently of each other. In a sample of 111 genes the following median values occur: leaders, 123 base pairs (bp); coding regions, 1242 bp; 3' untranslated regions (3'UTR), 246 bp; mRNAs, 1803 bp; 3' terminal exons 843 bp; and exons upstream of the last one 233 bp. Introns show a bimodal distribution with medians of 62 and 595 bp. Unexpected size correlations are evident for several of these elements. The size of the leader, for example, is correlated with the sizes of the coding region and the 3'UTR with very high levels of significance, and the size of the first intron is similarly correlated with the sizes of each of the individual components of the mature mRNA.

  7. Planar cell polarity in Drosophila

    PubMed Central

    Maung, Saw Myat Thanda W

    2011-01-01

    In all multicellular organisms, epithelial cells are not only polarized along the apical-basal axis, but also within the epithelial plane, giving cells a sense of direction. Planar cell polarity (PCP) signaling regulates establishment of polarity within the plane of an epithelium. The outcomes of PCP signaling are diverse and include the determination of cell fates, the generation of asymmetric but highly aligned structures, such as the stereocilia in the human inner ear or the hairs on a fly wing, or the directional migration of cells during convergence and extension during vertebrate gastrulation. In humans, aberrant PCP signaling can result in severe developmental defects, such as open neural tubes (spina bifida), and can cause cystic kidneys. In this review, we discuss the basic mechanism and more recent findings of PCP signaling focusing on Drosophila melanogaster, the model organism in which most key PCP components were initially identified. PMID:21983142

  8. Molecular neurobiology of Drosophila taste

    PubMed Central

    Freeman, Erica Gene; Dahanukar, Anupama

    2015-01-01

    Drosophila is a powerful model in which to study the molecular and cellular basis of taste coding. Flies sense tastants via populations of taste neurons that are activated by compounds of distinct categories. The past few years have borne witness to studies that define the properties of taste neurons, identifying functionally distinct classes of sweet and bitter taste neurons that express unique subsets of gustatory receptor (Gr) genes, as well as water, salt, and pheromone sensing neurons that express members of the pickpocket (ppk) or ionotropic receptor (Ir) families. There has also been significant progress in terms of understanding how tastant information is processed and conveyed to higher brain centers, and modulated by prior dietary experience or starvation. PMID:26102453

  9. Molecular neurobiology of Drosophila taste.

    PubMed

    Freeman, Erica Gene; Dahanukar, Anupama

    2015-10-01

    Drosophila is a powerful model in which to study the molecular and cellular basis of taste coding. Flies sense tastants via populations of taste neurons that are activated by compounds of distinct categories. The past few years have borne witness to studies that define the properties of taste neurons, identifying functionally distinct classes of sweet and bitter taste neurons that express unique subsets of gustatory receptor (Gr) genes, as well as water, salt, and pheromone sensing neurons that express members of the pickpocket (ppk) or ionotropic receptor (Ir) families. There has also been significant progress in terms of understanding how tastant information is processed and conveyed to higher brain centers, and modulated by prior dietary experience or starvation. Copyright © 2015. Published by Elsevier Ltd.

  10. Domain Specificity of MAP3K Family Members, MLK and Tak1, for JNK Signaling in Drosophila

    PubMed Central

    Stronach, Beth; Lennox, Ashley L.; Garlena, Rebecca A.

    2014-01-01

    A highly diverse set of protein kinases functions as early responders in the mitogen- and stress-activated protein kinase (MAPK/SAPK) signaling pathways. For instance, humans possess 14 MAPK kinase kinases (MAP3Ks) that activate Jun kinase (JNK) signaling downstream. A major challenge is to decipher the selective and redundant functions of these upstream MAP3Ks. Taking advantage of the relative simplicity of Drosophila melanogaster as a model system, we assessed MAP3K signaling specificity in several JNK-dependent processes during development and stress response. Our approach was to generate molecular chimeras between two MAP3K family members, the mixed lineage kinase, Slpr, and the TGF-β activated kinase, Tak1, which share 32% amino acid identity across the kinase domain but otherwise differ in sequence and domain structure, and then test the contributions of various domains for protein localization, complementation of mutants, and activation of signaling. We found that overexpression of the wild-type kinases stimulated JNK signaling in alternate contexts, so cells were capable of responding to both MAP3Ks, but with distinct outcomes. Relative to wild-type, the catalytic domain swaps compensated weakly or not at all, despite having a shared substrate, the JNK kinase Hep. Tak1 C-terminal domain-containing constructs were inhibitory in Tak1 signaling contexts, including tumor necrosis factor-dependent cell death and innate immune signaling; however, depressing antimicrobial gene expression did not necessarily cause phenotypic susceptibility to infection. These same constructs were neutral in the context of Slpr-dependent developmental signaling, reflecting differential subcellular protein localization and by inference, point of activation. Altogether, our findings suggest that the selective deployment of a particular MAP3K can be attributed in part to its inherent sequence differences, cellular localization, and binding partner availability. PMID:24429281

  11. The Ste20 Kinase Misshapen Regulates Both Photoreceptor Axon Targeting and Dorsal Closure, Acting Downstream of Distinct Signals

    PubMed Central

    Su, Yi-Chi; Maurel-Zaffran, Corinne; Treisman, Jessica E.; Skolnik, Edward Y.

    2000-01-01

    We have previously shown that the Ste20 kinase encoded by misshapen (msn) functions upstream of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase module in Drosophila. msn is required to activate the Drosophila JNK, Basket (Bsk), to promote dorsal closure of the embryo. A mammalian homolog of Msn, Nck interacting kinase, interacts with the SH3 domains of the SH2-SH3 adapter protein Nck. We now show that Msn likewise interacts with Dreadlocks (Dock), the Drosophila homolog of Nck. dock is required for the correct targeting of photoreceptor axons. We have performed a structure-function analysis of Msn in vivo in Drosophila in order to elucidate the mechanism whereby Msn regulates JNK and to determine whether msn, like dock, is required for the correct targeting of photoreceptor axons. We show that Msn requires both a functional kinase and a C-terminal regulatory domain to activate JNK in vivo in Drosophila. A mutation in a PXXP motif on Msn that prevents it from binding to the SH3 domains of Dock does not affect its ability to rescue the dorsal closure defect in msn embryos, suggesting that Dock is not an upstream regulator of msn in dorsal closure. Larvae with only this mutated form of Msn show a marked disruption in photoreceptor axon targeting, implicating an SH3 domain protein in this process; however, an activated form of Msn is not sufficient to rescue the dock mutant phenotype. Mosaic analysis reveals that msn expression is required in photoreceptors in order for their axons to project correctly. The data presented here genetically link msn to two distinct biological events, dorsal closure and photoreceptor axon pathfinding, and thus provide the first evidence that Ste20 kinases of the germinal center kinase family play a role in axonal pathfinding. The ability of Msn to interact with distinct classes of adapter molecules in dorsal closure and photoreceptor axon pathfinding may provide the flexibility that allows it to link to distinct

  12. The Ste20 kinase misshapen regulates both photoreceptor axon targeting and dorsal closure, acting downstream of distinct signals.

    PubMed

    Su, Y C; Maurel-Zaffran, C; Treisman, J E; Skolnik, E Y

    2000-07-01

    We have previously shown that the Ste20 kinase encoded by misshapen (msn) functions upstream of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase module in Drosophila. msn is required to activate the Drosophila JNK, Basket (Bsk), to promote dorsal closure of the embryo. A mammalian homolog of Msn, Nck interacting kinase, interacts with the SH3 domains of the SH2-SH3 adapter protein Nck. We now show that Msn likewise interacts with Dreadlocks (Dock), the Drosophila homolog of Nck. dock is required for the correct targeting of photoreceptor axons. We have performed a structure-function analysis of Msn in vivo in Drosophila in order to elucidate the mechanism whereby Msn regulates JNK and to determine whether msn, like dock, is required for the correct targeting of photoreceptor axons. We show that Msn requires both a functional kinase and a C-terminal regulatory domain to activate JNK in vivo in Drosophila. A mutation in a PXXP motif on Msn that prevents it from binding to the SH3 domains of Dock does not affect its ability to rescue the dorsal closure defect in msn embryos, suggesting that Dock is not an upstream regulator of msn in dorsal closure. Larvae with only this mutated form of Msn show a marked disruption in photoreceptor axon targeting, implicating an SH3 domain protein in this process; however, an activated form of Msn is not sufficient to rescue the dock mutant phenotype. Mosaic analysis reveals that msn expression is required in photoreceptors in order for their axons to project correctly. The data presented here genetically link msn to two distinct biological events, dorsal closure and photoreceptor axon pathfinding, and thus provide the first evidence that Ste20 kinases of the germinal center kinase family play a role in axonal pathfinding. The ability of Msn to interact with distinct classes of adapter molecules in dorsal closure and photoreceptor axon pathfinding may provide the flexibility that allows it to link to distinct

  13. A dp53/JNK-dependant feedback amplification loop is essential for the apoptotic response to stress in Drosophila

    PubMed Central

    Shlevkov, E; Morata, G

    2012-01-01

    Programmed cell death (apoptosis) is a conserved process aimed to eliminate unwanted cells. The key molecules are a group of proteases called caspases that cleave vital proteins, which leads to the death of cells. In Drosophila, the apoptotic pathway is usually represented as a cascade of events in which an initial stimulus activates one or more of the proapoptotic genes (hid, rpr, grim), which in turn activate caspases. In stress-induced apoptosis, the dp53 (Drosophila p53) gene and the Jun N-terminal kinase (JNK) pathway function upstream in the activation of the proapoptotic genes. Here we demonstrate that dp53 and JNK also function downstream of proapoptotic genes and the initiator caspase Dronc (Drosophila NEDD2-like caspase) and that they establish a feedback loop that amplifies the initial apoptotic stimulus. This loop plays a critical role in the apoptotic response because in its absence there is a dramatic decrease in the amount of cell death after a pulse of the proapoptotic proteins Hid and Rpr. Thus, our results indicate that stress-induced apoptosis in Drosophila is dependant on an amplification loop mediated by dp53 and JNK. Furthermore, they also demonstrate a mechanism of mutual activation of proapoptotic genes. PMID:21886179

  14. Orphan kinases turn eccentric

    PubMed Central

    Mikolcevic, Petra; Rainer, Johannes; Geley, Stephan

    2012-01-01

    PCTAIRE kinases (PCTK) are a highly conserved, but poorly characterized, subgroup of cyclin-dependent kinases (CDK). They are characterized by a conserved catalytic domain flanked by N- and C-terminal extensions that are involved in cyclin binding. Vertebrate genomes contain three highly similar PCTAIRE kinases (PCTK1,2,3, a.k.a., CDK16,17,18), which are most abundant in post-mitotic cells in brain and testis. Consistent with this restricted expression pattern, PCTK1 (CDK16) has recently been shown to be essential for spermatogenesis. PCTAIREs are activated by cyclin Y (CCNY), a highly conserved single cyclin fold protein. By binding to N-myristoylated CCNY, CDK16 is targeted to the plasma membrane. Unlike conventional cyclin-CDK interactions, binding of CCNY to CDK16 not only requires the catalytic domain, but also domains within the N-terminal extension. Interestingly, phosphorylation within this domain blocks CCNY binding, providing a novel means of cyclin-CDK regulation. By using these functional characteristics, we analyzed “PCTAIRE” sequence containing protein kinase genes in genomes of various organisms and found that CCNY and CCNY-dependent kinases are restricted to eumetazoa and possibly evolved along with development of a central nervous system. Here, we focus on the structure and regulation of PCTAIREs and discuss their established functions. PMID:22895054

  15. Conserved herpesvirus protein kinases

    PubMed Central

    Gershburg, Edward; Pagano, Joseph S.

    2008-01-01

    Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein-Barr Virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task. PMID:17881303

  16. Neuronal Cell Shape and Neurite Initiation Are Regulated by the Ndr Kinase SAX-1, a Member of the Orb6/COT-1/Warts Serine/Threonine Kinase Family

    PubMed Central

    Zallen, Jennifer A.; Peckol, Erin L.; Tobin, David M.; Bargmann, Cornelia I.

    2000-01-01

    The Caenorhabditis elegans sax-1 gene regulates several aspects of neuronal cell shape. sax-1 mutants have expanded cell bodies and ectopic neurites in many classes of neurons, suggesting that SAX-1 functions to restrict cell and neurite growth. The ectopic neurites in sensory neurons of sax-1 mutants resemble the defects caused by decreased sensory activity. However, the activity-dependent pathway, mediated in part by the UNC-43 calcium/calmodulin-dependent kinase II, functions in parallel with SAX-1 to suppress neurite initiation. sax-1 encodes a serine/threonine kinase in the Ndr family that is related to the Orb6 (Schizosaccharomyces pombe), Warts/Lats (Drosophila), and COT-1 (Neurospora) kinases that function in cell shape regulation. These kinases have similarity to Rho kinases but lack consensus Rho-binding domains. Dominant negative mutations in the C. elegans RhoA GTPase cause neuronal cell shape defects similar to those of sax-1 mutants, and genetic interactions between rhoA and sax-1 suggest shared functions. These results suggest that SAX-1/Ndr kinases are endogenous inhibitors of neurite initiation and cell spreading. PMID:10982409

  17. 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''.

  18. Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

    PubMed Central

    Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

    2005-01-01

    The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

  19. PAK family kinases

    PubMed Central

    Zhao, Zhuo-shen; Manser, Ed

    2012-01-01

    The p21-activated kinases (PAKs) are a family of Ser/Thr protein kinases that are represented by six genes in humans (PAK 1–6), and are found in all eukaryotes sequenced to date. Genetic and knockdown experiments in frogs, fish and mice indicate group I PAKs are widely expressed, required for multiple tissue development, and particularly important for immune and nervous system function in the adult. The group II PAKs (human PAKs 4–6) are more enigmatic, but their restriction to metazoans and presence at cell-cell junctions suggests these kinases emerged to regulate junctional signaling. Studies of protozoa and fungal PAKs show that they regulate cell shape and polarity through phosphorylation of multiple cytoskeletal proteins, including microtubule binding proteins, myosins and septins. This chapter discusses what we know about the regulation of PAKs and their physiological role in different model organisms, based primarily on gene knockout studies. PMID:23162738

  20. β-arrestin Kurtz inhibits MAPK and Toll signalling in Drosophila development

    PubMed Central

    Tipping, Marla; Kim, Yoosik; Kyriakakis, Phillip; Tong, Mei; Shvartsman, Stanislav Y; Veraksa, Alexey

    2010-01-01

    β-Arrestins have been implicated in the regulation of multiple signalling pathways. However, their role in organism development is not well understood. In this study, we report a new in vivo function of the Drosophila β-arrestin Kurtz (Krz) in the regulation of two distinct developmental signalling modules: MAPK ERK and NF-κB, which transmit signals from the activated receptor tyrosine kinases (RTKs) and the Toll receptor, respectively. Analysis of the expression of effectors and target genes of Toll and the RTK Torso in krz maternal mutants reveals that Krz limits the activity of both pathways in the early embryo. Protein interaction studies suggest a previously uncharacterized mechanism for ERK inhibition: Krz can directly bind and sequester an inactive form of ERK, thus preventing its activation by the upstream kinase, MEK. A simultaneous dysregulation of different signalling systems in krz mutants results in an abnormal patterning of the embryo and severe developmental defects. Our findings uncover a new in vivo function of β-arrestins and present a new mechanism of ERK inhibition by the Drosophila β-arrestin Krz. PMID:20802461

  1. 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

  2. Drosophila CHIP protects against mitochondrial dysfunction by acting downstream of Pink1 in parallel with Parkin.

    PubMed

    Chen, Jia; Xue, Jin; Ruan, Jingsong; Zhao, Juan; Tang, Beisha; Duan, Ranhui

    2017-08-04

    Mitochondrial kinase PTEN-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase Parkin function in a common pathway to regulate mitochondrial homeostasis contributing to the pathogenesis of Parkinson disease. The carboxyl terminus of Hsc70-interacting protein (CHIP) acts as a heat shock protein 70/heat shock protein 90 cochaperone to mediate protein folding or as an E3 ubiquitin ligase to target proteins for degradation. In this study, overexpression of Drosophila CHIP suppressed a range of Pink1 mutant phenotypes in flies, including abnormal wing posture, thoracic indentation, locomotion defects, muscle degeneration, and loss of dopaminergic neurons. Mitochondrial defects of Pink1 mutant, such as excessive fusion, reduced ATP content, and crista disorganization, were rescued by CHIP but not its ligase-dead mutants. Similar phenotypes and mitochondrial impairment were ameliorated in Parkin mutant flies by wild-type CHIP. Inactivation of CHIP with null fly mutants resulted in mitochondrial defects, such as reduced thoracic ATP content at 3 d old, decreased thoracic mitochondrial DNA content, and defective mitochondrial morphology at 60 d old. CHIP mutants did not exacerbate the phenotypes of Pink1 mutant flies but markedly shortened the life span of Parkin mutant flies. These results indicate that CHIP is involved in mitochondrial integrity and may act downstream of Pink1 in parallel with Parkin.-Chen, J., Xue, J., Ruan, J., Zhao, J., Tang, B., Duan, R. Drosophila CHIP protects against mitochondrial dysfunction by acting downstream of Pink1 in parallel with Parkin. © FASEB.

  3. Drosophila as a Model for Intractable Epilepsy: Gilgamesh Suppresses Seizures in parabss1 Heterozygote Flies

    PubMed Central

    Howlett, Iris C.; Rusan, Zeid M.; Parker, Louise; Tanouye, Mark A.

    2013-01-01

    Intractable epilepsies, that is, seizure disorders that do not respond to currently available therapies, are difficult, often tragic, neurological disorders. Na+ channelopathies have been implicated in some intractable epilepsies, including Dravet syndrome (Dravet 1978), but little progress has been forthcoming in therapeutics. Here we examine a Drosophila model for intractable epilepsy, the Na+ channel gain-of-function mutant parabss1 that resembles Dravet syndrome in some aspects (parker et al. 2011a). In particular, we identify second-site mutations that interact with parabss1, seizure enhancers, and seizure suppressors. We describe one seizure-enhancer mutation named charlatan (chn). The chn gene normally encodes an Neuron-Restrictive Silencer Factor/RE1-Silencing Transcription factor transcriptional repressor of neuronal-specific genes. We identify a second-site seizure-suppressor mutation, gilgamesh (gish), that reduces the severity of several seizure-like phenotypes of parabss1/+ heterozygotes. The gish gene normally encodes the Drosophila ortholog of casein kinase CK1g3, a member of the CK1 family of serine-threonine kinases. We suggest that CK1g3 is an unexpected but promising new target for seizure therapeutics. PMID:23797108

  4. Abi plays an opposing role to Abl in Drosophila axonogenesis and synaptogenesis.

    PubMed

    Lin, Tzu-Yang; Huang, Chiu-Hui; Kao, Hsiu-Hua; Liou, Gan-Guang; Yeh, Shih-Rung; Cheng, Chih-Ming; Chen, Mei-Hsin; Pan, Rong-Long; Juang, Jyh-Lyh

    2009-09-01

    Abl tyrosine kinase (Abl) regulates axon guidance by modulating actin dynamics. Abelson interacting protein (Abi), originally identified as a kinase substrate of Abl, also plays a key role in actin dynamics, yet its role with respect to Abl in the developing nervous system remains unclear. Here we show that mutations in abi disrupt axonal patterning in the developing Drosophila central nervous system (CNS). However, reducing abi gene dosage by half substantially rescues Abl mutant phenotypes in pupal lethality, axonal guidance defects and locomotion deficits. Moreover, we show that mutations in Abl increase synaptic growth and spontaneous synaptic transmission frequency at the neuromuscular junction. Double heterozygosity for abi and enabled (ena) also suppresses the synaptic overgrowth phenotypes of Abl mutants, suggesting that Abi acts cooperatively with Ena to antagonize Abl function in synaptogenesis. Intriguingly, overexpressing Abi or Ena alone in cultured cells dramatically redistributed peripheral F-actin to the cytoplasm, with aggregates colocalizing with Abi and/or Ena, and resulted in a reduction in neurite extension. However, co-expressing Abl with Abi or Ena redistributed cytoplasmic F-actin back to the cell periphery and restored bipolar cell morphology. These data suggest that abi and Abl have an antagonistic interaction in Drosophila axonogenesis and synaptogenesis, which possibly occurs through the modulation of F-actin reorganization.

  5. [18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity

    PubMed Central

    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.

    2016-01-01

    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—[18F]Clofarabine; 2-chloro-2′-deoxy-2′-[18F]fluoro-9-β-d-arabinofuranosyl-adenine ([18F]CFA) and 2′-deoxy-2′-[18F]fluoro-9-β-d-arabinofuranosyl-guanine ([18F]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, [18F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [18F]F-AraG is a better substrate for dGK than for dCK. [18F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [18F]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 [18F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [18F]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 [18F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [18F]CFA PET as a new cancer biomarker for treatment stratification and monitoring. PMID:27035974

  6. [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.

  7. 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…

  8. 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…

  9. Synaptic and genomic responses to JNK and AP-1 signaling in Drosophila neurons

    PubMed Central

    Etter, Paul D; Narayanan, Radhakrishnan; Navratilova, Zaneta; Patel, Chirag; Bohmann, Dirk; Jasper, Heinrich; Ramaswami, Mani

    2005-01-01

    Background The transcription factor AP-1 positively controls synaptic plasticity at the Drosophila neuromuscular junction. Although in motor neurons, JNK has been shown to activate AP-1, a positive regulator of growth and strength at the larval NMJ, the consequences of JNK activation are poorly studied. In addition, the downstream transcriptional targets of JNK and AP-1 signaling in the Drosophila nervous system have yet to be identified. Here, we further investigated the role of JNK signaling at this model synapse employing an activated form of JNK-kinase; and using Serial Analysis of Gene Expression and oligonucleotide microarrays, searched for candidate early targets of JNK or AP-1 dependent transcription in neurons. Results Temporally-controlled JNK induction in postembryonic motor neurons triggers synaptic growth at the NMJ indicating a role in developmental plasticity rather than synaptogenesis. An unexpected observation that JNK activation also causes a reduction in transmitter release is inconsistent with JNK functioning solely through AP-1 and suggests an additional, yet-unidentified pathway for JNK signaling in motor neurons. SAGE profiling of mRNA expression helps define the neural transcriptome in Drosophila. Though many putative AP-1 and JNK target genes arose from the genomic screens, few were confirmed in subsequent validation experiments. One potentially important neuronal AP-1 target discovered, CG6044, was previously implicated in olfactory associative memory. In addition, 5 mRNAs regulated by RU486, a steroid used to trigger conditional gene expression were identified. Conclusion This study demonstrates a novel role for JNK signaling at the larval neuromuscular junction and provides a quantitative profile of gene transcription in Drosophila neurons. While identifying potential JNK/AP-1 targets it reveals the limitations of genome-wide analyses using complex tissues like the whole brain. PMID:15932641

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

    PubMed

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

    2012-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  12. Drosophila chem mutations disrupt epithelial polarity in Drosophila embryos

    PubMed Central

    Zamudio-Arroyo, José M.

    2016-01-01

    Drosophila embryogenesis has proven to be an extremely powerful system for developmental gene discovery and characterization. We isolated five new EMS-induced alleles that do not complement the l(3R)5G83 lethal line isolated in the Nüsslein-Volhard and Wieschaus screens. We have named this locus chem. Lethality of the new alleles as homozygous zygotic mutants is not completely penetrant, and they have an extended phenocritical period. Like the original allele, a fraction of mutant embryos die with cuticular defects, notably head involution and dorsal closure defects. Embryonic defects are much more extreme in germline clones, where the majority of mutant embryos die during embryogenesis and do not form cuticle, implying a strong chem maternal contribution. chem mutations genetically interact with mutations in cytoskeletal genes (arm) and with mutations in the epithelial polarity genes coracle, crumbs, and yurt. chem mutants dorsal open defects are similar to those present in yurt mutants, and, likewise, they have epithelial polarity defects. chem1 and chem3 mutations suppress yurt3, and chem3 mutants suppress crumbs1 mutations. In contrast, chem1 and coracle2 mutations enhance each other. Compared to controls, in chem mutants in embryonic lateral epithelia Crumbs expression is mislocalized and reduced, Coracle is increased and mislocalized basally at embryonic stages 13–14, then reduced at stage 16. Arm expression has a similar pattern but levels are reduced. PMID:27920954

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

    USDA-ARS?s Scientific Manuscript database

    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...

  14. Phosphorylation of period is influenced by cycling physical associations of double-time, period, and timeless in the Drosophila clock.

    PubMed

    Kloss, B; Rothenfluh, A; Young, M W; Saez, L

    2001-06-01

    The clock gene double-time (dbt) encodes an ortholog of casein kinase Iepsilon that promotes phosphorylation and turnover of the PERIOD protein. Whereas the period (per), timeless (tim), and dClock (dClk) genes of Drosophila each contribute cycling mRNA and protein to a circadian clock, dbt RNA and DBT protein are constitutively expressed. Robust circadian changes in DBT subcellular localization are nevertheless observed in clock-containing cells of the fly head. These localization rhythms accompany formation of protein complexes that include PER, TIM, and DBT, and reflect periodic redistribution between the nucleus and the cytoplasm. Nuclear phosphorylation of PER is strongly enhanced when TIM is removed from PER/TIM/DBT complexes. The varying associations of PER, DBT and TIM appear to determine the onset and duration of nuclear PER function within the Drosophila clock.

  15. Genetic mechanisms regulating stem cell self-renewal and differentiation in the central nervous system of Drosophila

    PubMed Central

    Kim, Dongwook W

    2009-01-01

    Recent studies using the Drosophila central nervous system as a model have identified key molecules and mechanisms underlying stem cell self-renewal and differentiation. These studies suggest that proteins like Aurora-A, atypical protein kinase C, Prospero and Brain tumor act as key regulators in a tightly coordinated interplay between mitotic spindle orientation and asymmetric protein localization. These data also provide initial evidence that both processes are coupled to cell cycle progression and growth control, thereby regulating a binary switch between proliferative stem self-renewal and differentiative progenitor cell specification. Considering the evolutionary conservation of some of the mechanisms and molecules involved, these data provide a rationale and genetic model for understanding stem cell self-renewal and differentiation in general. The new data gained in Drosophila may therefore lead to conceptual advancements in understanding the aetiology and treatment of human neurological disorders such as brain tumor formation and neurodegenerative diseases. PMID:19421003

  16. Patch-clamping Drosophila sensory neurons.

    PubMed

    Kucher, Volodymyr; Eaton, Benjamin A; Stockand, James D; Boiko, Nina

    2013-01-01

    Electrophysiological studies provide essential clues about the regulation and physiological function of ion channel proteins. Probing ion channel activity in vivo, though, often is challenging. This can limit the usefulness of such model organisms as Drosophila for electrophysiological studies. This is unfortunate because these genetically tractable organisms represent powerful research tools that facilitate elaboration of complex questions of physiology. Here, we describe a recently developed method for recording ion channel activity in Drosophila sensory neurons. This approach is based on patch-clamping primary neuron cultures from Drosophila embryos. Such cultures allow the study of ion channels in different genetic backgrounds. In addition to describing how to prepare a primary neuronal cell culture from Drosophila embryos, we discuss, as an example of utility, analysis of Na(+) currents in cultured class IV multidendritic (md) sensory neurons with the patch clamp technique. Excitability of md sensory neurons, manifested as action potential firing, is revealed with whole-cell current-clamping. Voltage-clamping class IV md neurons revealed the activity of the voltage-gated Na(+) channel, paralytic. Moreover, challenging class IV md neurons with acidic pH activates acid-sensing inward Na(+) currents. Genetic manipulation of Drosophila combined with this electrophysiological readout of activity identifies pickpocket1 (Ppk1), a member of the Deg/ENaC channel family, as responsible for conducting an acid-sensing Na(+) current in class IV md sensory neurons.

  17. 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.

  18. Sleep and wakefulness in Drosophila melanogaster

    PubMed Central

    Cirelli, Chiara; Bushey, Daniel

    2009-01-01

    Summary Sleep is present and tightly regulated in every vertebrate species in which it has been carefully investigated, but what sleep is for remains a mystery. Sleep is also present in invertebrates, and an extensive analysis in Drosophila melanogaster has shown that sleep in fruit flies show most of the fundamental features that characterize sleep in mammals. In Drosophila, fly sleep consists of sustained periods of quiescence associated with an increased arousal threshold. Fly sleep is modulated by several of the same stimulants and hypnotics that affect mammalian sleep. Moreover, like in mammals, fly sleep shows remarkable interindividual variability. The expression of several genes involved in energy metabolism, synaptic plasticity, and the response to cellular stress varies in Drosophila between sleep and wakefulness, and the same occurs in rodents. Brain activity also changes in flies as a function of behavioral state. Furthermore, Drosophila sleep is tightly regulated in a circadian and homeostatic manner, and the homeostatic regulation is largely independent of the circadian regulation. After sleep deprivation recovery sleep in flies is longer in duration and more consolidated, as indicated by an increase in arousal threshold and fewer brief awakenings. Finally, sleep deprivation in flies impairs vigilance and performance. Because of the extensive similarities between flies and mammals, Drosophila is now being used as a promising model system for the genetic dissection of sleep. Over the last few years, mutagenesis screens have isolated several short sleeping mutants, a demonstration that that single genes can have a powerful effect on a complex trait like sleep. PMID:18591491

  19. Comparison of human and Drosophila atlastin GTPases.

    PubMed

    Wu, Fuyun; Hu, Xiaoyu; Bian, Xin; Liu, Xinqi; Hu, Junjie

    2015-02-01

    Formation of the endoplasmic reticulum (ER) network requires homotypic membrane fusion, which involves a class of atlastin (ATL) GTPases. Purified Drosophila ATL is capable of mediating vesicle fusion in vitro, but such activity has not been reported for any other ATLs. Here, we determined the preliminary crystal structure of the cytosolic segment of Drosophila ATL in a GDP-bound state. The structure reveals a GTPase domain dimer with the subsequent three-helix bundles associating with their own GTPase domains and pointing in opposite directions. This conformation is similar to that of human ATL1, to which GDP and high concentrations of inorganic phosphate, but not GDP only, were included. Drosophila ATL restored ER morphology defects in mammalian cells lacking ATLs, and measurements of nucleotide-dependent dimerization and GTPase activity were comparable for Drosophila ATL and human ATL1. However, purified and reconstituted human ATL1 exhibited no in vitro fusion activity. When the cytosolic segment of human ATL1 was connected to the transmembrane (TM) region and C-terminal tail (CT) of Drosophila ATL, the chimera still exhibited no fusion activity, though its GTPase activity was normal. These results suggest that GDP-bound ATLs may adopt multiple conformations and the in vitro fusion activity of ATL cannot be achieved by a simple collection of functional domains.

  20. 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

  1. 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.

  2. Pantothenate - kinase associated neurodegeneration.

    PubMed

    Parmar, Alpana; Khare, Shruti; Srivastav, Vipul

    2012-04-01

    Neurodegeneration with brain iron accumulation is a group of disorders, the commonest of which is PKAN (Pantothenate kinase associated neurodegeneration). We present here, a case of 18 year old boy with progressive dementia, pyramidal and extrapyramidal involvement, dysarthria, seizures and myoclonus. The patient was diagnosed as PKAN (formerly Hallervorden Spatz disease) after "eye of tiger" appearance on neuro-imaging.

  3. Visualizing autophosphorylation in histidine kinases.

    PubMed

    Casino, Patricia; Miguel-Romero, Laura; Marina, Alberto

    2014-01-01

    Reversible protein phosphorylation is the most widespread regulatory mechanism in signal transduction. Autophosphorylation in a dimeric sensor histidine kinase is the first step in two-component signalling, the predominant signal-transduction device in bacteria. Despite being the most abundant sensor kinases in nature, the molecular bases of the histidine kinase autophosphorylation mechanism are still unknown. Furthermore, it has been demonstrated that autophosphorylation can occur in two directions, cis (intrasubunit) or trans (intersubunit) within the dimeric histidine kinase. Here, we present the crystal structure of the complete catalytic machinery of a chimeric histidine kinase. The structure shows an asymmetric histidine kinase dimer where one subunit is caught performing the autophosphorylation reaction. A structure-guided functional analysis on HK853 and EnvZ, two prototypical cis- and trans-phosphorylating histidine kinases, has allowed us to decipher the catalytic mechanism of histidine kinase autophosphorylation, which seems to be common independently of the reaction directionality.

  4. Increased expression of a cGMP-dependent protein kinase in rotation-adapted western corn rootworm (Diabrotica virgifera virgifera L.).

    PubMed

    Garabagi, Freydoun; Wade French, B; Schaafsma, Arthur W; Peter Pauls, K

    2008-07-01

    A new 'variant' behavior in western corn rootworm (WCR) has resulted in egg-laying into non-cornfields, compared to 'normal' deposition of eggs in cornfields, allowing these insects to circumvent crop rotation. No morphological or genetic characteristics have been defined to differentiate between the normal and variant biotypes. Cyclic GMP-dependent protein kinases (PKG) have been implicated in the regulation of behaviors in vertebrates, insects, and nematodes, including foraging behavior in Drosophila. A cDNA with homology to the Drosophila melanogaster foraging gene (called Dvfor1) was cloned from WCR. The deduced DvFOR1 protein is approximately 70% similar to FOR proteins in Drosophila, silkworm (Bombyx mori) and honeybee (Apis mellifera). It contains a coiled-coil region, two tandem cyclic nucleotide-binding domains, a serine/threonine kinase catalytic domain, and a serine/threonine kinase catalytic domain extension, which are all characteristically found in PKG proteins. Real-time PCR assays of foraging transcript levels in heads of normal and rotation adapted females of WCR obtained from lab-reared insect colonies indicated that the variants had higher levels (25%) of PKG expression than normals. The magnitude of this increase is similar to that observed in Drosophila rover phenotypes compared to sitter phenotypes. However, Diabrotica contains at least two different foraging gene transcripts, which complicates establishing a direct link between the level of gene expression and insect behavior.

  5. Phosphorylation of DNA topoisomerase II by casein kinase II: modulation of eukaryotic topoisomerase II activity in vitro.

    PubMed Central

    Ackerman, P; Glover, C V; Osheroff, N

    1985-01-01

    The phosphorylation of Drosophila melanogaster DNA topoisomerase II by purified casein kinase II was characterized in vitro. Under the conditions used, the kinase incorporated a maximum of 2-3 molecules of phosphate per homodimer of topoisomerase II. No autophosphorylation of the topoisomerase was observed. The only amino acid residue modified by casein kinase II was serine. Apparent Km and Vmax values for the phosphorylation reaction were 0.4 microM topoisomerase II and 3.3 mumol of phosphate incorporated per min per mg of kinase, respectively. Phosphorylation stimulated the DNA relaxation activity of topoisomerase II by 3-fold over that of the dephosphorylated enzyme, and the effects of modification could be reversed by treatment with alkaline phosphatase. Therefore, this study demonstrates that post-translational enzymatic modifications can be used to modulate the interaction between topoisomerase II and DNA. Images PMID:2987912

  6. Kinase Inhibitors from Marine Sponges

    PubMed Central

    Skropeta, Danielle; Pastro, Natalie; Zivanovic, Ana

    2011-01-01

    Protein kinases play a critical role in cell regulation and their deregulation is a contributing factor in an increasing list of diseases including cancer. Marine sponges have yielded over 70 novel compounds to date that exhibit significant inhibitory activity towards a range of protein kinases. These compounds, which belong to diverse structural classes, are reviewed herein, and ordered based upon the kinase that they inhibit. Relevant synthetic studies on the marine natural product kinase inhibitors have also been included. PMID:22073013

  7. C. elegans STK39/SPAK ortholog-mediated inhibition of ClC anion channel activity is regulated by WNK-independent ERK kinase signaling.

    PubMed

    Falin, Rebecca A; Miyazaki, Hiroaki; Strange, Kevin

    2011-03-01

    Mammalian Ste20-like proline/alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) kinases phosphorylate and regulate cation-coupled Cl(-) cotransporter activity in response to cell volume changes. SPAK and OSR1 are activated via phosphorylation by upstream with-no-lysine (WNK) kinases. In Caenorhabditis elegans, the SPAK/OSR1 ortholog germinal center kinase (GCK)-3 binds to and regulates the activity of the cell volume- and meiotic cell cycle-dependent ClC anion channel CLH-3b. We tested the hypothesis that WNK kinases function in the GCK-3/CLH-3b signaling cascade. CLH-3b heterologously expressed in human embryonic kidney (HEK) cells was unaffected by coexpression with the single C. elegans WNK kinase, WNK-1, or kinase-dead WNK-1 dominant-negative mutants. RNA interference (RNAi) knockdown of the single Drosophila WNK kinase had no effect on the activity of CLH-3b expressed in Drosophila S2 cells. Similarly, RNAi silencing of C. elegans WNK-1 had no effect on basal or cell volume-sensitive activity of CLH-3b expressed endogenously in worm oocytes. Previous yeast 2-hybrid studies suggested that ERK kinases may function upstream of GCK-3. Pharmacological inhibition of ERK signaling disrupted CLH-3b activity in HEK cells in a GCK-3-dependent manner. RNAi silencing of the C. elegans ERK kinase MPK-1 or the ERK phosphorylating/activating kinase MEK-2 constitutively activated native CLH-3b. MEK-2 and MPK-1 play important roles in regulating the meiotic cell cycle in C. elegans oocytes. Cell cycle-dependent changes in MPK-1 correlate with the pattern of CLH-3b activation observed during oocyte meiotic maturation. We postulate that MEK-2/MPK-1 functions upstream from GCK-3 to regulate its activity during cell volume and meiotic cell cycle changes.

  8. C. elegans STK39/SPAK ortholog-mediated inhibition of ClC anion channel activity is regulated by WNK-independent ERK kinase signaling

    PubMed Central

    Falin, Rebecca A.; Miyazaki, Hiroaki

    2011-01-01

    Mammalian Ste20-like proline/alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) kinases phosphorylate and regulate cation-coupled Cl− cotransporter activity in response to cell volume changes. SPAK and OSR1 are activated via phosphorylation by upstream with-no-lysine (WNK) kinases. In Caenorhabditis elegans, the SPAK/OSR1 ortholog germinal center kinase (GCK)-3 binds to and regulates the activity of the cell volume- and meiotic cell cycle-dependent ClC anion channel CLH-3b. We tested the hypothesis that WNK kinases function in the GCK-3/CLH-3b signaling cascade. CLH-3b heterologously expressed in human embryonic kidney (HEK) cells was unaffected by coexpression with the single C. elegans WNK kinase, WNK-1, or kinase-dead WNK-1 dominant-negative mutants. RNA interference (RNAi) knockdown of the single Drosophila WNK kinase had no effect on the activity of CLH-3b expressed in Drosophila S2 cells. Similarly, RNAi silencing of C. elegans WNK-1 had no effect on basal or cell volume-sensitive activity of CLH-3b expressed endogenously in worm oocytes. Previous yeast 2-hybrid studies suggested that ERK kinases may function upstream of GCK-3. Pharmacological inhibition of ERK signaling disrupted CLH-3b activity in HEK cells in a GCK-3-dependent manner. RNAi silencing of the C. elegans ERK kinase MPK-1 or the ERK phosphorylating/activating kinase MEK-2 constitutively activated native CLH-3b. MEK-2 and MPK-1 play important roles in regulating the meiotic cell cycle in C. elegans oocytes. Cell cycle-dependent changes in MPK-1 correlate with the pattern of CLH-3b activation observed during oocyte meiotic maturation. We postulate that MEK-2/MPK-1 functions upstream from GCK-3 to regulate its activity during cell volume and meiotic cell cycle changes. PMID:21160027

  9. Lim kinase regulates the development of olfactory and neuromuscular synapses.

    PubMed

    Ang, Lay-Hong; Chen, Weitao; Yao, Ying; Ozawa, Rie; Tao, Enxiang; Yonekura, Junichiro; Uemura, Tadashi; Keshishian, Haig; Hing, Huey

    2006-05-01

    Lim Kinase (Limk) belongs to a phylogenetically conserved family of serine/threonine kinases, which have been shown to be potent regulators of the actin cytoskeleton. Despite accumulating evidence of its biochemical actions, its in vivo function has remained poorly understood. The association of the Limk1 gene with Williams Syndrome indicates that proteins of this family play a role in the nervous system. To unravel the cellular and molecular functions of Limk, we have either knocked out or activated the Limk gene in Drosophila. At the neuromuscular junction, loss of Limk leads to enlarged terminals, while increasing the activity of Limk leads to stunted terminals with fewer synaptic boutons. In the antennal lobe, loss of Limk abolishes the ability of p21-activated kinase (Pak) to alter glomerular development. In contrast, increase in Limk function leads to ectopic glomeruli, a phenotype suppressible by the coexpression of a hyperactive Cofilin gene. These results establish Limk as a critical regulator of Cofilin function and synapse development, and a downstream effector of Pak in vivo.

  10. Integrin adhesions suppress syncytium formation in the Drosophila larval epidermis

    PubMed Central

    Wang, Yan; Antunes, Marco; Anderson, Aimee E.; Kadrmas, Julie L.; Jacinto, Antonio; Galko, Michael J.

    2015-01-01

    Summary Integrins are critical for barrier epithelial architecture. Integrin loss in vertebrate skin leads to blistering and wound healing defects. However, how Integrins and associated proteins maintain the regular morphology of epithelia is not well understood. We found that targeted knockdown of the integrin focal adhesion (FA) complex components βIntegrin, PINCH, and Integrin-linked kinase (ILK), caused formation of multinucleate epidermal cells within the Drosophila larval epidermis. This phenotype was specific to the Integrin FA complex and not due to secondary effects on polarity or junctional structures. The multinucleate cells resembled the syncytia caused by physical wounding. Live imaging of wound-induced syncytium formation in the pupal epidermis suggested direct membrane breakdown leading to cell-cell fusion and consequent mixing of cytoplasmic contents. Activation of Jun N-terminal kinase (JNK) signaling, which occurs upon wounding, also correlated with syncytium formation induced by PINCH knockdown. Further, ectopic JNK activation directly caused epidermal syncytium formation. No mode of syncytium formation including that induced by wounding, genetic loss-of FA-proteins, or local JNK hyperactivation, involved misregulation of mitosis or apoptosis. Finally, the mechanism of epidermal syncytium formation following JNK hyperactivation and wounding appeared to be direct disassembly of FA complexes. In conclusion, the loss of function phenotype of Integrin FA components in the larval epidermis resembles a wound. Integrin FA loss in mouse and human skin also causes a wound-like appearance. Our results reveal a novel and unexpected role for proper Integrin-based adhesion in suppressing larval epidermal cell-cell fusion– a role that may be conserved in other epithelia. PMID:26255846

  11. Drosophila melanogaster in the Study of Human Neurodegeneration

    PubMed Central

    Hirth, Frank

    2010-01-01

    Human neurodegenerative diseases are devastating illnesses that predominantly affect elderly people. The majority of the diseases are associated with pathogenic oligomers from misfolded proteins, eventually causing the formation of aggregates and the progressive loss of neurons in the brain and nervous system. Several of these proteinopathies are sporadic and the cause of pathogenesis remains elusive. Heritable forms are associated with genetic defects, suggesting that the affected protein is causally related to disease formation and/or progression. The limitations of human genetics, however, make it necessary to use model systems to analyse affected genes and pathways in more detail. During the last two decades, research using the genetically amenable fruitfly has established Drosophila melanogaster as a valuable model system in the study of human neurodegeneration. These studies offer reliable models for Alzheimer’s, Parkinson’s, and motor neuron diseases, as well as models for trinucleotide repeat expansion diseases, including ataxias and Huntington’s disease. As a result of these studies, several signalling pathways including phosphatidylinositol 3-kinase (PI3K)/Akt and target of rapamycin (TOR), c-Jun N-terminal kinase (JNK) and bone morphogenetic protein (BMP) signalling, have been shown to be deregulated in models of proteinopathies, suggesting that two or more initiating events may trigger disease formation in an age-related manner. Moreover, these studies also demonstrate that the fruitfly can be used to screen chemical compounds for their potential to prevent or ameliorate the disease, which in turn can directly guide clinical research and the development of novel therapeutic strategies for the treatment of human neurodegenerative diseases. PMID:20522007

  12. 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.

  13. Axon and dendrite pruning in Drosophila.

    PubMed

    Yu, Fengwei; Schuldiner, Oren

    2014-08-01

    Pruning, a process by which neurons selectively remove exuberant or unnecessary processes without causing cell death, is crucial for the establishment of mature neural circuits during animal development. Yet relatively little is known about molecular and cellular mechanisms that govern neuronal pruning. Holometabolous insects, such as Drosophila, undergo complete metamorphosis and their larval nervous systems are replaced with adult-specific ones, thus providing attractive models for studying neuronal pruning. Drosophila mushroom body and dendritic arborization neurons have been utilized as two appealing systems to elucidate the underlying mechanisms of axon and dendrite pruning, respectively. In this review we highlight recent developments and discuss some similarities and differences in the mechanisms that regulate these two distinct modes of neuronal pruning in Drosophila.

  14. 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

  15. 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.

  16. Drosophila melanogaster: Deciphering Alzheimer’s Disease

    PubMed Central

    Tan, Florence Hui Ping; Azzam, Ghows

    2017-01-01

    Alzheimer’s disease (AD) is the most widespread neurodegenerative disorder worldwide. Its pathogenesis involves two hallmarks: aggregation of amyloid beta (Aβ) and occurrence of neurofibrillary tangles (NFTs). The mechanism behind the disease is still unknown. This has prompted the use of animal models to mirror the disease. The fruit fly, Drosophila melanogaster has garnered considerable attention as an organism to recapitulate human disorders. With the ability to monopolise a multitude of traditional and novel genetic tools, Drosophila is ideal for studying not only cellular aspects but also physiological and behavioural traits of human neurodegenerative diseases. Here, we discuss the use of the Drosophila model in understanding AD pathology and the insights gained in discovering drug therapies for AD. PMID:28894399

  17. A taste of the Drosophila gustatory receptors.

    PubMed

    Montell, Craig

    2009-08-01

    Insects such as the fruit fly, Drosophila melanogaster, rely on contact chemosensation to detect nutrient-rich foods, to avoid consuming toxic chemicals, and to select mates and hospitable zones to deposit eggs. Flies sense tastants and nonvolatile pheromones through gustatory bristles and pegs distributed on multiple body parts including the proboscis, wing margins, legs, and ovipositor. The sensilla house gustatory receptor neurons, which express members of the family of 68 gustatory receptors (GRs). In contrast to mammalian chemosensation or Drosophila olfaction, which are initiated by receptors composed of dimers of one or two receptor types, the functional Drosophila GRs may include three or more subunits. Several GRs appear to be expressed in multiple cell types that are not associated with contact chemosensation raising the possibility that these proteins may have roles that extend beyond the detection of tastants and pheromones.

  18. [When Tribolium complements the genetics of Drosophila].

    PubMed

    Bonneton, François

    2010-03-01

    With its recently sequenced genome, the red flour beetle Tribolium castaneum became one of the few model organisms with all the main genetic tools. As a coleoptera, it belongs to the most species-rich order of animals. Tribolium is also a worldwide pest for stored dried foods. Regarding developmental biology, Tribolium offers a complementary model to the highly derived Drosophila. For example, the function of many gap and pair-rule segmentation genes is different in both species. These differences reveal the evolutionary plasticity between two modes of development, with a long germ band in fly and a short one in Tribolium. This beetle allowed the identification of a new type of ecdysone receptor for holometabolous insects. Finally, in the search for the juvenile hormone receptor, a crucial result was obtained with experiments that could be performed only with Tribolium, and not with Drosophila. Tribolium, in association with Drosophila, should help to understand the general rules of development in insects.

  19. 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

  20. DREF plays multiple roles during Drosophila development.

    PubMed

    Tue, Nguyen Trong; Yoshioka, Yasuhide; Mizoguchi, Megumi; Yoshida, Hideki; Zurita, Mario; Yamaguchi, Masamitsu

    2017-06-01

    DREF was originally identified as a transcription factor that coordinately regulates the expression of DNA replication- and proliferation-related genes in Drosophila. Subsequent studies demonstrated that DREF is involved in tumor suppressor pathways including p53 and Hippo signaling. DREF also regulates the expression of genes encoding components of the JNK and EGFR pathways during Drosophila development. DREF itself is under the control of the TOR pathway during cell and tissue growth responding to nutrition. Recent studies revealed that DREF plays a role in chromatin organization including insulator function, chromatin remodeling, and telomere maintenance. DREF is also involved in the regulation of genes related to mitochondrial biogenesis, linking it to cellular proliferation. Thus, DREF is now emerging as not only a transcription factor, but also a multi-functional protein. In this review, we summarize current advances in studies on the novel functions of Drosophila DREF. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Sexual Behavior of Drosophila suzukii.

    PubMed

    Revadi, Santosh; Lebreton, Sébastien; Witzgall, Peter; Anfora, Gianfranco; Dekker, Teun; Becher, Paul G

    2015-03-09

    A high reproductive potential is one reason for the rapid spread of Drosophila suzukii in Europe and in the United States. In order to identify mechanisms that mediate mating and reproduction in D. suzukii we studied the fly's reproductive behavior, diurnal mating activity and sexual maturation. Furthermore, we studied the change of female cuticular hydrocarbons (CHCs) with age and conducted a preliminary investigation on the role of female-derived chemical signals in male mating behavior. Sexual behavior in D. suzukii is characterized by distinct elements of male courtship leading to female acceptance for mating. Time of day and age modulate D. suzukii mating activity. As with other drosophilids, female sexual maturity is paralleled by a quantitative increase in CHCs. Neither female CHCs nor other olfactory signals were required to induce male courtship, however, presence of those signals significantly increased male sexual behavior. With this pilot study we hope to stimulate research on the reproductive biology of D. suzukii, which is relevant for the development of pest management tools.

  2. Flavin reduction activates Drosophila cryptochrome.

    PubMed

    Vaidya, Anand T; Top, Deniz; Manahan, Craig C; Tokuda, Joshua M; Zhang, Sheng; Pollack, Lois; Young, Michael W; Crane, Brian R

    2013-12-17

    Entrainment of circadian rhythms in higher organisms relies on light-sensing proteins that communicate to cellular oscillators composed of delayed transcriptional feedback loops. The principal photoreceptor of the fly circadian clock, Drosophila cryptochrome (dCRY), contains a C-terminal tail (CTT) helix that binds beside a FAD cofactor and is essential for light signaling. Light reduces the dCRY FAD to an anionic semiquinone (ASQ) radical and increases CTT proteolytic susceptibility but does not lead to CTT chemical modification. Additional changes in proteolytic sensitivity and small-angle X-ray scattering define a conformational response of the protein to light that centers at the CTT but also involves regions remote from the flavin center. Reduction of the flavin is kinetically coupled to CTT rearrangement. Chemical reduction to either the ASQ or the fully reduced hydroquinone state produces the same conformational response as does light. The oscillator protein Timeless (TIM) contains a sequence similar to the CTT; the corresponding peptide binds dCRY in light and protects the flavin from oxidation. However, TIM mutants therein still undergo dCRY-mediated degradation. Thus, photoreduction to the ASQ releases the dCRY CTT and promotes binding to at least one region of TIM. Flavin reduction by either light or cellular reductants may be a general mechanism of CRY activation.

  3. Flavin reduction activates Drosophila cryptochrome

    PubMed Central

    Vaidya, Anand T.; Top, Deniz; Manahan, Craig C.; Tokuda, Joshua M.; Zhang, Sheng; Pollack, Lois; Young, Michael W.; Crane, Brian R.

    2013-01-01

    Entrainment of circadian rhythms in higher organisms relies on light-sensing proteins that communicate to cellular oscillators composed of delayed transcriptional feedback loops. The principal photoreceptor of the fly circadian clock, Drosophila cryptochrome (dCRY), contains a C-terminal tail (CTT) helix that binds beside a FAD cofactor and is essential for light signaling. Light reduces the dCRY FAD to an anionic semiquinone (ASQ) radical and increases CTT proteolytic susceptibility but does not lead to CTT chemical modification. Additional changes in proteolytic sensitivity and small-angle X-ray scattering define a conformational response of the protein to light that centers at the CTT but also involves regions remote from the flavin center. Reduction of the flavin is kinetically coupled to CTT rearrangement. Chemical reduction to either the ASQ or the fully reduced hydroquinone state produces the same conformational response as does light. The oscillator protein Timeless (TIM) contains a sequence similar to the CTT; the corresponding peptide binds dCRY in light and protects the flavin from oxidation. However, TIM mutants therein still undergo dCRY-mediated degradation. Thus, photoreduction to the ASQ releases the dCRY CTT and promotes binding to at least one region of TIM. Flavin reduction by either light or cellular reductants may be a general mechanism of CRY activation. PMID:24297896

  4. Sexual Behavior of Drosophila suzukii

    PubMed Central

    Revadi, Santosh; Lebreton, Sébastien; Witzgall, Peter; Anfora, Gianfranco; Dekker, Teun; Becher, Paul G.

    2015-01-01

    A high reproductive potential is one reason for the rapid spread of Drosophila suzukii in Europe and in the United States. In order to identify mechanisms that mediate mating and reproduction in D. suzukii we studied the fly’s reproductive behavior, diurnal mating activity and sexual maturation. Furthermore, we studied the change of female cuticular hydrocarbons (CHCs) with age and conducted a preliminary investigation on the role of female-derived chemical signals in male mating behavior. Sexual behavior in D. suzukii is characterized by distinct elements of male courtship leading to female acceptance for mating. Time of day and age modulate D. suzukii mating activity. As with other drosophilids, female sexual maturity is paralleled by a quantitative increase in CHCs. Neither female CHCs nor other olfactory signals were required to induce male courtship, however, presence of those signals significantly increased male sexual behavior. With this pilot study we hope to stimulate research on the reproductive biology of D. suzukii, which is relevant for the development of pest management tools. PMID:26463074

  5. Evolution of Polypyrimidines in Drosophila

    PubMed Central

    Cseko, Y. M. T.; Dower, N. A.; Minoo, P.; Lowenstein, L.; Smith, G. R.; Stone, J.; Sederoff, R.

    1979-01-01

    We surveyed 101 different Drosophila species for the presence of a particular highly repetitive DNA sequence containing long tracts of polypyrimidine/polypurine DNA, first found in D. melanogaster. Out of 55 tested species in the melanogaster group, only the sibling species D. simulans and D. mauritiana, as well as one distant relative in the ananassae subgroup, D. varians, contained the same sequence. All four of these species have long pyrimidine tracts as shown by acid hydrolysis of labelled DNA. All four species have the same sequence, but the amount of this polypyrimidine/polypurine DNA varies greatly. Four other species in the hydei subgroup were found to contain a polypyrimidine/polypurine sequence, with an oligonucleotide composition different from that of D. melanogaster. This polypyrimidine DNA varies from as much as 10% of the total DNA in D. nigrohydei, to as little as 0.4% in D. neohydei. The long pyrimidine tracts in the hydei subgroup are often more than a thousand nucleotides in length, representing exceedingly homogeneous repetitious sequences.—These results show a rapid but discontinuous pattern of evolution for polypyrimidine/polypurine DNA. These sequences are not species specific, yet closely related species have greatly different amounts of polypyrimidines. Drastic changes occur in the amounts of these satellite type DNA sequences, as if the sequence had no continuous selective advantage in evolution. The implications of these results with regard to the general function and evolution of satellite DNA are discussed. PMID:114448

  6. Drosophila melanogaster Models of Galactosemia.

    PubMed

    Daenzer, J M I; Fridovich-Keil, J L

    2017-01-01

    The galactosemias are a family of autosomal recessive genetic disorders resulting from impaired function of the Leloir pathway of galactose metabolism. Type I, or classic galactosemia, results from profound deficiency of galactose-1-phosphate uridylyltransferase, the second enzyme in the Leloir pathway. Type II galactosemia results from profound deficiency of galactokinase, the first enzyme in the Leloir pathway. Type III galactosemia results from partial deficiency of UDP galactose 4'-epimerase, the third enzyme in the Leloir pathway. Although at least classic galactosemia has been recognized clinically for more than 100 years, and detectable by newborn screening for more than 50 years, all three galactosemias remain poorly understood. Early detection and dietary restriction of galactose prevent neonatal lethality, but many affected infants grow to experience a broad range of developmental and other disabilities. To date, there is no intervention known that prevents or reverses these long-term complications. Drosophila melanogaster provides a genetically and biochemically facile model for these conditions, enabling studies that address mechanism and open the door for novel approaches to intervention.

  7. Secondary kinase reactions catalyzed by yeast pyruvate kinase.

    PubMed

    Leblond, D J; Robinson, J L

    1976-06-07

    1. Yeast pyruvate kinase (EC 2.7.1.40) catalyzes, in addition to the primary, physiologically important reaction, three secondary kinase reactions, the ATP-dependent phosphorylations of fluoride (fluorokinase), hydroxylamine (hydroxylamine kinase) and glycolate (glycolate kinase). 2. These reactions are accelerated by fructose-1,6-bisphosphate, the allosteric activator of the primary reaction. Wth Mg2+ as the required divalent cation, none of these reactions are observed in the absence of fructose-biphosphate. With Mn2+, fructose-bisphosphate is required for the glycolate kinase reaction, but merely stimulates the other reactions. 3. The effect of other divalent cations and pH on three secondary kinase reactions was also examined. 4. Results are compared with those obtained from muscle pyruvate kinase and the implications of the results for the mechanism of the yeast enzyme are discussed.

  8. 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

  9. Drosophila DJ-1 Decreases Neural Sensitivity to Stress by Negatively Regulating Daxx-Like Protein through dFOXO

    PubMed Central

    Choi, Gahee; Suh, Yoon Seok; Han, Seung Yeop; Lee, Minjung; Park, Seung Hwan; Lee, Jang Ho; Lee, Soojin; Bang, Se Min; Jeong, Yuji; Chung, Won-Ju; Lee, Im-Soon; Jeong, Gilsang; Chung, Jongkyeong; Cho, Kyoung Sang

    2013-01-01

    DJ-1, a Parkinson's disease (PD)–associated gene, has been shown to protect against oxidative stress in Drosophila. However, the molecular mechanism underlying oxidative stress-induced phenotypes, including apoptosis, locomotive defects, and lethality, in DJ-1-deficient flies is not fully understood. Here we showed that Daxx-like protein (DLP), a Drosophila homologue of the mammalian Death domain-associated protein (Daxx), was upregulated under oxidative stress conditions in the loss-of-function mutants of Drosophila DJ-1β, a Drosophila homologue of DJ-1. DLP overexpression induced apoptosis via the c-Jun N-terminal kinase (JNK)/Drosophila forkhead box subgroup O (dFOXO) pathway, whereas loss of DLP increased resistance to oxidative stress and UV irradiation. Moreover, the oxidative stress-induced phenotypes of DJ-1β mutants were dramatically rescued by DLP deficiency, suggesting that enhanced expression of DLP contributes to the DJ-1β mutant phenotypes. Interestingly, we found that dFOXO was required for the increase in DLP expression in DJ-1β mutants and that dFOXO activity was increased in the heads of DJ-1β mutants. In addition, subcellular localization of DLP appeared to be influenced by DJ-1 expression so that cytosolic DLP was increased in DJ-1β mutants. Similarly, in mammalian cells, Daxx translocation from the nucleus to the cytosol was suppressed by overexpressed DJ-1β under oxidative stress conditions; and, furthermore, targeted expression of DJ-1β to mitochondria efficiently inhibited the Daxx translocation. Taken together, our findings demonstrate that DJ-1β protects flies against oxidative stress- and UV-induced apoptosis by regulating the subcellular localization and gene expression of DLP, thus implying that Daxx-induced apoptosis is involved in the pathogenesis of DJ-1-associated PD. PMID:23593018

  10. Defensive repertoire of Drosophila larvae in response to toxic fungi.

    PubMed

    Trienens, Monika; Kraaijeveld, Ken; Wertheim, Bregje

    2017-07-26

    Chemical warfare including insecticidal secondary metabolites is a well-known strategy for environmental microbes to monopolize a food source. Insects in turn have evolved behavioural and physiological defences to eradicate or neutralize the harmful microorganisms. We studied the defensive repertoire of insects in this interference competition by combining behavioural and developmental assays with whole-transcriptome time-series analysis. Confrontation with the toxic filamentous fungus Aspergillus nidulans severely reduced the survival of Drosophila melanogaster larvae. Nonetheless, the larvae did not behaviourally avoid the fungus, but aggregated at it. Confrontation with fungi strongly affected larval gene expression, including many genes involved in detoxification (e.g., CYP, GST and UGT genes) and the formation of the insect cuticle (e.g., Tweedle genes). The most strongly upregulated genes were several members of the insect-specific gene family Osiris, and CHK-kinase-like domains were over-represented. Immune responses were not activated, reflecting the competitive rather than pathogenic nature of the antagonistic interaction. While internal microbes are widely acknowledged as important, our study emphasizes the underappreciated role of environmental microbes as fierce competitors. © 2017 John Wiley & Sons Ltd.

  11. 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

  12. Ciliary Phosphoinositide Regulates Ciliary Protein Trafficking in Drosophila.

    PubMed

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

    2015-12-29

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

  13. 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. © 2015. Published by The Company of Biologists Ltd.

  14. Developmental profiles of PERIOD and DOUBLETIME in Drosophila melanogaster ovary.

    PubMed

    Kotwica, Joanna; Larson, Maureen K; Bebas, Piotr; Giebultowicz, Jadwiga M

    2009-05-01

    The clock protein PERIOD (PER) displays circadian cycles of accumulation, phosphorylation, nuclear translocation and degradation in Drosophila melanogaster clock cells. One exception to this pattern is in follicular cells enclosing previtellogenic ovarian egg chambers. In these cells, PER remains high and cytoplasmic at all times of day. Genetic evidence suggest that PER and its clock partner TIMELESS (TIM) interact in these cells, yet, they do not translocate to the nucleus. Here, we investigated the levels and subcellular localization of PER in older vitellogenic follicles. Cytoplasmic PER levels decreased in the follicular cells at the onset of vitellogenesis (stage 9). Interestingly, PER was observed in the nuclei of some follicular cells at this stage. PER signal disappeared in more advanced (stage 10) vitellogenic follicles. Since the phosphorylation state of PER is critical for the progression of circadian cycle, we investigated the status of PER phosphorylation in the ovary and the expression patterns of DOUBLETIME (DBT), a kinase known to affect PER in the clock cells. DBT was absent in previtellogenic follicular cells, but present in the cytoplasm of some stage 9 follicular cells. DBT was not distributed uniformly but was present in patches of adjacent cells, in a pattern resembling PER distribution at the same stage. Our data suggest that the absence of dbt expression in the follicular cells of previtellogenic egg chambers may be related to stable and cytoplasmic expression of PER in these cells. Onset of dbt expression in vitellogenic follicles coincides with nuclear localization of PER protein.

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

    PubMed

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

    2001-07-01

    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.

  16. Water taste transduction pathway is calcium dependent in Drosophila.

    PubMed

    Meunier, Nicolas; Marion-Poll, Frédéric; Lucas, Philippe

    2009-06-01

    In mammals, detection of osmolarity by the gustatory system was overlooked until recently. In insects, specific taste receptor neurons detect hypoosmotic stimuli and are commonly called "W" (water) cells. W cells are easy to access in vivo and represent a good model to study the transduction of hypoosmotic stimuli. Using pharmacological and genetic approaches in Drosophila, we show that tarsal W cell firing activity depends on the concentration of external calcium bathing the dendrite. This dependence was confirmed by the strong inhibition of W cell responses to hypoosmotic stimuli by lanthanum (IC(50) = 8 nM), an ion known to inhibit calcium-permeable channels. Downstream, the transduction pathway likely involves calmodulin because calmodulin antagonists such as W-7 (IC(50) = 2 microM) and fluphenazine (IC(50) = 30 microM) prevented the activation of the W cell by hypoosmotic stimuli. A protein kinase C (PKC) may also be involved as W cell responses were blocked by PKC inhibitors, chelerythrine (IC(50) = 20 microM) and staurosporine (IC(50) = 30 microM). It was also reduced when expressing an inhibitory pseudosubstrate of PKC in gustatory receptor neurons. In the rat, the transduction pathway underlying low osmolarity detection involves aquaporin and swelling-activated ion channels. Our study suggests that the transduction pathway of hypoosmotic stimuli in insects differs from mammals.

  17. Mapping signaling pathway cross-talk in Drosophila cells

    PubMed Central

    Ammeux, Noemie; Housden, Benjamin E.; Georgiadis, Andrew; Hu, Yanhui; Perrimon, Norbert

    2016-01-01

    During development and homeostasis, cells integrate multiple signals originating either from neighboring cells or systemically. In turn, responding cells can produce signals that act in an autocrine, paracrine, or endocrine manner. Although the nature of the signals and pathways used in cell–cell communication are well characterized, we lack, in most cases, an integrative view of signaling describing the spatial and temporal interactions between pathways (e.g., whether the signals are processed sequentially or concomitantly when two pathways are required for a specific outcome). To address the extent of cross-talk between the major metazoan signaling pathways, we characterized immediate transcriptional responses to either single- or multiple pathway stimulations in homogeneous Drosophila cell lines. Our study, focusing on seven core pathways, epidermal growth factor receptor (EGFR), bone morphogenetic protein (BMP), Jun kinase (JNK), JAK/STAT, Notch, Insulin, and Wnt, revealed that many ligands and receptors are primary targets of signaling pathways, highlighting that transcriptional regulation of genes encoding pathway components is a major level of signaling cross-talk. In addition, we found that ligands and receptors can integrate multiple pathway activities and adjust their transcriptional responses accordingly. PMID:27528688

  18. The Hippo pathway regulates hematopoiesis in Drosophila melanogaster.

    PubMed

    Milton, Claire C; Grusche, Felix A; Degoutin, Joffrey L; Yu, Eefang; Dai, Qi; Lai, Eric C; Harvey, Kieran F

    2014-11-17

    The Salvador-Warts-Hippo (Hippo) pathway is an evolutionarily conserved regulator of organ growth and cell fate. It performs these functions in epithelial and neural tissues of both insects and mammals, as well as in mammalian organs such as the liver and heart. Despite rapid advances in Hippo pathway research, a definitive role for this pathway in hematopoiesis has remained enigmatic. The hematopoietic compartments of Drosophila melanogaster and mammals possess several conserved features. D. melanogaster possess three types of hematopoietic cells that most closely resemble mammalian myeloid cells: plasmatocytes (macrophage-like cells), crystal cells (involved in wound healing), and lamellocytes (which encapsulate parasites). The proteins that control differentiation of these cells also control important blood lineage decisions in mammals. Here, we define the Hippo pathway as a key mediator of hematopoiesis by showing that it controls differentiation and proliferation of the two major types of D. melanogaster blood cells, plasmatocytes and crystal cells. In animals lacking the downstream Hippo pathway kinase Warts, lymph gland cells overproliferated, differentiated prematurely, and often adopted a mixed lineage fate. The Hippo pathway regulated crystal cell numbers by both cell-autonomous and non-cell-autonomous mechanisms. Yorkie and its partner transcription factor Scalloped were found to regulate transcription of the Runx family transcription factor Lozenge, which is a key regulator of crystal cell fate. Further, Yorkie or Scalloped hyperactivation induced ectopic crystal cells in a non-cell-autonomous and Notch-pathway-dependent fashion.

  19. Functional dissection of phosphorylation of Dishevelled in Drosophila

    PubMed Central

    Yanfeng, Wang A.; Berhane, Hebist; Mola, Marion; Singh, Jaskirat; Jenny, Andreas; Mlodzik, Marek

    2011-01-01

    Dishevelled/Dsh proteins (Dvl in mammals) are core components of both Wnt/Wg-signaling pathways: canonical β-catenin signaling and Frizzled (Fz)-planar cell polarity (PCP) signaling. Although Dsh is a key cytoplasmic component of both Wnt/Fz-pathways, regulation of its signaling specificity is not well understood. Dsh is phosphorylated, but the functional significance of its phosphorylation remains unclear. We have systematically investigated the phosphorylation of Dsh by combining mass-spectrometry analyses, biochemical studies, and in vivo genetic methods in Drosophila. Our approaches identified multiple phospho-residues of Dsh in vivo. Our data define three novel and unexpected conclusions: (1) Strikingly and in contrast to common assumptions, all conserved serines/threonines are non-essential for Dsh function in either pathway; (2) phosphorylation of conserved Tyrosine473 in the DEP domain is critical for PCP-signaling - DshY473F behaves like a PCP-specific allele; and (3) defects associated with the PCP specific dsh1 allele, DshK417M, located within a putative Protein Kinase C consensus site, are likely due to a post-translational modification requirement of Lys417, rather than phosphorylation nearby. In summary, our combined data indicate that while many Ser/Thr and Tyr residues are indeed phosphorylated in vivo, strikingly most of these phosphorylation events are not critical for Dsh function with the exception of DshY473. PMID:21963539

  20. 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

  1. Asymmetric stem cell division: lessons from Drosophila.

    PubMed

    Wu, Pao-Shu; Egger, Boris; Brand, Andrea H

    2008-06-01

    Asymmetric cell division is an important and conserved strategy in the generation of cellular diversity during animal development. Many of our insights into the underlying mechanisms of asymmetric cell division have been gained from Drosophila, including the establishment of polarity, orientation of mitotic spindles and segregation of cell fate determinants. Recent studies are also beginning to reveal the connection between the misregulation of asymmetric cell division and cancer. What we are learning from Drosophila as a model system has implication both for stem cell biology and also cancer research.

  2. Monoclonal Antibodies against the Drosophila Nervous System

    NASA Astrophysics Data System (ADS)

    Fujita, Shinobu C.; Zipursky, Stephen L.; Benzer, Seymour; Ferrus, Alberto; Shotwell, Sandra L.

    1982-12-01

    A panel of 148 monoclonal antibodies directed against Drosophila neural antigens has been prepared by using mice immunized with homogenates of Drosophila tissue. Antibodies were screened immunohistochemically on cryostat sections of fly heads. A large diversity of staining patterns was observed. Some antigens were broadly distributed among tissues; others were highly specific to nerve fibers, neuropil, muscle, the tracheal system, cell nuclei, photoreceptors, or other structures. The antigens for many of the antibodies have been identified on immunoblots. Monoclonal antibodies that identify specific molecules within the nervous system should prove useful in the study of the molecular genetics of neural development.

  3. Genetics and neurobiology of aggression in Drosophila

    PubMed Central

    Zwarts, Liesbeth; Versteven, Marijke; Callaerts, Patrick

    2012-01-01

    Aggressive behavior is widely present throughout the animal kingdom and is crucial to ensure survival and reproduction. Aggressive actions serve to acquire territory, food, or mates and in defense against predators or rivals; while in some species these behaviors are involved in establishing a social hierarchy. Aggression is a complex behavior, influenced by a broad range of genetic and environmental factors. Recent studies in Drosophila provide insight into the genetic basis and control of aggression. The state of the art on aggression in Drosophila and the many opportunities provided by this model organism to unravel the genetic and neurobiological basis of aggression are reviewed. PMID:22513455

  4. LIM-kinase1.

    PubMed

    Stanyon, C A; Bernard, O

    1999-01-01

    LIM-kinase1 (LIMK1) is a serine-only protein kinase that contains LIM and PDZ protein-protein interaction domains which is highly expressed in neurons. Overexpression of LIMK1 in cultured cells results in accumulation of filamentous (F-) actin. LIMK1 phosphorylates cofilin, an actin depolymerisation factor, which is then unable to bind and depolymerise F-actin. Rac-GTP enhances phosphorylation of LIMK1 and cofilin, which leads to accumulation of F-actin, while Rac-GDP and PMA reduce these effects. LIMK1 is therefore a key component of a signal transduction network that connects extracellular stimuli to changes in cytoskeletal structure. Control of cell morphology and mobility via LIMK1 activity may provide novel approaches to cancer therapy.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  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; 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.

  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; 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.

  13. 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.

  14. 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.

  15. 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

  16. Tricornered Kinase Regulates Synapse Development by Regulating the Levels of Wiskott-Aldrich Syndrome Protein.

    PubMed

    Natarajan, Rajalaxmi; Barber, Kara; Buckley, Amanda; Cho, Phillip; Egbejimi, Anuoluwapo; Wairkar, Yogesh P

    2015-01-01

    Precise regulation of synapses during development is essential to ensure accurate neural connectivity and function of nervous system. Many signaling pathways, including the mTOR (mechanical Target of Rapamycin) pathway operate in neurons to maintain genetically determined number of synapses during development. mTOR, a kinase, is shared between two functionally distinct multi-protein complexes- mTORC1 and mTORC2, that act downstream of Tuberous Sclerosis Complex (TSC). We and others have suggested an important role for TSC in synapse development at the Drosophila neuromuscular junction (NMJ) synapses. In addition, our data suggested that the regulation of the NMJ synapse numbers in Drosophila largely depends on signaling via mTORC2. In the present study, we further this observation by identifying Tricornered (Trc) kinase, a serine/threonine kinase as a likely mediator of TSC signaling. trc genetically interacts with Tsc2 to regulate the number of synapses. In addition, Tsc2 and trc mutants exhibit a dramatic reduction in synaptic levels of WASP, an important regulator of actin polymerization. We show that Trc regulates the WASP levels largely, by regulating the transcription of WASP. Finally, we show that overexpression of WASP (Wiskott-Aldrich Syndrome Protein) in trc mutants can suppress the increase in the number of synapses observed in trc mutants, suggesting that WASP regulates synapses downstream of Trc. Thus, our data provide a novel insight into how Trc may regulate the genetic program that controls the number of synapses during development.

  17. Automated measurement of Drosophila wings.

    PubMed

    Houle, David; Mezey, Jason; Galpern, Paul; Carter, Ashley

    2003-12-11

    Many studies in evolutionary biology and genetics are limited by the rate at which phenotypic information can be acquired. The wings of Drosophila species are a favorable target for automated analysis because of the many interesting questions in evolution and development that can be addressed with them, and because of their simple structure. We have developed an automated image analysis system (WINGMACHINE) that measures the positions of all the veins and the edges of the wing blade of Drosophilid flies. A video image is obtained with the aid of a simple suction device that immobilizes the wing of a live fly. Low-level processing is used to find the major intersections of the veins. High-level processing then optimizes the fit of an a priori B-spline model of wing shape. WINGMACHINE allows the measurement of 1 wing per minute, including handling, imaging, analysis, and data editing. The repeatabilities of 12 vein intersections averaged 86% in a sample of flies of the same species and sex. Comparison of 2400 wings of 25 Drosophilid species shows that wing shape is quite conservative within the group, but that almost all taxa are diagnosably different from one another. Wing shape retains some phylogenetic structure, although some species have shapes very different from closely related species. The WINGMACHINE system facilitates artificial selection experiments on complex aspects of wing shape. We selected on an index which is a function of 14 separate measurements of each wing. After 14 generations, we achieved a 15 S.D. difference between up and down-selected treatments. WINGMACHINE enables rapid, highly repeatable measurements of wings in the family Drosophilidae. Our approach to image analysis may be applicable to a variety of biological objects that can be represented as a framework of connected lines.

  18. Antigenotoxicity studies in Drosophila melanogaster.

    PubMed

    Graf, U; Abraham, S K; Guzmán-Rincón, J; Würgler, F E

    1998-06-18

    The fruit fly Drosophila melangaster with its well developed array of genotoxicity test systems has been used in a number of studies on antigenotoxicity of various compounds and mixtures. In recent years, the newly developed Somatic Mutation and Recombination Tests (SMART) have mainly been employed. These one-generation tests make use of the wing or eye imaginal disc cells in larvae and have proven to be very efficient and sensitive. They are based on the principle that the loss of heterozygosity of suitable recessive markers can lead to the formation of mutant clones of cells that are then expressed as spots on the wings or eyes of the adult flies. We have employed the wing spot test with the two markers multiple wing hairs (mwh,3-0.3) and flare (flr,3-38.8). Three-day-old larvae, trans-heterozygous for these markers, are treated chronically or acutely by oral administration with the test compound(s) or complex mixtures. For antigenotoxicity studies, chronic co-treatments can be used, as well as separate pre-treatments with an antigenotoxic agent followed by a chronic treatment with a genotoxin. After eclosion, the wings of the adult flies are scored for the presence of single and twin spots. These spots can be due to different genotoxic events: either mitotic recombination or mutation (deletion, point mutation, specific types of translocation, etc.). The analysis of two different genotypes (one with structurally normal chromosomes, one with a multiply inverted balancer chromosome) allows for a quantitative determination of the recombinagenic activity of genotoxins. Results of two separate studies presented: (1) instant coffee has antirecombinagenic but not antimutagenic activity in the wing spot test; and (2) ascorbic acid and catechin are able to protect against in vivo nitrosation products of methyl urea in combination with sodium nitrite.

  19. Symmetry Breaking During Drosophila Oogenesis

    PubMed Central

    Roth, Siegfried; Lynch, Jeremy A.

    2009-01-01

    The orthogonal axes of Drosophila are established during oogenesis through a hierarchical series of symmetry-breaking steps, most of which can be traced back to asymmetries inherent in the architecture of the ovary. Oogenesis begins with the formation of a germline cyst of 16 cells connected by ring canals. Two of these 16 cells have four ring canals, whereas the others have fewer. The first symmetry-breaking step is the selection of one of these two cells to become the oocyte. Subsequently, the germline cyst becomes surrounded by somatic follicle cells to generate individual egg chambers. The second symmetry-breaking step is the posterior positioning of the oocyte within the egg chamber, a process mediated by adhesive interactions with a special group of somatic cells. Posterior oocyte positioning is accompanied by a par gene-dependent repolarization of the microtubule network, which establishes the posterior cortex of the oocyte. The next two steps of symmetry breaking occur during midoogenesis after the volume of the oocyte has increased about 10-fold. First, a signal from the oocyte specifies posterior follicle cells, polarizing a symmetric prepattern present within the follicular epithelium. Second, the posterior follicle cells send a signal back to the oocyte, which leads to a second repolarization of the oocyte microtubule network and the asymmetric migration of the oocyte nucleus. This process again requires the par genes. The repolarization of the microtubule network results in the transport of bicoid and oskar mRNAs, the anterior and posterior determinants, respectively, of the embryonic axis, to opposite poles of the oocyte. The asymmetric positioning of the oocyte nucleus defines a cortical region of the oocyte where gurken mRNA is localized, thus breaking the dorsal–ventral symmetry of the egg and embryo. PMID:20066085

  20. Signaling by Drosophila capa neuropeptides.

    PubMed

    Davies, Shireen-A; Cabrero, Pablo; Povsic, Manca; Johnston, Natalie R; Terhzaz, Selim; Dow, Julian A T

    2013-07-01

    The capa peptide family, originally identified in the tobacco hawk moth, Manduca sexta, is now known to be present in many insect families, with increasing publications on capa neuropeptides each year. The physiological actions of capa peptides vary depending on the insect species but capa peptides have key myomodulatory and osmoregulatory functions, depending on insect lifestyle, and life stage. Capa peptide signaling is thus critical for fluid homeostasis and survival, making study of this neuropeptide family attractive for novel routes for insect control. In Dipteran species, including the genetically tractable Drosophila melanogaster, capa peptide action is diuretic; via elevation of nitric oxide, cGMP and calcium in the principal cells of the Malpighian tubules. The identification of the capa receptor (capaR) in several insect species has shown this to be a canonical GPCR. In D. melanogaster, ligand-activated capaR activity occurs in a dose-dependent manner between 10(-6) and 10(-12)M. Lower concentrations of capa peptide do not activate capaR, either in adult or larval Malpighian tubules. Use of transgenic flies in which capaR is knocked-down in only Malpighian tubule principal cells demonstrates that capaR modulates tubule fluid secretion rates and in doing so, sets the organismal response to desiccation. Thus, capa regulates a desiccation-responsive pathway in D. melanogaster, linking its role in osmoregulation and fluid homeostasis to environmental response and survival. The conservation of capa action between some Dipteran species suggests that capa's role in desiccation tolerance may not be confined to D. melanogaster.

  1. 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

  2. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    PubMed Central

    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.

    2016-01-01

    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

  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. 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…

  6. 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)

  7. 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…

  8. 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

  9. Organization of descending neurons in Drosophila melanogaster.

    PubMed

    Hsu, Cynthia T; Bhandawat, Vikas

    2016-02-03

    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.

  10. Isolation of Drosophila egg chambers for imaging.

    PubMed

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

    2010-04-01

    The fruit fly Drosophila melanogaster is an important model for basic research into the molecular mechanisms underlying cell function and development, as well as a major biomedical research tool. A significant advantage of Drosophila is the ability to apply live cell imaging to a variety of living tissues that can be dissected and imaged in vivo, ex vivo, or in vitro. Drosophila egg chambers, for example, have proven to be a useful model system for studying border cell migration, Golgi unit transport, the rapid movement of mRNA and protein particles, and the role of microtubules in meiosis and oocyte differentiation. A crucial first step before imaging is preparation of the experimental material to ensure physiological relevance and to achieve the best conditions for image quality. Early- to mid-stage egg chambers cannot be mounted in an aqueous-based medium, because this causes a change in microtubule organization and follicle cell morphology. Such egg chambers survive better in Halocarbon oil, which allows free diffusion of oxygen, has low viscosity, and thus prevents dehydration and hypoxia. With a refractive index similar to glycerol, Halocarbon oil also has good optical properties for imaging. It also provides a good environment for injection and is particularly useful for long-term imaging of embryos. However, unlike with aqueous solutions, changes in the medium are not possible. This protocol describes the isolation of Drosophila egg chambers.

  11. Mechanisms of nondisjunction induction in drosophila oocytes.

    PubMed

    Leigh, B

    1979-08-01

    Quantitative and qualitative studies on the induction of no-disjunction and related phenomena can be carried out using the germ cells of Drosophila. X-Irradiation breaks chromosomes and cold-shock disrupts spindles, these two treatments producing different spectra of nondisjunction in oocytes.

  12. 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.

  13. 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…

  14. 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)

  15. 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…

  16. 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…

  17. The taste response to ammonia in Drosophila

    PubMed Central

    Delventhal, R.; Menuz, K.; Joseph, R.; Park, J.; Sun, J. S.; Carlson, J. R.

    2017-01-01

    Ammonia is both a building block and a breakdown product of amino acids and is found widely in the environment. The odor of ammonia is attractive to many insects, including insect vectors of disease. The olfactory response of Drosophila to ammonia has been studied in some detail, but the taste response has received remarkably little attention. Here, we show that ammonia is a taste cue for Drosophila. Nearly all sensilla of the major taste organ of the Drosophila head house a neuron that responds to neutral solutions of ammonia. Ammonia is toxic at high levels to many organisms, and we find that it has a negative valence in two paradigms of taste behavior, one operating over hours and the other over seconds. Physiological and behavioral responses to ammonia depend at least in part on Gr66a+ bitter-sensing taste neurons, which activate a circuit that deters feeding. The Amt transporter, a critical component of olfactory responses to ammonia, is widely expressed in taste neurons but is not required for taste responses. This work establishes ammonia as an ecologically important taste cue in Drosophila, and shows that it can activate circuits that promote opposite behavioral outcomes via different sensory systems. PMID:28262698

  18. 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

  19. A high-throughput radiometric kinase assay

    PubMed Central

    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 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

  20. Aurora A kinase amplifies a midzone phosphorylation gradient to promote high fidelity cytokinesis

    PubMed Central

    Ye, Anna A.; Torabi, Julia; Maresca, Thomas J.

    2017-01-01

    Aurora B kinase (ABK) re-localizes from centromeres to the spindle midzone during cytokinesis where it is thought to provide a spatial cue for cytokinesis. While global ABK inhibition in Drosophila S2 cells results in macro- and multi-nucleated large cells, mis-localization of midzone ABK (mABK) by depletion of Subito (Drosophila MKLP2) does not cause notable cytokinesis defects. Subito depletion was; therefore, used to investigate the contribution of other molecules and redundant pathways to cytokinesis in the absence of mABK. Inhibiting potential polar relaxation pathways via removal of centrosomes (CNN RNAi) or a kinetochore-based phosphatase-gradient (Sds22 RNAi) did not result in cytokinesis defects on their own or in combination with loss of mABK. Disruption of Aurora A kinase (AAK) activity resulted in midzone assembly defects but did not significantly affect contractile ring positioning or cytokinesis. Live-cell imaging of a FRET-based aurora kinase phosphorylation sensor revealed that midzone substrates were less phosphorylated in AAK-inhibited cells, despite the fact that midzone levels of active phosphorylated ABK (pABK) were normal. Interestingly, an increased number of binucleated cells were observed following AAK inhibition in the absence of mABK. The data suggest that equatorial stimulation rather than polar relaxation mechanisms are the major determinants of contractile ring positioning and high-fidelity cytokinesis in Drosophila S2 cells. Furthermore, we propose that equatorial stimulation is mediated primarily by the delivery of factors to the cortex by non-centrosomal microtubules (MTs) as well as a midzone-derived phosphorylation gradient that is amplified by the concerted activities of mABK and a soluble pool of AAK. PMID:27638695