Drosophila in vision research. The Friedenwald Lecture.

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.

A genetic approach involving the use of ERG-defective Drosophila mutants, described in this article, was developed with the explicit goal of elucidating phototransduction and related photoreceptor events at the molecular level. Advances in the past 30 years have made this goal a reality. Although the phototransduction process in Drosophila is not yet fully understood, enormous strides are being made by many investigators now working in this field. In addition to elucidating phototransduction vents in invertebrate photoreceptors, works of these investigators are providing insights into the functional machinery of signaling systems in general. For example, mobilization of Ca2+ and replenishment of intracellular Ca2+ stores after their depletion is a current topic of intense interest (see, for example, references 93, 136 and 137). Study of trp mutants is providing insights into this process. Similarly, the mechanisms of inactivation of G-protein coupled receptors appear to be highly conserved in diverse transduction systems, and inactivation of Drosophila metarhodopsin is proving a valuable model system for this study. The identification of the NinaA protein and its role as chaperon and/or foldase for opsin would not have been possible without the genetic approach described in this article. In fact, study of the NinaA gene is providing fresh insights not only into the rhodopsin maturation process but also into the role of cyclophilins in general. Moreover, recent results suggest that related retina-specific proteins are also present in mammals. As valuable as the NinaA protein has been, it may be only the first of a number of such novel proteins or novel isozymes of known proteins required in the retina to be discovered through this approach. For two of the Drosophila proteins discussed in this article, NorpA and NinaA, related mammalian proteins expressed in photoreceptors have been identified, even though it seemed unlikely that such proteins would have any role in mammalian photoreceptors. A recent report suggests that the Trp protein has human homologs expressed most heavily in the brain (C. Montell, cited in reference 93). It may well be that almost any protein identified in the Drosophila retina has its counterpart(s) in mammals. Discovery of these mammalian homologs of Drosophila proteins will probably spur new lines of investigation in mammalian photoreceptor function. For example, because no role had been assigned to PLC in cone phototransduction, the discovery that NorpA-homologous PLCs are present in cone outer segments raises questions about what role(s) these PLCs might play in signal transduction in cone outer segments.

PMID: 7591624 [PubMed - indexed for MEDLINE]

Purification and partial amino acid sequences of phosphoinositide-specific phospholipase C of Drosophila eye.

Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.

To examine whether the norpA (no receptor potential A) gene encodes a phosphoinositide-specific phospholipase C (PLC) in the eye of Drosophila, a major PLC in the extract from normal Drosophila heads, which was absent in the extract from norpA mutant heads, and purified and its partial amino acid sequences were determined. The purification of the major PLC in KCl extract from normal Drosophila heads was achieved by sequential column chromatography on DEAE-Sepharose CL-6B, Mono Q, Superose 12, Mono S, second Mono S, and second Mono Q, followed by column chromatography on Superose 12 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 98,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme hydrolyzed both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). Interestingly, the calcium and pH requirements for activation of the crude enzyme (KCl extract) were quite different from those of partially purified enzyme (active fraction from second Mono Q column). The maximal activity for PIP2 hydrolysis was observed at calcium concentrations between 10(-7) and 10(-5) M for both the crude and partially purified enzymes. On the other hand, the activity for PI hydrolysis of the crude enzyme increased with increasing calcium concentrations, while that of the partially purified enzyme reached a maximum at calcium concentrations between 10(-6) and 10(-4) M, and decreased at millimollar concentration. The pH dependences for PI hydrolysis of the crude enzyme and the partially purified enzyme were similar. The crude enzyme hydrolyzed PIP2 over a broad pH range from 6 to 8.5, while the activity of the partially purified enzyme monotonously increased with increasing pH. The partial amino acid sequences were determined by treating the purified enzyme with endopeptidase Lys-C; the resultant peptide fragments were purified on a high performance liquid chromatography-reverse phase column and then sequenced with sequencer. The obtained sequences were found to be a part of the deduced amino acid sequences of cDNA which was suggested to be norpA gene.

PMID: 2168393 [PubMed - indexed for MEDLINE]

Multiple subtypes of phospholipase C are encoded by the norpA gene of Drosophila melanogaster.

Department of Biological Sciences, State University of New York, Buffalo, New York 14260, USA.

The norpA gene of Drosophila melanogaster encodes a phosphatidylinositol-specific phospholipase C that is essential for phototransduction. Besides being found abundantly in retina, norpA gene products are expressed in a variety of tissues that do not contain phototransduction machinery, implying that norpA is involved in signaling pathways in addition to phototransduction. We have identified a second subtype of norpA protein that is generated by alternative splicing of norpA RNA. The alternative splicing occurs at a single exon that is excluded from mature norpA transcripts when a substitute exon of equal size is retained. The net difference between the two subtypes of norpA protein is 14 amino acid substitutions occurring between amino acid positions 130 and 155 of the enzyme. Results from Northern analyses suggest that norpA subtype I transcripts are most abundantly expressed in adult retina, while subtype II transcripts are most abundant in adult body. Moreover, norpA subtype I RNA can be detected by the reverse transcription-polymerase chain reaction in extracts of adult head tissue but not adult body nor at earlier stages of Drosophila development. Conversely, norpA subtype II RNA can be detected by reverse transcription-polymerase chain reaction throughout development as well as in heads and bodies of adults. Furthermore, norpA subtype I RNA is easily detected in retina using tissue in situ hybridization analysis, while subtype II RNA is not detectable in retina but is found in brain. Since only norpA subtype I RNA is found in retina, we conclude that subtype I protein is utilized in phototransduction. Since norpA subtype II RNA is not found in retina but is expressed in a variety of tissues not known to contain phototransduction machinery, subtype II protein is likely to be utilized in signaling pathways other than phototransduction. The amino acid differences between the two subtypes of norpA protein may reflect the need for each subtype to interact with signaling components of different signal-generating pathways.

PMID: 7540168 [PubMed - indexed for MEDLINE]

Molecular, biochemical, and electrophysiological characterization of Drosophila norpA mutants.

Department of Biological Science, Purdue University, West Lafayette, Indiana 47907, USA.

Inositol phosphate signaling has been implicated in a wide variety of eukaryotic cellular processes. In Drosophila, the phototransduction cascade is mediated by a phosphoinositide-specific phospholipase C (PLC) encoded by the norpA gene. We have characterized eight norpA mutants by electroretinogram (ERG), Western, molecular, and in vitro PLC activity analyses. ERG responses of the mutants show allele-dependent reductions in amplitudes and retardation in kinetics. The mutants also exhibit allele-dependent reductions in in vitro PLC activity levels and greatly reduced or undetectable NorpA protein levels. Three carry a missense mutation and five carry a nonsense mutation within the norpA coding sequence. In missense mutants, the amino acid substitution occurs at residues highly conserved among PLCs. These substitutions reduce the levels of both the NorpA protein and the PLC activity, with the reduction in PLC activity being greater than can be accounted for simply by the reduction in protein. The effects of the mutations on the amount and activity of the protein are much greater than their effects on the ERG, suggesting an amplification of the transduction signal at the effector (NorpA) protein level. Transgenic flies were generated by germline transformation of a null norpA mutant using a P-element construct containing the wild-type norpA cDNA driven by the ninaE promoter. Transformed flies show rescue of the electrophysiological phenotype in R1-R6 photoreceptors, but not in R7 or R8. The degeneration phenotype of R1-R6 photoreceptors is also rescued.

PMID: 8617767 [PubMed - indexed for MEDLINE]

Drosophila phospholipase C-gamma expressed predominantly in blastoderm cells at cellularization and in endodermal cells during later embryonic stages.

Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.

A Drosophila gene encoding a gamma-type isozyme of phosphoinositide-specific phospholipase C (PLC) was isolated and characterized. The gene, termed plc-gamma d, was mapped at position 14B-C of the X chromosome. The encoded protein, termed PLC-gamma D, contains X and Y regions, common to all known PLC isozymes. The two regions are split by a Z region that comprises two src homology 2 and one src homology 3 domains and is characteristic of gamma-type mammalian PLC (PLC-gamma 1 and -gamma 2). The deduced amino acid sequence of PLC-gamma D shows overall similarity to mammalian PLC-gamma s; no large deletion was observed except the short C-terminal extended region. In particular, the two split catalytic domains (X and Y regions) and the regulatory Z region including the src homology 2 and src homology 3 domains are well conserved. The mRNA is expressed throughout development, but expression is relatively higher during the embryonic stage, suggesting fundamental and important roles in both cell proliferation and differentiation. Distribution of the mRNA during embryogenesis, as analyzed by whole amount in situ hybridization, revealed that the mRNA emerges and reaches maximum levels at the cellular blastoderm stage and then decreases rapidly to a lower level. In later embryonic stages, invaginated anterior and posterior midgut primordia show high levels of mRNA expression, and fused midgut also maintains a high level of expression. In other tissues and cells, the mRNA was detected at lower levels. These results indicate that Drosophila PLC-gamma may be involved in universal cellular processes mediated possibly by receptor tyrosine kinases during embryogenesis and may also play specific roles during cellularization and midgut differentiation.

PMID: 8034716 [PubMed - indexed for MEDLINE]

NorpA and itpr mutants reveal roles for phospholipase C and inositol (1,4,5)- trisphosphate receptor in Drosophila melanogaster renal function.

Institute of Biomedical and Life Sciences, Division of Molecular Genetics, University of Glasgow, Glasgow G11 6NU, UK.

Mutants of norpA, encoding phospholipase C beta (PLC beta), and itpr, encoding inositol (1,4,5)-trisphosphate receptor (IP(3)R), both attenuate response to diuretic peptides of Drosophila melanogaster renal (Malpighian) tubules. Intact tubules from norpA mutants severely reduced diuresis stimulated by the principal cell- and stellate cell-specific neuropeptides, CAP(2b) and Drosophila leucokinin (Drosokinin), respectively, suggesting a role for PLC beta in both these cell types. Measurement of IP(3) production in wild-type tubules and in Drosokinin-receptor-transfected S2 cells stimulated with CAP(2b) and Drosokinin, respectively, confirmed that both neuropeptides elevate IP(3) levels. In itpr hypomorphs, basal IP(3) levels are lower, although CAP(2b)-stimulated IP(3) levels are not significantly reduced compared with wild type. However, CAP(2b)-stimulated fluid transport is significantly reduced in itpr alleles. Rescue of the itpr(90B.0) allele with wild-type itpr restores CAP(2b)-stimulated fluid transport levels to wild type. Drosokinin-stimulated fluid transport is also reduced in homozygous and heteroallelic itpr mutants. Measurements of cytosolic calcium levels in intact tubules of wild-type and itpr mutants using targeted expression of the calcium reporter, aequorin, show that mutations in itpr attenuated both CAP(2b)- and Drosokinin-stimulated calcium responses. The reductions in calcium signals are associated with corresponding reductions in fluid transport rates. Thus, we describe a role for norpA and itpr in renal epithelia and show that both CAP(2b) and Drosokinin are PLC beta-dependent, IP(3)-mobilising neuropeptides in DROSOPHILA: IP(3)R contributes to the calcium signalling cascades initiated by these peptides in both principal and stellate cells.

PMID: 12547945 [PubMed - indexed for MEDLINE]

Larval optic nerve and adult extra-retinal photoreceptors sequentially associate with clock neurons during Drosophila brain development.

Institut de Neurobiologie Alfred Fessard, CNRS UPR 2216 (NGI), 91198 Gif-sur-Yvette, France.

The visual system is one of the input pathways for light into the circadian clock of the Drosophila brain. In particular, extra-retinal visual structures have been proposed to play a role in both larval and adult circadian photoreception. We have analyzed the interactions between extra-retinal structures of the visual system and the clock neurons during brain development. We first show that the larval optic nerve, or Bolwig nerve, already contacts clock cells (the lateral neurons) in the embryonic brain. Analysis of visual system-defective genotypes showed that the absence of the afferent Bolwig nerve resulted in a severe reduction of the lateral neurons dendritic arborization, and that the inhibition of nerve activity induced alterations of the dendritic morphology. During wild-type development, the loss of a functional Bolwig nerve in the early pupa was also accompanied by remodeling of the arborization of the lateral neurons. Approximately 1.5 days later, visual fibers that came from the Hofbauer-Buchner eyelet, a putative photoreceptive organ for the adult circadian clock, were seen contacting the lateral neurons. Both types of extra-retinal photoreceptors expressed rhodopsins RH5 and RH6, as well as the norpA-encoded phospholipase C. These data strongly suggest a role for RH5 and RH6, as well as NORPA, signaling in both larval and adult extra-retinal circadian photoreception. The Hofbauer-Buchner eyelet therefore does not appear to account for the previously described norpA-independent light input to the adult clock. This supports the existence of yet uncharacterized photoreceptive structures in Drosophila.

PMID: 11880353 [PubMed - indexed for MEDLINE]

Novel Gq alpha isoform is a candidate transducer of rhodopsin signaling in a Drosophila testes-autonomous pacemaker.

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.

DGq is the alpha subunit of the heterotrimeric GTPase (G alpha), which couples rhodopsin to phospholipase C in Drosophila vision. We have uncovered three duplicated exons in dgq by scanning the GenBank data base for unrecognized coding sequences. These alternative exons encode sites involved in GTPase activity and G beta-binding, NorpA (phospholipase C)-binding, and rhodopsin-binding. We examined the in vivo splicing of dgq in adult flies and find that, in all but the male gonads, only two isoforms are expressed. One, dgqA, is the original visual isoform and is expressed in eyes, ocelli, brain, and male gonads. The other, dgqB, has the three novel exons and is widely expressed. Remarkably, all three nonvisual B exons are highly similar (82% identity at the amino acid level) to the Gq alpha family consensus, from Caenorhabditis elegans to human, but all three visual A exons are divergent (61% identity). Intriguingly, we have found a third isoform, dgqC, which is specifically and abundantly expressed in male gonads, and shares the divergent rhodopsin-binding exon of dgqA. We suggest that DGqC is a candidate for the light-signal transducer of a testes-autonomous photosensory clock. This proposal is supported by the finding that rhodopsin 2 and arrestin 1, two photoreceptor-cell-specific genes, are also expressed in male gonads.

PMID: 8901571 [PubMed - indexed for MEDLINE]

PMCID: PMC37981

The transient receptor potential protein (Trp), a putative store-operated Ca2+ channel essential for phosphoinositide-mediated photoreception, forms a signaling complex with NorpA, InaC and InaD.

Zoological Institute I, University of Karlsruhe, Germany.

The transient receptor potential protein (Trp) is a putative capacitative Ca2+ entry channel present in fly photoreceptors, which use the inositol 1,4,5-trisphosphate (InsP3) signaling pathway for phototransduction. By immunoprecipitation studies, we find that Trp is associated into a multiprotein complex with the norpA-encoded phospholipase C, an eye-specific protein kinase C (InaC) and with the InaD protein (InaD). InaD is a putative substrate of InaC and contains two PDZ repeats, putative protein-protein interaction domains. These proteins are present in the photoreceptor membrane at about equimolar ratios. The Trp homolog analyzed here is isolated together with NorpA, InaC and InaD from blowfly (Calliphora) photoreceptors. Compared to Drosophila Trp, the Calliphora Trp homolog displays 77% amino acid identity. The highest sequence conservation is found in the region that contains the putative transmembrane domains S1-S6 (91% amino acid identity). As investigated by immunogold labeling with specific antibodies directed against Trp and InaD, the Trp signaling complex is located in the microvillar membranes of the photoreceptor cells. The spatial distribution of the signaling complex argues against a direct conformational coupling of Trp to an InsP3 receptor supposed to be present in the membrane of internal photoreceptor Ca2+ stores. It is suggested that the organization of signal transducing proteins into a multiprotein complex provides the structural basis for an efficient and fast activation and regulation of Ca2+ entry through the Trp channel.

PMID: 9003779 [PubMed - indexed for MEDLINE]

PMCID: PMC452529

TRP and the PDZ protein, INAD, form the core complex required for retention of the signalplex in Drosophila photoreceptor cells.

Department of Biological Chemistry and Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

The light response in Drosophila photoreceptor cells is mediated by a series of proteins that assemble into a macromolecular complex referred to as the signalplex. The central player in the signalplex is inactivation no afterpotential D (INAD), a protein consisting of a tandem array of five PDZ domains. At least seven proteins bind INAD, including the transient receptor potential (TRP) channel, which depends on INAD for localization to the phototransducing organelle, the rhabdomere. However, the determinants required for localization of INAD are not known. In this work, we showed that INAD was required for retention rather than targeting of TRP to the rhabdomeres. In addition, we demonstrated that TRP bound to INAD through the COOH terminus, and this interaction was required for localization of INAD. Other proteins that depend on INAD for localization, phospholipase C and protein kinase C, also mislocalized. However, elimination of any other member of the signalplex had no impact on the spatial distribution of INAD. A direct interaction between TRP and INAD did not appear to have a role in the photoresponse independent of localization of multiple signaling components. Rather, the primary function of the TRP/ INAD complex is to form the core unit required for localization of the signalplex to the rhabdomeres.

PMID: 10995445 [PubMed - indexed for MEDLINE]

PMCID: PMC2150714

Two distantly positioned PDZ domains mediate multivalent INAD-phospholipase C interactions essential for G protein-coupled signaling.

Departments of Physiology and Neuroscience, The Johns Hopkins University School of Medicine, 725 N.Wolfe Street, WBSB 216, Baltimore, MD 21205, USA.

Drosophila INAD, which contains five tandem protein interaction PDZ domains, plays an important role in the G protein-coupled visual signal transduction. Mutations in InaD alleles display mislocalization of signaling molecules of phototransduction which include the essential effector, phospholipase C-beta (PLC-beta), which is also known as NORPA. The molecular and biochemical details of this functional link are unknown. We report that INAD directly binds to NORPA via two terminally positioned PDZ1 and PDZ5 domains. PDZ1 binds to the C-terminus of NORPA, while PDZ5 binds to an internal region overlapping with the G box-homology region (a putative G protein-interacting site). The NORPA proteins lacking binding sites, which display normal basal PLC activity, can no longer associate with INAD in vivo. These truncations cause significant reduction of NORPA protein expression in rhabdomeres and severe defects in phototransduction. Thus, the two terminal PDZ domains of INAD, through intermolecular and/or intramolecular interactions, are brought into proximity in vivo. Such domain organization allows for the multivalent INAD-NORPA interactions which are essential for G protein-coupled phototransduction.

PMID: 9545241 [PubMed - indexed for MEDLINE]

PMCID: PMC1170572

Tissue-specific expression of phospholipase C encoded by the norpA gene of Drosophila melanogaster.

Department of Biological Sciences, State University of New York, Buffalo 14260.

Mutations in the norpA gene of Drosophila melanogaster severely affect the light-evoked photoreceptor potential with strong mutations rendering the fly blind. Molecular cloning of the norpA gene revealed that it encodes phosphatidylinositol-specific phospholipase C, which enzymes play a pivotal role in one of the largest classes of signaling pathways known. We have used Northern analysis, Western blots, phospholipase C activity assays, and immunohistochemical staining of tissues to examine the tissue-specific expression of the norpA gene and found that it is expressed in a variety of tissues besides the eye. Hybridization of norpA cRNA probe to blots of poly(A+) RNA reveals that the gene encodes at least four transcripts: a 7.5-kilobase (kb) transcript that is expressed in eye and 6.5-, 5.5-, and 5.0-kb transcripts that are expressed in adult body or early stages of development. Antiserum generated against the major gene product of norpA recognizes a 130-kDa protein that is abundant in eyes but severely reduced or absent in norpA mutants, a result which is consistent with previous conclusions that the norpA gene encodes an essential component of the visual system. However, the norpA antiserum also recognizes a 130-kDa protein in adult legs, thorax, and male abdomen, but not female abdomen. These localizations are supported by results of phospholipase C activity assays which show that the norpA mutation reduces phospholipase C activity in each of the tissues in which norpA protein can be detected. Furthermore, immunohistochemical staining of tissue sections with the norpA antiserum demonstrates that the norpA protein is abundant in the retina and ocelli and is present to a lesser extent in the brain and thoracic nervous system. Since some of the above mentioned tissues that express norpA (such as thorax, legs, and abdomen) have no known photoreceptor tissue, we conclude that the norpA gene product is also likely to have a role in signaling pathways other than phototransduction.

PMID: 8340420 [PubMed - indexed for MEDLINE]

Association of INAD with NORPA is essential for controlled activation and deactivation of Drosophila phototransduction in vivo.

Department of Pharmacology, Vanderbilt University, Nashville, TN 37232-6600, USA. shiehb@ctrvax.vanderbilt.edu

Visual transduction in Drosophila is a G protein-coupled phospholipase C-mediated process that leads to depolarization via activation of the transient receptor potential (TRP) calcium channel. Inactivation-no-afterpotential D (INAD) is an adaptor protein containing PDZ domains known to interact with TRP. Immunoprecipitation studies indicate that INAD also binds to eye-specific protein kinase C and the phospholipase C, no-receptor-potential A (NORPA). By overlay assay and site-directed mutagenesis we have defined the essential elements of the NORPA-INAD association and identified three critical residues in the C-terminal tail of NORPA that are required for the interaction. These residues, Phe-Cys-Ala, constitute a novel binding motif distinct from the sequences recognized by the PDZ domain in INAD. To evaluate the functional significance of the INAD-NORPA association in vivo, we generated transgenic flies expressing a modified NORPA, NORPAC1094S, that lacks the INAD interaction. The transgenic animals display a unique electroretinogram phenotype characterized by slow activation and prolonged deactivation. Double mutant analysis suggests a possible inaccessibility of eye-specific protein kinase C to NORPAC1094S, undermining the observed defective deactivation, and that delayed activation may similarly result from NORPAC1094S being unable to localize in close proximity to the TRP channel. We conclude that INAD acts as a scaffold protein that facilitates NORPA-TRP interactions required for gating of the TRP channel in photoreceptor cells.

PMID: 9356510 [PubMed - indexed for MEDLINE]

PMCID: PMC25084

Phospholipase C rescues visual defect in norpA mutant of Drosophila melanogaster.

Department of Biological Sciences, State University of New York, Buffalo 14260, USA.

Mutations in the norpA gene of Drosophila melanogaster severely affect the light-evoked photoreceptor potential with strong mutations rendering the fly blind. The norpA gene has been proposed to encode phosphatidylinositol-specific phospholipase C (PLC), which enzymes play a pivotal role in one of the largest classes of signaling pathways known. A chimeric norpA minigene was constructed by placing the norpA cDNA behind an R1-6 photoreceptor cell-specific rhodopsin promoter. This minigene was transferred into norpAP24 mutant by P-element-mediated germline transformation to determine whether it could rescue the phototransduction defect concomitant with restoring PLC activity. Western blots of head homogenates stained with norpA antiserum show that norpA protein is restored in heads of transformed mutants. Moreover, transformants exhibit a large amount of measurable PLC activity in heads, whereas heads of norpAP24 mutant exhibit very little to none. Immunohistochemical staining of tissue sections using norpA antiserum confirm that expression of norpA protein in transformants localizes in the retina, more specifically in rhabdomeres of R1-6 photoreceptor cells, but not R7 or R8 photoreceptor cells. Furthermore, electrophysiological analyses reveal that transformants exhibit a restoration of light-evoked photoreceptor responses in R1-6 photoreceptor cells, but not in R7 or R8 photoreceptor cells. This is the strongest evidence thus far supporting the hypothesis that the norpA gene encodes phospholipase C that is utilized in phototransduction.

PMID: 7768926 [PubMed - indexed for MEDLINE]

Genomic organization of a Drosophila phospholipase C, norpA, and molecular lesions in two temperature-sensitive mutants.

Department of Physics, Faculty of Science, University of Tokyo.

The Drosophila mutant no receptor potential A (norpA) is the phototransduction-defective mutant which lacks phosphatidylinositol-specific phospholipase C (PI-PLC) activity. Recently, norpA cDNA was isolated and its homology to a bovine PI-PLC was demonstrated [Bloomquist, B.T. et al. (1988) Cell 54, 723-733]. On the basis of its cDNA, we determined the genomic organization of the norpA gene and revealed that the norpA gene consists of 13 coding exons spreading over a 15 kb genomic area. Furthermore, we identified the mutational sites of two temperature-sensitive (ts) mutants. The analysis of norpAH52 revealed that the single amino acid change from Ser-551 to Tyr causes the PI-PLC activity to become temperature-sensitive. The other allele, norpAKO50, has two substitutions from Ser-406 to Phe and from Gly-451 to Ser. These three mutations are located in regions highly conserved in other mammalian PI-PLC molecules. This suggests that these regions are important for PI-PLC catalytic activity.

PMID: 1939007 [PubMed - indexed for MEDLINE]

Temperature synchronization of the Drosophila circadian clock.

Institut für Zoologie, Universität Regensburg, 93040 Regensburg, Germany.

BACKGROUND: Circadian clocks are synchronized by both light:dark cycles and by temperature fluctuations. Although it has long been known that temperature cycles can robustly entrain Drosophila locomotor rhythms, nothing is known about the molecular mechanisms involved. RESULTS: We show here that temperature cycles induce synchronized behavioral rhythms and oscillations of the clock proteins PERIOD and TIMELESS in constant light, a situation that normally leads to molecular and behavioral arrhythmicity. We show that expression of the Drosophila clock gene period can be entrained by temperature cycles in cultured body parts and isolated brains. Further, we show that the phospholipase C encoded by the norpA gene contributes to thermal entrainment, suggesting that a receptor-coupled transduction cascade signals temperature changes to the circadian clock. We initiated the further genetic dissection of temperature-entrainment and isolated the novel Drosophila mutation nocte, which is defective in molecular and behavioral entrainment by temperature cycles but synchronizes normally to light:dark cycles. CONCLUSIONS: We conclude that temperature synchronization of the circadian clock is a tissue-autonomous process that is able to override the arrhythmia-inducing effects of constant light. Our data suggest that it involves a cell-autonomous signal-transduction cascade from a thermal receptor to the circadian clock. This process includes the function of phospholipase C and the product specified by the novel mutation nocte.

PMID: 16085487 [PubMed - indexed for MEDLINE]

Comment in:

Neuron. 2008 Jan 10;57(1):1-2.

The SOCS box protein STOPS is required for phototransduction through its effects on phospholipase C.

Department of Biological Chemistry, Center for Sensory Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Phosphoinositide-specific phospholipase C (PLC) isozymes play roles in a diversity of processes including Drosophila phototransduction. In fly photoreceptor cells, the PLCbeta encoded by norpA is critical for activation of TRP channels. Here, we describe a PLCbeta regulator, STOPS, which encodes a SOCS box protein. Mutation of stops resulted in a reduced concentration of NORPA and a defect in stopping signaling following cessation of the light stimulus. NORPA has been proposed to have dual roles as a PLC- and GTPase-activating protein (GAP). We found that the slow termination resulting from expressing low levels of wild-type NORPA was suppressed by addition of normal amounts of an altered NORPA, which had wild-type GAP activity, but no PLC activity. STOPS is the first protein identified that specifically regulates PLCbeta protein concentration. Moreover, this work demonstrates that a PLCbeta derivative that does not promote TRP channel activation, still contributes to signaling in vivo.

PMID: 18184564 [PubMed - indexed for MEDLINE]

PMCID: PMC2253723

Limited role of developmental programmed cell death pathways in Drosophila norpA retinal degeneration.

Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556-0369, USA.

We examined the role of programmed cell death (PCD) pathways in retinal degeneration caused by a mutation in the norpA gene. norpA degeneration shows morphological hallmarks of programmed cell death, specifically cytoplasmic condensation and engulfment of the dying photoreceptor cells by neighboring retinal pigment cells. However, genetic mosaic analysis of adult photoreceptors lacking rpr, hid, and grim show that these PCD inducers are not required for norpA degeneration. We showed previously that ectopic expression of either rpr or hid triggers rapid PCD in adult photoreceptors, and this is completely suppressed by the coexpression of the baculoviral P35 caspase inhibitor. In contrast, expression of P35 does not suppress norpA retinal degeneration, although a small delay in the rate of degeneration is observed in low light-low temperature conditions. P35 does not alter the morphological characteristics of norpA cell death. Overexpression of the Drosophila inhibitor of apoptosis Diap1 or a dominant-negative form of the Dronc caspase, even when coexpressed with P35, does not dramatically alter the time course of norpA degeneration. These results establish that the pathways responsible for PCD in development do not play a major role in adult retinal degeneration caused by norpA.

PMID: 14724249 [PubMed - indexed for MEDLINE]

The Drosophila dgq gene encodes a G alpha protein that mediates phototransduction.

Department of Biological Sciences, University of Notre Dame, Indiana 46556.

We examined the roles of the Drosophila Gq alpha proteins (DGq) in the phototransduction pathway. The DGq proteins immunolocalized to the ocelli and all eight retinular photoreceptor cell rhabdomeres. An affinity-purified anti-DGq alpha immunoglobulin blocked the light-dependent GTP hydrolysis activity associated with Drosophila head membranes in vitro, suggesting that rhodopsin stimulated DGq. Dominantly active DGq1 mutants exhibited a light-independent GTPase activity and abnormal electrophysiological light responses, such as reduced retinal sensitivity and slow response kinetics compared with wild-type flies. Dominant DGq2 mutants exhibited a light-independent GTPase activity with normal electrophysiological light responses. Retinas of double mutants of DGq1, but not DGq2, with the light-dependent retinal degeneration mutant rdgB degenerated even in the dark. DGq1 stimulation of rdgB retinal degeneration in the dark was norpA-dependent. These results indicate that DGq1 mediates the stimulation by light-activated rhodopsin of the norpA-encoded phospholipase C in the visual transduction cascade.

PMID: 7946351 [PubMed - indexed for MEDLINE]

G protein control of Drosophila photoreceptor phospholipase C.

Howard Hughes Medical Institute, University of Washington, Seattle 98195, USA.

Light stimulates phosphatidylinositol bisphosphate phospholipase C (PLC) activity in Drosophila photoreceptors. We have investigated the mechanism of this reaction by assaying PLC activity in Drosophila head membranes using exogenous phospholipid substrates. PLC activation depends on the photoconversion of rhodopsin to metarhodopsin and is reduced in norpAEE5 PLC and ninaEP332 rhodopsin mutants. NorpA PLC is stimulated by light at free Ca2+ concentrations between 10 nM and 1 microM. This finding is consistent with a Ca(2+)-mediated positive feedback mechanism that contributes to the rapid temporal response of invertebrate photoreceptor cells. The guanyl nucleotide dependence of light-stimulated PLC activity indicates that a G protein regulates NorpA. This was confirmed by the observation that light stimulation of PLC activity is deficient in mutants that lack the eye-specific G protein beta subunit G beta e. These results indicate that G beta e functions as the beta subunit of the G protein coupling rhodopsin to NorpA PLC.

PMID: 7759511 [PubMed - indexed for MEDLINE]