William L. Pak

Isolation of a Putative Phospholipase C Gene of Drosophila, norpA, and Its Role in Phototransduction

Severe norpA mutations in Drosophila eliminate the photoreceptor potential and render the fly completely blind. Recent biochemical analyses have shown that norpA mutants lack phospholipase C (PLC) activity in the eye. A combination of chromosomal walking and transposon-mediated mutagenesis was used to clone the norpA gene. This gene encodes a 7.5 kb RNA that is expressed in the adult head. In situ hybridizations of norpA cDNA to adult tissue sections show that this gene is expressed abundantly in the retina. The putative norpA protein is composed of 1095 amino acid residues and has extensive sequence similarity to a PLC amino acid sequence from bovine brain. We suggest that the norpA gene encodes a PLC expressed in the eye of Drosophila and that PLC is an essential component of the Drosophila phototransduction pathway.

The Drosophila ninaE gene encodes an opsin

The Drosophila ninaE gene was isolated by a multistep protocol on the basis of its homology to bovine opsin cDNA. The gene encodes the major visual pigment protein (opsin) contained in Drosophila photoreceptor cells R1-R6. The coding sequence is interrupted by four short introns. The positions of three introns are conserved with respect to positions in mammalian opsin genes. The nucleotide sequence has intermittent regions of homology to bovine opsin coding sequences. The deduced amino acid sequence reveals significant homology to vertebrate opsins; there is strong conservation of the retinal binding site and two other regions. The predicted protein secondary structure strikingly resembles that of mammalian opsins. We conclude the Drosophila and vertebrate opsin genes are derived from a common ancestor.

cGMP-dependent changes in phototaxis: a possible role for the foraging gene in honey bee division of labor

SUMMARY Division of labor in honey bee colonies is influenced by the foraging gene (Amfor), which encodes a cGMP-dependent protein kinase (PKG). Amfor upregulation in the bee brain is associated with the age-related transition from working in the hive to foraging for food outside, and cGMP treatment (which increases PKG activity) causes precocious foraging. We present two lines of evidence in support of the hypothesis that Amfor affects division of labor by modulating phototaxis. We first show that a subset of worker bees involved in the removal of corpses from the hive had forager-like brain levels of Amfor brain expression despite being middle aged; age-matched food-handlers, who do not leave the hive to perform their job, had low levels of Amfor expression. This finding suggests that occupations that involve working outside the hive are associated with high levels of Amfor in brain. Secondly, foragers were much more positively phototactic than hive bees in a laboratory assay, and cGMP treatment caused a precocious onset of positive phototaxis. The cGMP effect was not due to a general increase in behavioral activity; cGMP treatment had no effect on locomotor activity under either constant darkness or a light:dark regime. The cGMP effect also was not due to changes in circadian rhythmicity; cGMP treatment had no effect on age at onset of locomotor circadian rhythmicity or the period of rhythmicity. The effects of Amfor on phototaxis are not related to peripheral processing; electroretinogram analysis revealed no effect of cGMP treatment on photoreceptor activity and no differences between untreated hive bees and foragers. The cAMP/PKA pathway does not appear to be playing a similar role to cGMP/PKG in the honey bee; cAMP treatment did not affect phototaxis and gene expression analysis revealed task-related differences only for the gene encoding the regulatory subunit, but not the catalytic subunit, of PKA. Our findings implicate one neural process associated with honey bee division of labor that can be affected by naturally occurring changes in the expression of Amfor.

Genetic and molecular identification of a Drosophila histidine decarboxylase gene required in photoreceptor transmitter synthesis.

Drosophila mutants of a single complementation group with defective on‐/off‐transients of the electroretinogram (ERG) were found to be deficient in synthesis of the photoreceptor transmitter, histamine, in a gene‐dosage dependent manner, suggesting that the gene identified by the mutants (hdc) might be the structural gene for Drosophila histidine decarboxylase (HDC). A rat HDC cDNA was used to isolate a Drosophila homolog which shows approximately 60% sequence identity with mammalian HDCs over a region of 476 amino acids. In RNA blots, the Drosophila homolog detects four transcripts that are expressed primarily in the eye and are severely reduced in hdc mutants. The cloned Drosophila cDNA hybridizes to the 46F region of the chromosome, to which hdc mutations have been mapped, and rescues the hdc mutant phenotype in transgenic flies generated by P element‐mediated germline transformation. The results thus show that the Drosophila homolog corresponds to the histidine decarboxylase gene, identified by the hdc mutants, and that mutations in the gene disrupt photoreceptor synaptic transmission.

Isolation of light-induced response of the central retinula cells from the electroretinogram ofDrosophila

SummaryAn intense blue light stimulus induces a prolonged depolarizing after-potential (PDA) in the peripheral retinula cells, but not in the central retinula cells, of theDrosophila ommatidia, providing the fly has been dark or red adapted and the screening pigments have been genetically removed from the compound eye. Thus, the PDA saturates only the peripheral retinula cells, allowing one to isolate and study extracellularly the summed receptor potentials (SR) of the central retinula cells (Fig. 1). The following lines of evidence support these ideas: a)The on and off transients, typical of the normal electroretinogram (ERG), are not present in the SR (Fig. 1).b)The intensity response curve of the intracellularly recorded responses of the peripheral retinula cells fits that of the ERG only after subtraction of the SR contribution from the latter (Fig. 2).c)The SR action spectrum is different from that of the dark adapted ERG and appears to arise from more than one spectral mechanism (Fig. 3).

Phosrestin I undergoes the earliest light-induced phosphorylation by a calcium/calmodulin-dependent protein kinase in drosophila photoreceptors

Activation of PI-PLC initiates two independent branches of protein phosphorylation cascades catalyzed by either PKC or Ca2+/calmodulin-dependent protein kinase (CaMK). We find that phosrestin I (PRI), a Drosophila homolog of vertebrate photoreceptor arrestin, undergoes light-induced phosphorylation on a subsecond time scale which is faster than that of any other protein in vivo. We determine that a CaMK activity is responsible for in vitro PRI phosphorylation at Ser366 in the C-terminal tryptic segment, MetLysSer(P)IleGluGlnHisArg, in which Ser(P) represents phosphoserine366. We also demonstrate that Ser366 is the phosphorylation site of PRI in vivo by identifying the molecular species resulting from in-gel tryptic digestion of purified phospho-PRI using HPLC-electrospray ionization tandem quadrupole mass spectroscopy. From these data, we conclude that the CaMK pathway, not the PKC pathway, is responsible for the earliest protein phosphorylation event following activation of PI-PLC in living Drosophila photoreceptors.

The Target of Drosophila Photoreceptor Synaptic Transmission Is a Histamine-gated Chloride Channel Encoded byort (hclA)

By screening Drosophila mutants that are potentially defective in synaptic transmission between photoreceptors and their target laminar neurons, L1/L2, (lack of electroretinogram on/off transients), we identified ort as a candidate gene encoding a histamine receptor subunit on L1/L2. We provide evidence that the ort gene corresponds toCG7411 (referred to as hclA), identified in the Drosophila genome data base, by P-element-mediated germ line rescue of the ort phenotype using clonedhclA cDNA and by showing that several ortmutants exhibit alterations in hclA regulatory or coding sequences and/or allele-dependent reductions inhclA transcript levels. Other workers have shown thathclA, when expressed in Xenopus oocytes, forms histamine-sensitive chloride channels. However, the connection between these chloride channels and photoreceptor synaptic transmission was not established. We show unequivocally that hclA-encoded channels are the channels required in photoreceptor synaptic transmission by 1) establishing the identity between hclAand ort and 2) showing that ort mutants are defective in photoreceptor synaptic transmission. Moreover, the present work shows that this function of the HCLA (ORT) protein is its native function in vivo.

DAG Lipase Activity Is Necessary for TRP Channel Regulation in Drosophila Photoreceptors

In Drosophila, a phospholipase C-mediated signaling cascade links photoexcitation of rhodopsin to the opening of the TRP/TRPL channels. A lipid product of the cascade, diacylglycerol (DAG) and its metabolite(s), polyunsaturated fatty acids (PUFAs), have both been proposed as potential excitatory messengers. A crucial enzyme in the understanding of this process is likely to be DAG lipase (DAGL). However, DAGLs that might fulfill this role have not been previously identified in any organism. In this work, the Drosophila DAGL gene, inaE, has been identified from mutants that are defective in photoreceptor responses to light. The inaE-encoded protein isoforms show high sequence similarity to known mammalian DAG lipases, exhibit DAG lipase activity in vitro, and are highly expressed in photoreceptors. Analyses of norpA inaE double mutants and severe inaE mutants show that normal DAGL activity is required for the generation of physiologically meaningful photoreceptor responses.

Morphological Defects in orajk84 Photoreceptors Caused by Mutation in R1-6 Opsin Gene of Drosophila

The Drosophila mutant, oraJK84, lacks rhabdomeres in the major (R1-6) class of photoreceptors because these rhabdomeres rapidly degenerate in young flies. Genetic analysis reveals that oraJK84 actually contains two mutations (a ninaE and an ort allele) that affect the visual process. The mutation in ort appears to have no effect on photoreceptor structure. The other mutation occurs within the ninaE gene, which encodes the species of rhodopsin found in the R1-6 class of photoreceptors. Our analysis shows that this mutation is responsible for R1-6 rhabdomere degeneration in oraJK84 mutants. We also examined a ninaE mutant, denoted ninaEo117, that produces no ninaE transcript. The morphological phenotype observed in ninaEo117 is similar to that seen in oraJK84 mutants. We conclude that rhodopsin plays a vital role in maintaining photoreceptor structure in Drosophila.

Drosophila rhodopsin: Photochemistry, extraction and differences in the norp AP12 phototransduction mutant

Summary The visual pigments of Drosophila melanogaster eyes were studied by spectrophotometric measurements in whole eyes and digitonin extracts. The main visual pigment transition appears to be RHODOPSIN480 METARHODOPSIN580. Under most conditions the products are thermally stable and reversible by illumination. The norp AP12 phototransduction mutant appears to contain approximately one-third of the total visual pigment found in the wild type eye. The rhodopsin480 is greatly reduced in concentration. A rhodopsin420 may be dominant and a metarhodopsin480 may also be present. These species may also be present in the wild type eye. The reduced visual pigment concentration of norp AP12 does not appear to explain the small visual receptor potentials of this mutant and one must consider a more complex visual transduction mechanism.