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Nat Neurosci. Author manuscript; available in PMC Feb 21, 2013.
Published in final edited form as:
PMCID: PMC3578554
NIHMSID: NIHMS420575
Visual neurotransmission in Drosophila requires expression of Fic in glial capitate projections
Mokhlasur Rahman,1 Hyeilin Ham,2 Xinran Liu,1 Yoshie Sugiura,1 Kim Orth,2 and Helmut Krämer1,3
1Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas, USA
2Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
3Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
Correspondence should be addressed to H.K. (helmut.kramer/at/utsouthwestern.edu)
Fic domains can catalyze the addition of adenosine monophosphate to target proteins. To date, the function of Fic domain proteins in eukaryotic physiology remains unknown. We generated genetic models of the single Drosophila Fic domain–containing protein, Fic. Flies lacking Fic were viable and fertile, but blind. Photoreceptor cells depolarized normally following light stimulation, but failed to activate postsynaptic neurons, as indicated by the loss of ON transients in electroretinograms, consistent with a neurotransmission defect. Functional rescue of neurotransmission required expression of enzymatically active Fic on capitate projections of glia cells, but not neurons, supporting a role in the recycling of the visual neurotransmitter histamine. Histamine levels were reduced in the lamina of Fic null flies, and dietary histamine partially restored ON transients. These findings establish a previously unknown regulatory mechanism in visual neurotransmission and provide, to the best of our knowledge, the first evidence for a role of glial capitate projections in neurotransmitter recycling.
Reversible post-translational modifications have a pivotal role in eukaryotic signaling and are therefore exploited by bacterial pathogens. Studies on a Vibrio virulence factor, VopS, demonstrated that its Fic domain is used to modify Rho-GTPases with adenosine monophosphate (AMP)1. This modification was first observed over 40 years ago when covalent modification of a tyrosine with AMP reversibly regulated a bacterial protein glutamine synthetase2,3. Only recently have new enzymes, including the Fic domain–containing proteins and nucleosidyl transferases, been discovered that are not restricted to bacteria, but are also found in eukaryotes1,4,5. The former domain is found in human Huntingtin-interacting protein E and the Drosophila Fic protein (Flybase: CG9523) and both have been shown to catalyze AMPylation reactions5,6, but the physiological contexts in which they operate have not been defined yet. To explore the role of these proteins, we focused on the Drosophila Fic protein.
We reasoned that a regulatory role of Fic might be revealed by genetically manipulating its expression in flies. Notably, high-level overexpression of UAS-Fic by the da-Gal4 driver was lethal (Supplementary Table 1). This lethality depended on the enzymatic activity of Fic, as no phenotypes were detected following expression of the catalytically inactive FicH375A mutant, which lacks AMPylation activity6. Flies expressing wild-type Fic or mutant UAS-Fic transgenes at low levels under arm-Gal4 control did not exhibit externally visible phenotypes or lethality (Supplementary Table 1). These data support the hypothesis that AMPylation may regulate important cellular processes.
To explore this proposal, we generated Fic loss-of-function deletion mutations by excision of a nearby P element (Fig. 1a). One of the resulting alleles, Fic55, lacked the 5′ UTR and the first 260 of 493 amino acids, including the highly conserved tetratricopeptide repeat domain. Western analysis confirmed the loss of Fic protein (Fig. 1b). Flies homozygous for Fic55 were viable and fertile. Their behavior, however, seemed sluggish compared with wild-type flies. This deficit was quantified in a fast-phototaxis assay7,8, which revealed that the Fic55 mutants were not attracted to a light source, that is, they were blind (Fig. 1c). This phenotype was specific for Fic55 flies, as it was also observed in flies expressing a UAS-Fic dsRNA transgene under da-Gal4 control, and wild-type behavior was restored by a genomic Fic transgene (Fig. 1c).
Figure 1
Figure 1
Fic is required for visual neurotransmission. (a) Map of Fic gene and protein, including predicted35 transmembrane domain (TM), potential N-glycosylation site Asn288, tetratricopeptide repeat domain and Fic domain with active site at His375 (ref. 6). (more ...)
This altered behavior could reflect a deficit in motor output, light reception or the processing of visual information. To distinguish between these possibilities, we measured the electrical response to a light pulse using electroretinograms (ERGs). ERGs revealed normal light-induced depolarization in wild-type and Fic55 flies, arguing against a substantial defect in light reception in Fic55 photoreceptors (Fig. 1d). ERGs of Fic55 flies missed, however, the characteristic ON transient of the response to light. ON transients in ERGs are a reflection of synaptic activation of the postsynaptic laminar neurons9,10. This defect was specific for the loss of Fic function, as it was also observed in Fic55/Df(2L)BSC296 hemizygotes and after Fic knockdown (UAS-Fic dsRNA; da-Gal4). Furthermore ON transients were restored by a genomic Fic transgene or by expression of a Fic cDNA under the control of the low-level arm-Gal4 driver (Fig. 1e). Similarly, expression of transgenes that encoded Fic fused at its C terminus to GFP or horseradish peroxidase (HRP) restored ON transients, indicating that these chimeric proteins are functional. Notably, expression of the catalytically inactive FicH375A mutant6 was not able to restore ON transients (Fig. 1e), indicating that Fic enzymatic activity is necessary for normal activity of photoreceptor synapses required for vision.
Loss of ON transients in ERG recordings reflects the loss of synaptic transmission from photoreceptor cells. This defect might occur as a consequence of many different physiological deficiencies, such as the loss of pre- or postsynaptic proteins affecting synapse function11,12, or changes in the distribution of organelles, including mitochondria or synaptic vesicles13,14. When photoreceptors and their synaptic connections were evaluated by electron microscopy (Fig. 2a–f), no difference was evident in the arrangement of photoreceptor axons (Fig. 2a,b) or rhabdomeres (Fig. 2g,h) or in the synaptic ultrastructure, as mitochondria, synaptic vesicles and active zone T bars appeared unchanged (Fig. 2a–f). These finding argue against Fic function being required as a structural element of synapses.
Figure 2
Figure 2
Visual neurotransmission requires Fic enzymatic activity in glia cells. (ah) Electron micrographs of sections of wild-type (a,c,e,g) and Fic55 (b,d,f,h) lamina reveal the presence of mitochondria (M), capitate projections (CP), synaptic vesicles (more ...)
To further narrow the focus of Fic activity, we explored which cells require its expression for normal visual neurotransmission. To test the requirement in photoreceptor cells, we generated chimeric flies11 with whole-eye clones homozygous for Fic55 (ey3.5-Flp; FRT Fic55). These flies still exhibited ON transients (Fig. 2i), as did flies with photoreceptor-specific knockdown of Fic (GMR-Gal4/UAS-Fic dsRNA). Furthermore, expression of Fic in both photoreceptors and their lamina target neurons under control of the elav-Gal4 neuronal driver15 did not restore ON transients in Fic55 mutants (Fig. 2i). Together these data argue against a requirement of Fic in photoreceptors or lamina neurons.
In contrast, glia-specific expression of Fic under the control of the repo-Gal4 driver16 was sufficient to restore ON transients (Fig. 2i). Moreover, glia-specific knockdown of Fic (UAS-Fic dsRNAi; repo-Gal4) indicated that Fic is necessary for normal ON transients in glia cells. To test the relevance of these electrophysiological responses to behavior, we evaluated flies in the fast-phototaxis assay. Consistent with the ERG recordings, loss of Fic function in whole-eye clones or its photoreceptor-specific knockdown (GMR-Gal4/UAS-Fic dsRNA) did not eliminate fast phototaxis. Furthermore, neuronal Fic expression (elav-Gal4; UAS-Fic) did not restore normal behavior in Fic55 mutants (Fig. 2j). In contrast, repo-Gal4–driven knockdown of Fic indicated its requirement in glia cells for fast-phototaxis. Moreover, glia-specific expression of Fic (repo-Gal4; UAS-Fic) significantly improved fast phototaxis of Fic55 mutants (P < 0.001; Fig. 2j), although it did not fully restore normal behavior, indicating a requirement in additional cells of the phototaxis circuits17. Notably, expression of the catalytically inactive FicH375A mutant in glia cells did not rescue the characteristic ON transient of the response to light (Fig. 2i) or improve the fast-phototaxis behavior of Fic55 flies (Fig. 2j). Together, these data indicate that Fic enzymatic activity in glia cells is necessary and sufficient for normal synaptic transmission in the visual system.
One important role of glia cells in the Drosophila lamina is the recycling of histamine, the neurotransmitter released by photoreceptors1820. Histamine is synthesized in photoreceptors by histidine decarboxylase (Hdc)21. However, Hdc activity covers only a fraction of the histamine requirement for their normal synaptic activity; the balance of histamine is provided by recycling22. After release by photoreceptor synapses, histamine is removed from the synaptic cleft by active uptake into glia cells. There, a β-alanyl-histamine synthetase encoded by ebony catalyzes the condensation of histamine with β-alanine23,24. The resulting product, carcinine, is extruded from glia cells and taken up by photoreceptor cells in a poorly understood process18,25,26. In photoreceptors, histamine is recovered from carcinine by a β-alanyl-histamine hydrolase encoded by tan20. Interruption of this cycle results in the loss of visual transmission, as indicated by behavioral deficits and the loss of ON transients in ERG recordings10,18,19,21.
Notably, phenotypes caused by mutants interfering with histamine metabolism can be partially rescued by dietary histamine or carcinine, which as a result of the recycling pathway, can be taken up into photoreceptor cells18,19. When Fic55 flies were fed a diet enriched with histamine or carcinine, we observed partial restoration of ON transients (Fig. 3a), although the level of rescue was not sufficient to restore normal behavioral in the fast-phototaxis assay (Fig. 3b). To test whether histamine distribution is altered, we stained sections of wild-type and Fic55 heads with antibodies to histamine25. Wild-type flies exhibited some variable staining in photoreceptor cell bodies and strong staining in the layer of epithelial glia cells just proximal to photoreceptors and in photoreceptor axon terminals (Fig. 3c). In contrast, histamine immunoreactivity in epithelial glia was strongly reduced in Fic55 flies (Fig. 3d), although staining was still observed in photoreceptor cell bodies. Staining in epithelial glia cells was partially restored by glia-specific expression of Fic (Fig. 3e). Specificity of staining for histamine was indicated by its absence in HdcP218 heads21 (Fig. 3f). Loss of histamine staining in Fic55 lamina is reminiscent of the changes in histamine distribution in other mutants that disrupt histamine recycling in photoreceptor synapses, including tan, ebony and Vmat20,23,25,27. Together, these data are consistent with the notion that histamine metabolism is one of the physiological processes regulated by the enzymatic activity of Fic in glia cells.
Figure 3
Figure 3
Fic is required for recycling of the histamine neurotransmitter. (a,b) Quantification of ON transients from ERG recordings (a) and fast phototaxis response (b) of wild-type or Fic55 flies fed food supplemented with histamine (HA) or carcinine (CA) or (more ...)
To gain further insight into the mechanism by which histamine metabolism is affected by Fic, we analyzed its subcellular localization. Subcellular fractionation of S2 cells showed Fic partitioning with the endoplasmic reticulum marker BiP in the P2 particulate fraction, indicating that Fic may be a membrane-associated or transmembrane protein (Fig. 4a). To test whether Fic is secreted, we analyzed whether Fic was N-glycosylated, a modification that occurs in the lumen of the endoplasmic reticulum. When Fic was expressed using an in vitro translation system, the addition of microsomes caused a shift in mobility consistent with N-glycosylation of Fic. Mutation of Asn288 at the only predicted N-glycosylation site suppressed this shift in FicN288Q (Fig. 4b). We reasoned that if Fic is secreted into the endoplasmic reticulum lumen, then its glycosylation might be sensitive to endoglycosidase H (EndoH), which specifically cleaves the high-mannose sugars. To test this, we treated Fic protein purified from Sf21 cells or endogenously present in S2 cell lysates with EndoH. Both Fic preparations exhibited a shift in their mobility that was consistent with a loss of high-mannose modifications (Fig. 4c). To assess whether part or all of Fic was secreted, we performed a protease protection assay. In vitro–translated Fic was readily degraded by proteinase K, but, after translation in the presence of microsomes, a ~48-kDa protein was protected from degradation (Fig. 4c). These data suggest that Fic is a type II transmembrane protein with its single N-terminal hydrophobic stretch (Fig. 1a) functioning as a transmembrane domain and its N-glycosylation site and catalytic domain being secreted into the lumen of the secretory pathway.
Figure 4
Figure 4
Fic active site is extracellular and localizes to capitate projections. (a) Differential centrifugation of S2 cell homogenates at 500g (P1), 10,000g (P2) and 100,000g (P3). Fic co-fractionated with endoplasmic reticulum marker BiP, but not nuclear histone (more ...)
To determine the localization of Fic in glia cells (Fig. 4d–r), where it is required for normal visual neurotransmission, we expressed a functionally active Fic-HRP fusion protein (Fig. 1e) either under the control of the endogenous Fic promoter and enhancer elements (Fig. 4d,f) or under control of the repo-Gal4 driver (Fig. 4h,j,l–o). HRP activity of the chimeric protein was used to detect Fic-HRP by electron microscopy28. Fic-HRP was expressed on the surface of glia cells and prominently enriched in capitate projections that epithelial glia cells insert into synaptic endings of photoreceptors (Fig. 4d,f,h,j,l–o). HRP activity was strongest in the head of capitate projections, as revealed by the electron-dense DAB precipitate in the extracellular space between glia cell and neuron (Fig. 4d,f,h,j,l–o). Control sections of flies lacking Fic-HRP expression showed no such DAB accumulation (Fig. 4e,g,i), instead revealing the slightly expanded extracellular space typical for capitate projections (Figs. 2e,f and 4k,p–r)14,29. Prompted by this localization of Fic, we quantified the appearance of capitate projections. In Fic55 mutant heads, the frequency of capitate projections was slightly reduced; we observed 4.3 ± 0.3 capitate projections per synaptic cross-section in wild type compared with 2.8 ± 0.2 in Fic55 (mean ± s.d., n = 3 heads each, P = 0.012, t = 4.32, F = 3.0). However, the shape (Fig. 2e,f) and average diameter of capitate projections were not altered in Fic55 mutants (wild type, 184 ± 5.8 nm; Fic55, 190 ± 4.0 nm (mean ± s.d.); n = 3 heads each, P = 0.4, t = 0.846, F = 2.079), indicating that Fic activity is not necessary for their formation. Notably, capitate projections have been speculated to be the site of neurotransmitter recycling into photoreceptor cells14, although no transporter or other transmembrane protein, besides Fic, is known to specifically localize to these specialized synaptic organelles.
Our data are consistent with the idea that Fic’s enzymatic activity is involved in the regulation of the distribution of the visual neurotransmitter histamine, which is critical for the function of photoreceptor synapses and vision. Proteins in the histamine metabolism and secretion pathway that are sensitive to Fic-mediated AMPylation are not yet known. A first hint at possible targets comes from Fic’s requirement in glia cells, where its target may participate in the uptake of histamine or its subsequent modification to carcinine. This latter reaction is catalyzed by Ebony, a cytoplasmic protein23,24, which is therefore an unlikely substrate for the secreted Fic active site. Thus, the more likely targets in glia cells are the unknown transporters that facilitate the uptake of histamine or its recycling to photoreceptors in the form of carcinine.
Strong support for the latter model arises from the specific localization of Fic in glia cells, where this enzyme is found on the cell surface at capitate projections (Fig. 4l–o) These invaginations of glia cells into the synaptic endings of photoreceptor cells are dynamic organelles that can occur as shallow indentations (Fig. 4k,o) or with extended necks with one (Fig. 4h,m,p) or multiple heads (Fig. 4r). On the basis of this intriguing morphology, changes in their number in tan and ebony mutants affecting histamine recycling30, and the close association with sites of active endocytosis, capitate projections have long been speculated to be sites of neurotransmitter recycling14,26. The localization of Fic to these organelles provides, to the best of our knowledge, the first functional data to support this speculation. The localization of Fic in glia cells may reflect a direct interaction with transporters that are targeted to capitate projections and serve as targets of Fic.
What function could Fic-mediated modification of these transporters serve? One model comes from classic studies in which the active site of the enzyme glutamine synthetase is regulated by reversible modification of tyrosine residue with AMP2. In a more recent example, AMPylation regulates Rab1 activation during Legionella pneumophila invasion into cells. Early during invasion, Rab1 is activated by the effector protein SidM, which subsequently catalyzes Rab1 AMPylation4. This modification transiently locks Rab1 in the activated GTP-bound state, which promotes the fusion of secretory vesicles with the vacuole containing the Legionella bacteria. Only release of a second bacterial effector, SidD, removes the AMP residue and thereby unlocks Rab1 so that it can be inactivated31,32. Similarly, AMPylation may modify transporter activity at capitate projections depending on changes in metabolites or other cellular signals. Although Fic domains have been found to use cytidine diphosphorylcholine as a substrate for modifying proteins with phosphocholine33, structural predictions support the hypothesis that most Fic domains will use ATP34. The requirement of a phosphotransferase activity adds another unexpected layer of complexity to visual neurotransmission.
Antibodies and reagents
Antibodies against a C-terminal peptide of Fic were raised and affinity purified by Open Biosystems. Other antibodies used were against BiP (MAC 143, Babraham Institute), histamine (ImmunoStar), Histon2B (Millipore), tubulin (D, M1A, Sigma). Other reagents were histamine (Sigma) and carcinine (Bachem).
Molecular biology
Full-length Fic cDNAs encoding wild-type or N288Q mutant Fic were cloned into pET23a using BamHI and NotI sites for in vitro transcription/translation experiment or into a modified pBacPAK8 vector with His/FLAG tags inserted at the N terminus (provided by B. Li, University of Texas Southwestern) using KpnI and NotI sites for purification from Sf21 cells. Transgenic vectors for Fic expression in flies under UAS control were generated in pUASt by inserting cDNAs encoding wild-type Fic, FicH375A (ref. 6), or versions encoding Fic fused at its C terminus to GFP or HRP28. For expression of Fic-HRP under endogenous control, PCR fragments encompassing 2.2 kb of Fic upstream region and the 1.8-kb Fic coding region, the HRP coding region, or 3 kb of the Fic 3′ region were combined in a modified pAttb transformation vector. For all transgenes, expression of proteins of the expected sizes was confirmed by western blots (data not shown).
Fly work
Flies were maintained using standard conditions. The Fic dsRNA line KK105634 was obtained from the Vienna Drosophila RNAi Center. The Bloomington stock center provided repo-Gal4, da-Gal4, GMR-Gal4 and elav-Gal4 driver lines, Df(2L)BSC296, and the P element insertions k11101 and k07502b. Both of these insertions lines were listed as lethal in Flybase. We found, however, that they were viable over each other or over a deficiency of the area (Df(2L)BSC296). Thus, P element k11101 was mobilized using delta 2/3 transposase. Candidate deletions were identified by altered eye color and chromosomes with deletions in the Fic gene were mapped by PCR. All three deletions identified were homozygous viable. For genomic rescue, Pacman vector CmR-BW CH321-69B6 containing the Fic gene was inserted at chromosomal position 65B2 (Best Gene). The same landing site was used for insertion of the transgene encoding genomic Fic-HRP. ERGs were recorded as described previously36, with at least 20 flies per genotype and five traces per fly. Phototaxis was measured in a T maze with one arm exposed to ultraviolet light and another in the dark. Flies were placed in the center chamber of the T maze and had the choice between ultraviolet light and the dark. After 30 s, the percentage of flies that had moved toward the light was determined. At least 300 flies of each genotype were tested. In each set of experiment, around 45 flies were placed in the center of the chamber and tested three times each. To test nutritional supplements, we placed a yeast paste including histamine (25%, wt/wt) or carcinine (12%, wt/wt) on top of normal food in vials and newly enclosed wild-type adults were allowed to feed for 5 d on this histamine- or carcinine-enriched food.
Western blotting
Western blots were performed as described37. For detection of endogenous Fic from flies, the protein was first immunoprecipitated using antibody to Fic linked to magnetic beads. Proteins were eluted using SDS loading buffer, separated by SDS-PAGE and detected on western blots with Fic primary antibodies and IRdye 800Cw-labeled Protein-A on an Odyssey Imager (Li-Cor).
Subcellular fractionation
S2 cells were harvested and hypotonically lysed in HNMEK lysis buffer (20 mM HEPES (pH 7.6), 150 mM NaCl, 2 mM MgCl2, 2 mM EDTA, 10 mM KCl, 0.5 mM EGTA, 1 mM DTT and protease inhibitor cocktail tablet from Roche) on ice for 20 min. The cells were homogenized using a glass douncer and the total lysate was centrifuged at 500g for 10 min to pellet nuclei (P1). The supernatant was recovered and centrifuged at 10,000g for 10 min to pellet the particulate fraction enriched in organelles such as endoplasmic reticulum and Golgi (P2). The supernatant was then centrifuged at 100,000g for 1 h to separate membrane (P3) and cytosolic fraction (S3). Samples were analyzed by SDS-PAGE and western blotting.
In vitro translation with microsomes
Full-length Fic protein was generated in vitro using TNT Quick Coupled Transcription/Translation Systems (Promega) following the manufacturer’s instructions. To analyze glycosylation, we added canine pancreatic microsomal membranes (Promega) to the reaction. Briefly, 0.25 μg of pET23a-Fic (wild type or N288Q) was mixed with TNT Quick mastermix, [35S]methionine (10 mCi ml−1), microsomal membranes and nuclease-free water to a final volume of 25 μl and incubated at 30 °C for 1 h.
The protease protection assay was modified from a previously described protocol38. Briefly, 9 μl of in vitro–translated reaction was mixed with either 1 μl of proteinase K (10 mg ml−1) or buffer (100 mM potassium acetate, 2 mM magnesium acetate, 50 mM HEPES, pH 7.4) and incubated on ice for 60 min. Proteolysis was terminated by treatment with 0.2 μl of 0.25 M PMSF followed by the addition of 50 μl of preheated PK-kill buffer (1% SDS (wt/vol), 0.1 M Tris, pH 8.0). All reactions samples were boiled for 1 to 2 min at 100 °C and analyzed by SDS-PAGE and autoradiography.
EndoH treatment
Recombinant Fic purified from Sf21 cells and the endogenous Fic from S2 cells were treated with EndoH (New England Biolabs), as described by the manufacturers. N-terminal His/Flag-tagged Fic was expressed in Sf21 cells using a baculovirus expression system (Clontech) according to the manufacturer’s instructions and affinity purified using nickel beads (Sigma). For EndoH treatment, 5 μg of recombinant Fic or 50 μg of S2 subcellular fraction enriched in endogenous Fic was denatured at 100 °C for 10 min with denaturing buffer. 2 μl of EndoH and G5 reaction buffer were added to the reaction and incubated at 37 °C for 1 h. Following SDS-PAGE, recombinant Fic was analyzed by Coomassie Blue staining and endogenous Fic was detected by western blotting.
Histology
Histamine immunohistochemistry was performed as described previously25. In short, fly heads were fixed for 4 h in ice-cold solution of 4% 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide (wt/vol, Sigma) in 0.1 M phosphate buffer, washed overnight in 25% sucrose in phosphate buffer (wt/vol), embedded in optimal cutting temperature compound, frozen in liquid nitrogen and sectioned at 20-μm thickness on a cryostat microtome (Hacker-Bright, England). Sections were incubated overnight with antibodies to histamine (1:500, Immunostar, cat# 22939). Secondary antibodies were labeled with Alexa488 (1:500, Molecular Probes, cat# A-11008). Images were captured using Zeiss LSM510 confocal microscope with a 20× NA 0.75 or a 63× NA 1.4 lens on an inverted confocal microscope (LSM510 Meta; Carl Zeiss) at 21–23 °C.
Electron micrographs of 3-d-old fly heads and detection of Fic-HRP by HRP activity was carried out as described previously28. Size and frequency of capitate projections were determined from images of cross sections of photoreceptor synaptic endings. At least 100 synapses were scored in three lamina each for mutant and wild type. All digital images were imported into Photoshop (Adobe) and adjusted for gain, contrast and gamma settings.
Statistics
One-way ANOVA was used to determine statistical significance of differences in ERGs recordings or phototaxis assays, followed by Tukey’s multiple comparison test. For all ANOVA tests, F values were larger than 150. For these measurements, the Kolmogorov-Smirnov test indicated a normal distribution (alpha = 0.05). All bar graphs resulting from these comparisons show means ± s.d. Numbers of ERG measurements on individual flies and the number of phototaxis tests (each using 45 to 50 flies per genotype) are indicated in bar graphs for each genotype or supplemental condition. Sample sizes were chosen to be close to previously published examples14. Average diameters and frequency of capitate projections in wild type and Fic55 were compared by two-tailed Student’s t test. Statistical significance was determined using the Prism software.
Supplementary Material
Supplement
Acknowledgments
We are grateful to R. Hiesinger for help and advice with the ERG measurements and the use of his ERG setup, W. Pak, B. Hoveman and the Bloomington Stock center for fly lines, M. Buszczak for antibodies, C. Gilpin from the Molecular and Cellular Imaging Facility at UT Southwestern, S.H. Kim and B. Tu for technical help, and to E. Kavalali and A. Haberman for helpful discussions. This work was supported by grants to H.K. from the US National Institutes of Health National Eye Institute (EY10199 and EY021922) and Visual Science Core grant EY020799. K.O. and H.H. are supported by grants from the US National Institutes of Health (Allergy and Infectious Disease, R01-AI056404 and R01-AI087808) and the Welch Foundation (I-1561). K.O. is a Burroughs Wellcome Investigator in Pathogenesis of Infectious Disease and a W.W. Caruth Jr. Biomedical Scholar.
Footnotes
AUTHOR CONTRIBUTIONS
M.R. and H.K. performed the fly genetic studies. X.L. and Y.S. performed the electron microscopy studies. H.H. performed the molecular biology and biochemistry studies. H.K., M.R. and K.O. designed the study and analyzed the data. H.K. and K.O. wrote the paper. All of the authors discussed the results and commented on the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.
Supplementary information is available in the online version of the paper.
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