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Rolling blackout, a newly identified PIP2-DAG pathway lipase required for Drosophila phototransduction

Nature Neuroscience volume 7, pages 1070–1078 (2004)Cite this article

Abstract

The rolling blackout (rbo) gene encodes an integral plasma membrane lipase required for Drosophila phototransduction. Photoreceptors are enriched for the RBO protein, and temperature-sensitive rbo mutants show reversible elimination of phototransduction within minutes, demonstrating an acute requirement for the protein. The block is activity dependent, indicating that the action of RBO is use dependent. Conditional rbo mutants show activity-dependent depletion of diacylglycerol and concomitant accumulation of phosphatidylinositol phosphate and phosphatidylinositol 4,5-bisphosphate within minutes of induction, suggesting rapid downregulation of phospholipase C (PLC) activity. The RBO requirement identifies an essential regulatory step in G-protein-coupled, PLC-dependent inositol lipid signaling mediating activation of TRP and TRPL channels during phototransduction.

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Figure 1: Mapping, identification and genomic rescue of rbo, and hydrophobicity and membrane association of RBO protein.
Figure 2: RBO protein is enriched in neuropil and photoreceptors.
Figure 3: Blockade of phototransduction in rbo mutants.
Figure 4: Activity-dependent blockade of phototransduction in rbo mutants.
Figure 5: Sustained anoxia response in rbo mutants.
Figure 6: Reduction in the slope of initial ERG response in rbo mutants.
Figure 7: Activity-dependent elevation of PIP/PIP2 levels in rbo mutants.
Figure 8: Activity-dependent depletion of DAG levels in rbo mutants.

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References

  1. Bloomquist, B.T. et al. Isolation of a putative phospholipase C gene of Drosophila, norpA, and its role in phototransduction. Cell 54, 723–733 (1988).

    Article  CAS  Google Scholar 

  2. Niemeyer, B.A., Suzuki, E., Scott, K., Jalink, K. & Zuker, C.S. The Drosophila light-activated conductance is composed of the two channels TRP and TRPL. Cell 85, 651–659 (1996).

    Article  CAS  Google Scholar 

  3. Scott, K., Becker, A., Sun, Y., Hardy, R. & Zuker, C. Gqα protein function in vivo: genetic dissection of its role in photoreceptor cell physiology. Neuron 15, 919–927 (1995).

    Article  CAS  Google Scholar 

  4. Hardie, R.C. Phototransduction in Drosophila melanogaster. J. Exp. Biol. 204, 3403–3409 (2001).

    CAS  PubMed  Google Scholar 

  5. Montell, C. Visual transduction in Drosophila. Annu. Rev. Cell Dev. Biol. 15, 231–268 (1999).

    Article  CAS  Google Scholar 

  6. Estacion, M., Sinkins, W.G. & Schilling, W.P. Regulation of Drosophila transient receptor potential-like (TrpL) channels by phospholipase C-dependent mechanisms. J. Physiol. (Lond.) 530, 1–19 (2001).

    Article  CAS  Google Scholar 

  7. Minke, B. & Cook, B. TRP channel proteins and signal transduction. Physiol. Rev. 82, 429–472 (2002).

    Article  CAS  Google Scholar 

  8. Hardie, R.C. Regulation of trp channels via lipid second messengers. Annu. Rev. Physiol. 65, 735–759 (2003).

    Article  CAS  Google Scholar 

  9. Hardie, R.C. & Minke, B. The trp gene is essential for a light-activated Ca2+ channel in Drosophila photoreceptors. Neuron 8, 643–651 (1992).

    Article  CAS  Google Scholar 

  10. Acharya, J.K., Jalink, K., Hardy, R.W., Hartenstein, V. & Zuker, C.S. InsP3 receptor is essential for growth and differentiation but not for vision in Drosophila. Neuron 18, 881–887 (1997).

    Article  CAS  Google Scholar 

  11. Raghu, P. et al. Constitutive activity of the light-sensitive channels TRP and TRPL in the Drosophila diacylglycerol kinase mutant, rdgA. Neuron 26, 169–179 (2000).

    Article  CAS  Google Scholar 

  12. Smith, D.P. et al. Photoreceptor deactivation and retinal degeneration mediated by a photoreceptor-specific protein kinase C. Science 254, 1478–1484 (1991).

    Article  CAS  Google Scholar 

  13. Huber, A., Sander, P., Bahner, M. & Paulsen, R. The TRP Ca2+ channel assembled in a signaling complex by the PDZ domain protein INAD is phosphorylated through the interaction with protein kinase C (ePKC). FEBS Lett. 425, 317–322 (1998).

    Article  CAS  Google Scholar 

  14. Liu, M., Parker, L.L., Wadzinski, B.E. & Shieh, B.H. Reversible phosphorylation of the signal transduction complex in Drosophila photoreceptors. J. Biol. Chem. 275, 12194–12199 (2000).

    Article  CAS  Google Scholar 

  15. Chyb, S., Raghu, P. & Hardie, R.C. Polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL. Nature 397, 255–259 (1999).

    Article  CAS  Google Scholar 

  16. Hilgemann, D.W., Feng, S. & Nasuhoglu, C. The complex and intriguing lives of PIP2 with ion channels and transporters. Sci. STKE RE19 (2001).

  17. Ordway, R.W., Pallanck, L. & Ganetzky, B. Neurally expressed Drosophila genes encoding homologs of the NSF and SNAP secretory proteins. Proc. Natl Acad. Sci. USA 91, 5715–5719 (1994).

    Article  CAS  Google Scholar 

  18. Loughney, K., Kreber, R. & Ganetzky, B. Molecular analysis of the para locus, a sodium channel gene in Drosophila. Cell 58, 1143–1154 (1989).

    Article  CAS  Google Scholar 

  19. van der Bliek, A.M. & Meyerowitz, E.M. Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic. Nature 351, 411–414 (1991).

    Article  CAS  Google Scholar 

  20. Shyngle, J. & Sharma, R.P. Studies on paralysis and development of second chromosome temperature sensitive paralytic mutants of Drosophila melanogaster. Indian J. Exp. Biol. 23, 235–240 (1985).

    CAS  PubMed  Google Scholar 

  21. Kumar, M., Joseph, M. & Chandrashekaran, S. Effects of mutations at the stambh A locus of Drosophila melanogaster. J. Genet. 80, 83–95 (2001).

    Article  CAS  Google Scholar 

  22. Faulkner, D.L., Dockendorff, T.C. & Jongens, T.A. Clonal analysis of cmp44E, which encodes a conserved putative transmembrane protein, indicates a requirement for cell viability in Drosophila. Dev. Genet. 23, 264–274 (1998).

    Article  CAS  Google Scholar 

  23. Martinez, C., De Geus, P., Lauwereys, M., Matthyssens, G. & Cambillau, C. Fusarium solani cutinase is a lipolytic enzyme with a catalytic serine accessible to solvent. Nature 356, 615–618 (1992).

    Article  CAS  Google Scholar 

  24. Cygler, M. & Schrag, J.D. Structure as basis for understanding interfacial properties of lipases. Methods Enzymol. 284, 3–27 (1997).

    Article  CAS  Google Scholar 

  25. Bisogno, T. et al. Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain. J. Cell Biol. 163, 463–468 (2003).

    Article  CAS  Google Scholar 

  26. Hotta, Y. & Benzer, S. Abnormal electroretinograms in visual mutants of Drosophila. Nature 222, 354–356 (1969).

    Article  CAS  Google Scholar 

  27. Pak, W.L., Grossfield, J. & White, N.V. Nonphototactic mutants in a study of vision in Drosophila. Nature 222, 351–354 (1969).

    Article  CAS  Google Scholar 

  28. Agam, K. et al. Metabolic stress reversibly activates the Drosophila light-sensitive channels TRP and TRPL in vivo. J. Neurosci. 20, 5748–5755 (2000).

    Article  CAS  Google Scholar 

  29. Pearn, M.T., Randall, L.L., Shortridge, R.D., Burg, M.G. & Pak, W.L. Molecular, biochemical, and electrophysiological characterization of Drosophila norpA mutants. J. Biol. Chem. 271, 4937–4945 (1996).

    Article  CAS  Google Scholar 

  30. Juusola, M. & Hardie, R.C. Light adaptation in Drosophila photoreceptors: II. Rising temperature increases the bandwidth of reliable signaling. J. Gen. Physiol. 117, 27–42 (2001).

    Article  CAS  Google Scholar 

  31. McKay, R.R., Zhu, L. & Shortridge, R.D. Membrane association of phospholipase C encoded by the norpA gene of Drosophila melanogaster. Neuroscience 61, 141–148 (1994).

    Article  CAS  Google Scholar 

  32. Yoshioka, T., Inoue, H. & Hotta, Y. Absence of phosphatidylinositol phosphodiesterase in the head of a Drosophila visual mutant, norpA (no receptor potential A). J. Biochem. 97, 1251–1254 (1985).

    Article  CAS  Google Scholar 

  33. Preiss, J. et al. Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells. J. Biol. Chem. 261, 8597–8600 (1986).

    CAS  PubMed  Google Scholar 

  34. Hardie, R.C. & Raghu, P. Visual transduction in Drosophila. Nature 413, 186–193 (2001).

    Article  CAS  Google Scholar 

  35. Minke, B. & Selinger, Z. Inositol lipid pathway in fly photoreceptors: excitation, calcium mobilization and retinal degeneration. Prog. Retin. Res. 11, 99–124 (1991).

    Article  CAS  Google Scholar 

  36. Wu, L., Niemeyer, B., Colley, N., Socolich, M. & Zuker, C.S. Regulation of PLC-mediated signalling in vivo by CDP-diacylglycerol synthase. Nature 373, 216–222 (1995).

    Article  CAS  Google Scholar 

  37. Hardie, R.C. et al. Calcium influx via TRP channels is required to maintain PIP2 levels in Drosophila photoreceptors. Neuron 30, 149–159 (2001).

    Article  CAS  Google Scholar 

  38. Rodriguez de Turco, E.B. et al. Diacylglycerol kinase epsilon regulates seizure susceptibility and long-term potentiation through arachidonoyl-inositol lipid signaling. Proc. Natl Acad. Sci. USA 98, 4740–4745 (2001).

    Article  CAS  Google Scholar 

  39. Yoshioka, T., Inoue, H. & Hotta, Y. Absence of diglyceride kinase activity in the photoreceptor cells of Drosophila mutants. Biochem. Biophys. Res. Commun. 119, 389–395 (1984).

    Article  CAS  Google Scholar 

  40. Inoue, H., Yoshioka, T. & Hotta, Y. Diacylglycerol kinase defect in a Drosophila retinal degeneration mutant rdgA. J. Biol. Chem. 264, 5996–6000 (1989).

    CAS  PubMed  Google Scholar 

  41. Broadie, K. Functional assays of the peripheral and central nervous systems. in Drosophila Protocols (ed. W.A.M. Sullivan & R.S. Hawley) 297–312 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2000).

    Google Scholar 

  42. Fukami, K. et al. Antibody to phosphatidylinositol 4,5-bisphosphate inhibits oncogene-induced mitogenesis. Proc. Natl Acad. Sci. USA 85, 9057–9061 (1988).

    Article  CAS  Google Scholar 

  43. Bligh, E.G. & Dyer, W.J. A rapid method of total lipid extraction and purification. Can J Med Sci 37, 911–917 (1959).

    CAS  Google Scholar 

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Acknowledgements

We thank the Drosophila Bloomington Stock Center for supplying stocks; T. Jongens for cmp44E RFII pBluescript vector; T. Jongens, S. Chandrashekaran, C. Montell, H. Li, R. Hardie and W. Pak for flies, including cmp44E, stmA, trp343, trpl302 and norpA mutants; C. Sanders for DAG kinase; R. Shortridge for advice about PLC assays; R. Mohrmann for discussions, useful comments and help in generating figures and L. Yang for assistance in sequencing mutant alleles. This study was supported by US National Institutes of Health grant GM54544 to K.B.

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Author notes

  1. Fu-De Huang and Heinrich J G Matthies: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, Vanderbilt Kennedy Center, Vanderbilt Brain Institute, Vanderbilt University, Nashville, 37235-1634, Tennessee, USA

    Fu-De Huang, Heinrich J G Matthies & Kendal Broadie

  2. Department of Biology, University of Utah, Salt Lake City, 84112, Utah, USA

    Sean D Speese & Mark A Smith

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Correspondence to Kendal Broadie.

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Supplementary information

Supplementary Fig. 1

stmA, cmp44E and rbo mutants are allelic to each other, based on both lethality and conditional blindness complementation studies. (a)"*" indicates phenotype was fully rescued by genomic rbo-egfp expression. A single naming strategy was adopted for all mutant alleles of the locus, as indicated. (b) Representative traces showing the loss (top) and genomic rescue (bottom) of phototransduction in rbo mutants. (PDF 266 kb)

Supplementary Fig. 2

RBO dendrogram. A dendrogram generated by Clustal W representing the relationship of RBO homologs from multiple species. A single rbo gene is present in both Drosophila melanogaster (Dmrbo) and Anapheles gambiae (Agrbo). The closest homologs are two genes present in both human (Hsrbo1,2) and mouse (Mmrbo1,2), and, more distantly, a single gene in C. elegans (Cerbo). Homologs are present in yeast (Scrbo). The outlier groups contain homologs from Arabidopsis (Atrbo) and rice (Osrbo). (PDF 300 kb)

Supplementary Fig. 3

RBO is a predicted lipase. The RBO protein contains the domain structure of a lipolytic enzyme. Three highly-conserved lipase functional motifs (His, Ser and Asp/Glu active sites) contain the catalytic triad of His, Ser, and Asp/Glu residues, respectively. The consensus motif "GXSXG" is found in the Ser active site. The mutation site in rbots1 is indicated. (PDF 40 kb)

Supplementary Fig. 4

PLC activity is normal in rbo mutant head extracts. (a) Schematic diagram of the Drosophila phototransduction pathway. Light activation of rhodopsin (Rh) activates Gq, which activates PLC. PLC hydrolyzes PIP2 into DAG and IP3 in the microvillar membrane. DAG either directly activates TRP/TRPL channels or is further hydrolyzed by DAG lipase to produce PUFAs, which in turn activate TRP/TRPL channels. DAG Kinase (DGK; I) converts DAG to phosphatidic acid (PA), which is then recycled to synthesize CDP-DAG via CDP-DAG synthase (II) in the subrhabdomeric cisternae. CDP-DAG is converted to PI by phosphatidylinositol synthase (III) to form PI. PI is then transported back to the microvillar membrane by PI transport protein (IV) and serially phosphorylated by PI kinase and PIP kinase (V,VI) to reproduce PIP2. Dotted lines indicate hypothetical pathways. (b) PLC activity assay from head extracts (see Methods) of wildtype (WT), rbots1/rbo2 mutants (rbo) and PLC norpA null mutants (norpAP24) at 25°C and 37°C, as indicated. Higher temperature caused a consistent increase in PLC activity levels in both WT and rbo mutants. The two genotypes were indistinguishable. The PLC null norpAP24 had negligible PLC activity, <1% of either WT or rbo mutants. (PDF 230 kb)

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Huang, FD., Matthies, H., Speese, S. et al. Rolling blackout, a newly identified PIP2-DAG pathway lipase required for Drosophila phototransduction. Nat Neurosci 7, 1070–1078 (2004). https://doi.org/10.1038/nn1313

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