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Regulation of phototransduction responsiveness and retinal degeneration by a phospholipase D-generated signaling lipid.

LaLonde MM, Janssens H, Rosenbaum E, Choi SY, Gergen JP, Colley NJ, Stark WS, Frohman MA - J. Cell Biol. (2005)

Bottom Line: Drosophila melanogaster phototransduction proceeds via a phospholipase C (PLC)-triggered cascade of phosphatidylinositol (PI) lipid modifications, many steps of which remain undefined.We describe the involvement of the lipid phosphatidic acid and the enzyme that generates it, phospholipase D (Pld), in this process.Pld() flies exhibit decreased light sensitivity as well as a heightened susceptibility to retinal degeneration.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular and Cellular Biology, Center for Developmental Genetics, Stony Brook University, Stony Brook, NY 11794, USA.

ABSTRACT
Drosophila melanogaster phototransduction proceeds via a phospholipase C (PLC)-triggered cascade of phosphatidylinositol (PI) lipid modifications, many steps of which remain undefined. We describe the involvement of the lipid phosphatidic acid and the enzyme that generates it, phospholipase D (Pld), in this process. Pld() flies exhibit decreased light sensitivity as well as a heightened susceptibility to retinal degeneration. Pld overexpression rescues flies lacking PLC from light-induced, metarhodopsin-mediated degeneration and restores visual signaling in flies lacking the PI transfer protein, which is a key player in the replenishment of the PI 4,5-bisphosphate (PIP2) substrate used by PLC to transduce light stimuli into neurological signals. Altogether, these findings suggest that Pld facilitates phototransduction by maintaining adequate levels of PIP2 and by protecting the visual system from metarhodopsin-induced, low light degeneration.

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Pld does not directly transduce light signals, but does morphologically rescue retinal degeneration in the norpA7 mutant. ERGs were obtained from flies raised under continuous light conditions for 1 d: (A) Canton-S, (B) norpA7, and (C) norpA7; P{UAS–Pld}/ Rh1. Retinal tissue sections were prepared from flies raised under a 12-h light/12-h dark cycle for 21 d: (D) Canton-S, (E) norpA7, and (F) norpA7; P{UAS-Pld}/ Rh1. (D) All 7 photoreceptor cells in all 12 of the complete ommatidia are present in the wild-type Canton-S section, whereas the norpA7 flies (E) display an irregular ommatidial array that is characterized by intracellular vacuolation (arrows) and missing photoreceptor cells. Only five of the nine complete ommatidia within this section contained seven intact photoreceptor cells. However, a more normal ommatidial structure was observed in norpA7 flies that overexpressed Pld (F). Although some vacuolation was observed (arrow), all eight complete ommatidia in this section contained seven photoreceptor cells. The experiment was performed three times, and two eyes were sectioned per experimental condition with equivalent results. The entire set of sections was examined, and representative sections were selected for the figures. (G) Western blot analysis of rhodopsin protein levels using a mouse monoclonal antirhodopsin antibody. Tubulin was used as the loading control. Protein was extracted from the following adult heads: Canton-S (lane 1), norpA7 (lane 2), P{UAS-Pld}/ Rh1 (lane 3), norpA7; P{UAS-Pld}/ Rh1 (lane 4). Rhodopsin levels were not decreased by Pld overexpression. Sections were representative of three experiments performed. Slightly elevated levels of rhodopsin in the P{UAS-Pld}/ Rh1 sample were observed in this experiment, but this was not a consistent finding based on the other experiments (not depicted).
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fig4: Pld does not directly transduce light signals, but does morphologically rescue retinal degeneration in the norpA7 mutant. ERGs were obtained from flies raised under continuous light conditions for 1 d: (A) Canton-S, (B) norpA7, and (C) norpA7; P{UAS–Pld}/ Rh1. Retinal tissue sections were prepared from flies raised under a 12-h light/12-h dark cycle for 21 d: (D) Canton-S, (E) norpA7, and (F) norpA7; P{UAS-Pld}/ Rh1. (D) All 7 photoreceptor cells in all 12 of the complete ommatidia are present in the wild-type Canton-S section, whereas the norpA7 flies (E) display an irregular ommatidial array that is characterized by intracellular vacuolation (arrows) and missing photoreceptor cells. Only five of the nine complete ommatidia within this section contained seven intact photoreceptor cells. However, a more normal ommatidial structure was observed in norpA7 flies that overexpressed Pld (F). Although some vacuolation was observed (arrow), all eight complete ommatidia in this section contained seven photoreceptor cells. The experiment was performed three times, and two eyes were sectioned per experimental condition with equivalent results. The entire set of sections was examined, and representative sections were selected for the figures. (G) Western blot analysis of rhodopsin protein levels using a mouse monoclonal antirhodopsin antibody. Tubulin was used as the loading control. Protein was extracted from the following adult heads: Canton-S (lane 1), norpA7 (lane 2), P{UAS-Pld}/ Rh1 (lane 3), norpA7; P{UAS-Pld}/ Rh1 (lane 4). Rhodopsin levels were not decreased by Pld overexpression. Sections were representative of three experiments performed. Slightly elevated levels of rhodopsin in the P{UAS-Pld}/ Rh1 sample were observed in this experiment, but this was not a consistent finding based on the other experiments (not depicted).

Mentions: To maximize potential light-stimulated, Pld phototransduction events, we used the UAS–GAL4 system (Brand and Perrimon, 1993) with the ninaE-GAL4 (Rh1) driver to ectopically overexpress wild-type Pld cDNA in photoreceptor cells R1–6. To eliminate PLC-mediated phototransduction events in parallel and, thus, permit the isolated detection of any potential Pld-generated signal, the experiment was performed in flies lacking the eye-enriched isoform of PLC (no receptor potential A [norpA]), which eliminates PIP2 hydrolysis and overt light-stimulated signals (Fig. 4 B). Light-stimulated transduction was not observed in norpA7 flies that overexpressed Pld (Fig. 4 C), indicating that the contribution of Pld to phototransduction responses is not mediated by the rhodopsin-triggered activation of Pld and conversion of the ensuing PA to DAG. This finding only addresses whether light stimulation elicits a Pld-mediated change in membrane potential. It does not indicate whether Pld-generated PA is converted to DAG and subsequently activates TRP at a significant steady-state level, because ERGs only report changes in electrical activity; i.e., they do not provide a means to assess differences in baseline current between different individuals.


Regulation of phototransduction responsiveness and retinal degeneration by a phospholipase D-generated signaling lipid.

LaLonde MM, Janssens H, Rosenbaum E, Choi SY, Gergen JP, Colley NJ, Stark WS, Frohman MA - J. Cell Biol. (2005)

Pld does not directly transduce light signals, but does morphologically rescue retinal degeneration in the norpA7 mutant. ERGs were obtained from flies raised under continuous light conditions for 1 d: (A) Canton-S, (B) norpA7, and (C) norpA7; P{UAS–Pld}/ Rh1. Retinal tissue sections were prepared from flies raised under a 12-h light/12-h dark cycle for 21 d: (D) Canton-S, (E) norpA7, and (F) norpA7; P{UAS-Pld}/ Rh1. (D) All 7 photoreceptor cells in all 12 of the complete ommatidia are present in the wild-type Canton-S section, whereas the norpA7 flies (E) display an irregular ommatidial array that is characterized by intracellular vacuolation (arrows) and missing photoreceptor cells. Only five of the nine complete ommatidia within this section contained seven intact photoreceptor cells. However, a more normal ommatidial structure was observed in norpA7 flies that overexpressed Pld (F). Although some vacuolation was observed (arrow), all eight complete ommatidia in this section contained seven photoreceptor cells. The experiment was performed three times, and two eyes were sectioned per experimental condition with equivalent results. The entire set of sections was examined, and representative sections were selected for the figures. (G) Western blot analysis of rhodopsin protein levels using a mouse monoclonal antirhodopsin antibody. Tubulin was used as the loading control. Protein was extracted from the following adult heads: Canton-S (lane 1), norpA7 (lane 2), P{UAS-Pld}/ Rh1 (lane 3), norpA7; P{UAS-Pld}/ Rh1 (lane 4). Rhodopsin levels were not decreased by Pld overexpression. Sections were representative of three experiments performed. Slightly elevated levels of rhodopsin in the P{UAS-Pld}/ Rh1 sample were observed in this experiment, but this was not a consistent finding based on the other experiments (not depicted).
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Related In: Results  -  Collection

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fig4: Pld does not directly transduce light signals, but does morphologically rescue retinal degeneration in the norpA7 mutant. ERGs were obtained from flies raised under continuous light conditions for 1 d: (A) Canton-S, (B) norpA7, and (C) norpA7; P{UAS–Pld}/ Rh1. Retinal tissue sections were prepared from flies raised under a 12-h light/12-h dark cycle for 21 d: (D) Canton-S, (E) norpA7, and (F) norpA7; P{UAS-Pld}/ Rh1. (D) All 7 photoreceptor cells in all 12 of the complete ommatidia are present in the wild-type Canton-S section, whereas the norpA7 flies (E) display an irregular ommatidial array that is characterized by intracellular vacuolation (arrows) and missing photoreceptor cells. Only five of the nine complete ommatidia within this section contained seven intact photoreceptor cells. However, a more normal ommatidial structure was observed in norpA7 flies that overexpressed Pld (F). Although some vacuolation was observed (arrow), all eight complete ommatidia in this section contained seven photoreceptor cells. The experiment was performed three times, and two eyes were sectioned per experimental condition with equivalent results. The entire set of sections was examined, and representative sections were selected for the figures. (G) Western blot analysis of rhodopsin protein levels using a mouse monoclonal antirhodopsin antibody. Tubulin was used as the loading control. Protein was extracted from the following adult heads: Canton-S (lane 1), norpA7 (lane 2), P{UAS-Pld}/ Rh1 (lane 3), norpA7; P{UAS-Pld}/ Rh1 (lane 4). Rhodopsin levels were not decreased by Pld overexpression. Sections were representative of three experiments performed. Slightly elevated levels of rhodopsin in the P{UAS-Pld}/ Rh1 sample were observed in this experiment, but this was not a consistent finding based on the other experiments (not depicted).
Mentions: To maximize potential light-stimulated, Pld phototransduction events, we used the UAS–GAL4 system (Brand and Perrimon, 1993) with the ninaE-GAL4 (Rh1) driver to ectopically overexpress wild-type Pld cDNA in photoreceptor cells R1–6. To eliminate PLC-mediated phototransduction events in parallel and, thus, permit the isolated detection of any potential Pld-generated signal, the experiment was performed in flies lacking the eye-enriched isoform of PLC (no receptor potential A [norpA]), which eliminates PIP2 hydrolysis and overt light-stimulated signals (Fig. 4 B). Light-stimulated transduction was not observed in norpA7 flies that overexpressed Pld (Fig. 4 C), indicating that the contribution of Pld to phototransduction responses is not mediated by the rhodopsin-triggered activation of Pld and conversion of the ensuing PA to DAG. This finding only addresses whether light stimulation elicits a Pld-mediated change in membrane potential. It does not indicate whether Pld-generated PA is converted to DAG and subsequently activates TRP at a significant steady-state level, because ERGs only report changes in electrical activity; i.e., they do not provide a means to assess differences in baseline current between different individuals.

Bottom Line: Drosophila melanogaster phototransduction proceeds via a phospholipase C (PLC)-triggered cascade of phosphatidylinositol (PI) lipid modifications, many steps of which remain undefined.We describe the involvement of the lipid phosphatidic acid and the enzyme that generates it, phospholipase D (Pld), in this process.Pld() flies exhibit decreased light sensitivity as well as a heightened susceptibility to retinal degeneration.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular and Cellular Biology, Center for Developmental Genetics, Stony Brook University, Stony Brook, NY 11794, USA.

ABSTRACT
Drosophila melanogaster phototransduction proceeds via a phospholipase C (PLC)-triggered cascade of phosphatidylinositol (PI) lipid modifications, many steps of which remain undefined. We describe the involvement of the lipid phosphatidic acid and the enzyme that generates it, phospholipase D (Pld), in this process. Pld() flies exhibit decreased light sensitivity as well as a heightened susceptibility to retinal degeneration. Pld overexpression rescues flies lacking PLC from light-induced, metarhodopsin-mediated degeneration and restores visual signaling in flies lacking the PI transfer protein, which is a key player in the replenishment of the PI 4,5-bisphosphate (PIP2) substrate used by PLC to transduce light stimuli into neurological signals. Altogether, these findings suggest that Pld facilitates phototransduction by maintaining adequate levels of PIP2 and by protecting the visual system from metarhodopsin-induced, low light degeneration.

Show MeSH
Related in: MedlinePlus