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A dPIP5K dependent pool of phosphatidylinositol 4,5 bisphosphate (PIP2) is required for G-protein coupled signal transduction in Drosophila photoreceptors.

Chakrabarti P, Kolay S, Yadav S, Kumari K, Nair A, Trivedi D, Raghu P - PLoS Genet. (2015)

Bottom Line: Loss of dPIP5K causes profound defects in the electrical response to light and light-induced PIP2 dynamics at the photoreceptor membrane.These results provide evidence for the existence of a unique dPIP5K dependent pool of PIP2 required for normal Drosophila phototransduction.Our results define the existence of multiple pools of PIP2 in photoreceptors generated by distinct lipid kinases and supporting specific molecular processes at neuronal membranes.

View Article: PubMed Central - PubMed

Affiliation: Inositide Laboratory, Babraham Institute, Cambridge, United Kingdom.

ABSTRACT
Multiple PIP2 dependent molecular processes including receptor activated phospholipase C activity occur at the neuronal plasma membranes, yet levels of this lipid at the plasma membrane are remarkably stable. Although the existence of unique pools of PIP2 supporting these events has been proposed, the mechanism by which they are generated is unclear. In Drosophila photoreceptors, the hydrolysis of PIP2 by G-protein coupled phospholipase C activity is essential for sensory transduction of photons. We identify dPIP5K as an enzyme essential for PIP2 re-synthesis in photoreceptors. Loss of dPIP5K causes profound defects in the electrical response to light and light-induced PIP2 dynamics at the photoreceptor membrane. Overexpression of dPIP5K was able to accelerate the rate of PIP2 synthesis following light induced PIP2 depletion. Other PIP2 dependent processes such as endocytosis and cytoskeletal function were unaffected in photoreceptors lacking dPIP5K function. These results provide evidence for the existence of a unique dPIP5K dependent pool of PIP2 required for normal Drosophila phototransduction. Our results define the existence of multiple pools of PIP2 in photoreceptors generated by distinct lipid kinases and supporting specific molecular processes at neuronal membranes.

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Related in: MedlinePlus

dPIP5K is required to support rdgB dependent function in photoreceptors.(A) Representative ERG traces depicting the response of control, dPIP5K18, rdgB9 and rdgB9; dPIP5K18. Responses to single 2s flash of green light (intensity 5 cf. X-axis of Fig. 2D) are depicted. The genotypes corresponding to each trace are indicated on the top of the graph. Scale bar at the bottom shows the axis; X-axis represents time in seconds and Y-axis represents amplitude of the response in mV. The duration of the light pulse is indicated. (B) Comparison of maximum amplitude of the light response among control, dPIP5K18, rdgB9 and rdgB9; dPIP5K18. Y-axis represents mean +/− S.D of the peak amplitude from five flies. (C) Representative optical neutralization images from control, rdgB9, dPIP5K18 and rdgB9; dPIP5K18 showing the exacerbation of degeneration in rdgB9; dPIP5K18 compared to rdgB9, dPIP5K18 alone does not show any degeneration. The representative images shown are collected from one-day-old flies maintained in L/D cycle (900 lux). (D) Quantification of the effect of dPIP5K18 loss of function on photoreceptor structure in rdgB9. (E) Schematic diagram showing the light induced PIP2 cycle in Drosophila photoreceptors. Genes encoding a given enzyme activity where identified are marked in italics. Notations used: PI(4,5)P2-phosphatidylinositol 4,5 bisphosphate, norpA- no receptor potential A, PLCβ-phospholipase C beta, DAG- diacylglycerol, DGK- diacylglycerol kinase, rdgA- retinal degeneration A, laza-lipid phosphate phosphohydrolase (PA phosphatase), PA-phosphatidic acid, CDP-DAG- Cytidine diphosphate diacylglycerol; CDS CDP-DAG synthase, PI- phosphatidylinositol, rdgB- retinal degeneration B, PITP- phosphatidylinositol transfer protein, PI(4)P- phosphatidylinositol 4-phosphate (F) Schematic representation of the pools of PIP2 in Drosophila photoreceptor membranes. Representations are only semi-quantitative. The total pool of PIP2 in the plasma membrane is shown bounded by the solid black line. The PLC sensitive PIP2 pool sensitive to light induced PLC activity is shown in the rectangle bounded by the broken/dashed line indicating that PLC will likely also use a non-dPIP5K dependent pool. The basal PIP2 pool is indicated in green. The major function of each pool is indicated. Enzymes responsible for the synthesis of each pool are marked.
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pgen.1004948.g007: dPIP5K is required to support rdgB dependent function in photoreceptors.(A) Representative ERG traces depicting the response of control, dPIP5K18, rdgB9 and rdgB9; dPIP5K18. Responses to single 2s flash of green light (intensity 5 cf. X-axis of Fig. 2D) are depicted. The genotypes corresponding to each trace are indicated on the top of the graph. Scale bar at the bottom shows the axis; X-axis represents time in seconds and Y-axis represents amplitude of the response in mV. The duration of the light pulse is indicated. (B) Comparison of maximum amplitude of the light response among control, dPIP5K18, rdgB9 and rdgB9; dPIP5K18. Y-axis represents mean +/− S.D of the peak amplitude from five flies. (C) Representative optical neutralization images from control, rdgB9, dPIP5K18 and rdgB9; dPIP5K18 showing the exacerbation of degeneration in rdgB9; dPIP5K18 compared to rdgB9, dPIP5K18 alone does not show any degeneration. The representative images shown are collected from one-day-old flies maintained in L/D cycle (900 lux). (D) Quantification of the effect of dPIP5K18 loss of function on photoreceptor structure in rdgB9. (E) Schematic diagram showing the light induced PIP2 cycle in Drosophila photoreceptors. Genes encoding a given enzyme activity where identified are marked in italics. Notations used: PI(4,5)P2-phosphatidylinositol 4,5 bisphosphate, norpA- no receptor potential A, PLCβ-phospholipase C beta, DAG- diacylglycerol, DGK- diacylglycerol kinase, rdgA- retinal degeneration A, laza-lipid phosphate phosphohydrolase (PA phosphatase), PA-phosphatidic acid, CDP-DAG- Cytidine diphosphate diacylglycerol; CDS CDP-DAG synthase, PI- phosphatidylinositol, rdgB- retinal degeneration B, PITP- phosphatidylinositol transfer protein, PI(4)P- phosphatidylinositol 4-phosphate (F) Schematic representation of the pools of PIP2 in Drosophila photoreceptor membranes. Representations are only semi-quantitative. The total pool of PIP2 in the plasma membrane is shown bounded by the solid black line. The PLC sensitive PIP2 pool sensitive to light induced PLC activity is shown in the rectangle bounded by the broken/dashed line indicating that PLC will likely also use a non-dPIP5K dependent pool. The basal PIP2 pool is indicated in green. The major function of each pool is indicated. Enzymes responsible for the synthesis of each pool are marked.

Mentions: Photoreceptors of the Drosophila rdgB mutant show defects in the electrical response to light as well as light dependent degeneration. rdgB encodes a large multi domain protein including an N-terminal phosphatidylinositol transfer protein (PITP) domain [reviewed in [35]]. In vitro the PITP domain can bind and transfer phosphatidylinositol (PI) between two membrane bound compartments and it is presumed, though not demonstrated that the PI delivered to the acceptor compartment is the substrate for phosphorylation by PIPKs that generate phosphorylated versions of PI. In the case of PIP2 this would include the sequential phosphorylation of PI and PI4P by PI4K and PIP5K respectively. Although the precise molecular function of RDGB in photoreceptors in unknown, it has previously been shown that rdgB mutant photoreceptors have a defect in restoring the level of microvillar PIP2 following transduction triggered by a bright flash of light [36]. Thus rdgB mutants represent an opportunity to test the importance of a potential PIP5K that might generate microvillar PIP2 required for phototransduction. To test the relevance of dPIP5K in generating PIP2 required for G-protein coupled PLCβ activity, we generated photoreceptors that are double mutant rdgB9; dPIP5K18; importantly we used the rdgB9 allele that is a strong hypomorph and expresses a small amount of this protein and therefore has a residual response to light. We compared the light response of rdgB9 photoreceptors with those of rdgB9; dPIP5K18 (Fig. 7A). Under similar conditions, while rdgB9 photoreceptors have peak ERG amplitudes of ca. 1.5 mV (Fig. 7A), rdgB9; dPIP5K18 photoreceptors respond with a amplitude of only 0.4 mV (Fig. 7B). This observation suggests that dPIP5K function is required to support the residual light response in rdgB9 photoreceptors. We also studied a second phenotype of rdgB9 namely light dependent retinal degeneration and found that, rdgB9; dPIP5K18 photoreceptors degenerated faster than rdgB9 alone (Fig. 7C,D). By contrast loss of dPIP4K or sktl did not exacerbate the electrical response to light or the retinal degeneration phenotype of rdgB9.


A dPIP5K dependent pool of phosphatidylinositol 4,5 bisphosphate (PIP2) is required for G-protein coupled signal transduction in Drosophila photoreceptors.

Chakrabarti P, Kolay S, Yadav S, Kumari K, Nair A, Trivedi D, Raghu P - PLoS Genet. (2015)

dPIP5K is required to support rdgB dependent function in photoreceptors.(A) Representative ERG traces depicting the response of control, dPIP5K18, rdgB9 and rdgB9; dPIP5K18. Responses to single 2s flash of green light (intensity 5 cf. X-axis of Fig. 2D) are depicted. The genotypes corresponding to each trace are indicated on the top of the graph. Scale bar at the bottom shows the axis; X-axis represents time in seconds and Y-axis represents amplitude of the response in mV. The duration of the light pulse is indicated. (B) Comparison of maximum amplitude of the light response among control, dPIP5K18, rdgB9 and rdgB9; dPIP5K18. Y-axis represents mean +/− S.D of the peak amplitude from five flies. (C) Representative optical neutralization images from control, rdgB9, dPIP5K18 and rdgB9; dPIP5K18 showing the exacerbation of degeneration in rdgB9; dPIP5K18 compared to rdgB9, dPIP5K18 alone does not show any degeneration. The representative images shown are collected from one-day-old flies maintained in L/D cycle (900 lux). (D) Quantification of the effect of dPIP5K18 loss of function on photoreceptor structure in rdgB9. (E) Schematic diagram showing the light induced PIP2 cycle in Drosophila photoreceptors. Genes encoding a given enzyme activity where identified are marked in italics. Notations used: PI(4,5)P2-phosphatidylinositol 4,5 bisphosphate, norpA- no receptor potential A, PLCβ-phospholipase C beta, DAG- diacylglycerol, DGK- diacylglycerol kinase, rdgA- retinal degeneration A, laza-lipid phosphate phosphohydrolase (PA phosphatase), PA-phosphatidic acid, CDP-DAG- Cytidine diphosphate diacylglycerol; CDS CDP-DAG synthase, PI- phosphatidylinositol, rdgB- retinal degeneration B, PITP- phosphatidylinositol transfer protein, PI(4)P- phosphatidylinositol 4-phosphate (F) Schematic representation of the pools of PIP2 in Drosophila photoreceptor membranes. Representations are only semi-quantitative. The total pool of PIP2 in the plasma membrane is shown bounded by the solid black line. The PLC sensitive PIP2 pool sensitive to light induced PLC activity is shown in the rectangle bounded by the broken/dashed line indicating that PLC will likely also use a non-dPIP5K dependent pool. The basal PIP2 pool is indicated in green. The major function of each pool is indicated. Enzymes responsible for the synthesis of each pool are marked.
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Related In: Results  -  Collection

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Show All Figures
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pgen.1004948.g007: dPIP5K is required to support rdgB dependent function in photoreceptors.(A) Representative ERG traces depicting the response of control, dPIP5K18, rdgB9 and rdgB9; dPIP5K18. Responses to single 2s flash of green light (intensity 5 cf. X-axis of Fig. 2D) are depicted. The genotypes corresponding to each trace are indicated on the top of the graph. Scale bar at the bottom shows the axis; X-axis represents time in seconds and Y-axis represents amplitude of the response in mV. The duration of the light pulse is indicated. (B) Comparison of maximum amplitude of the light response among control, dPIP5K18, rdgB9 and rdgB9; dPIP5K18. Y-axis represents mean +/− S.D of the peak amplitude from five flies. (C) Representative optical neutralization images from control, rdgB9, dPIP5K18 and rdgB9; dPIP5K18 showing the exacerbation of degeneration in rdgB9; dPIP5K18 compared to rdgB9, dPIP5K18 alone does not show any degeneration. The representative images shown are collected from one-day-old flies maintained in L/D cycle (900 lux). (D) Quantification of the effect of dPIP5K18 loss of function on photoreceptor structure in rdgB9. (E) Schematic diagram showing the light induced PIP2 cycle in Drosophila photoreceptors. Genes encoding a given enzyme activity where identified are marked in italics. Notations used: PI(4,5)P2-phosphatidylinositol 4,5 bisphosphate, norpA- no receptor potential A, PLCβ-phospholipase C beta, DAG- diacylglycerol, DGK- diacylglycerol kinase, rdgA- retinal degeneration A, laza-lipid phosphate phosphohydrolase (PA phosphatase), PA-phosphatidic acid, CDP-DAG- Cytidine diphosphate diacylglycerol; CDS CDP-DAG synthase, PI- phosphatidylinositol, rdgB- retinal degeneration B, PITP- phosphatidylinositol transfer protein, PI(4)P- phosphatidylinositol 4-phosphate (F) Schematic representation of the pools of PIP2 in Drosophila photoreceptor membranes. Representations are only semi-quantitative. The total pool of PIP2 in the plasma membrane is shown bounded by the solid black line. The PLC sensitive PIP2 pool sensitive to light induced PLC activity is shown in the rectangle bounded by the broken/dashed line indicating that PLC will likely also use a non-dPIP5K dependent pool. The basal PIP2 pool is indicated in green. The major function of each pool is indicated. Enzymes responsible for the synthesis of each pool are marked.
Mentions: Photoreceptors of the Drosophila rdgB mutant show defects in the electrical response to light as well as light dependent degeneration. rdgB encodes a large multi domain protein including an N-terminal phosphatidylinositol transfer protein (PITP) domain [reviewed in [35]]. In vitro the PITP domain can bind and transfer phosphatidylinositol (PI) between two membrane bound compartments and it is presumed, though not demonstrated that the PI delivered to the acceptor compartment is the substrate for phosphorylation by PIPKs that generate phosphorylated versions of PI. In the case of PIP2 this would include the sequential phosphorylation of PI and PI4P by PI4K and PIP5K respectively. Although the precise molecular function of RDGB in photoreceptors in unknown, it has previously been shown that rdgB mutant photoreceptors have a defect in restoring the level of microvillar PIP2 following transduction triggered by a bright flash of light [36]. Thus rdgB mutants represent an opportunity to test the importance of a potential PIP5K that might generate microvillar PIP2 required for phototransduction. To test the relevance of dPIP5K in generating PIP2 required for G-protein coupled PLCβ activity, we generated photoreceptors that are double mutant rdgB9; dPIP5K18; importantly we used the rdgB9 allele that is a strong hypomorph and expresses a small amount of this protein and therefore has a residual response to light. We compared the light response of rdgB9 photoreceptors with those of rdgB9; dPIP5K18 (Fig. 7A). Under similar conditions, while rdgB9 photoreceptors have peak ERG amplitudes of ca. 1.5 mV (Fig. 7A), rdgB9; dPIP5K18 photoreceptors respond with a amplitude of only 0.4 mV (Fig. 7B). This observation suggests that dPIP5K function is required to support the residual light response in rdgB9 photoreceptors. We also studied a second phenotype of rdgB9 namely light dependent retinal degeneration and found that, rdgB9; dPIP5K18 photoreceptors degenerated faster than rdgB9 alone (Fig. 7C,D). By contrast loss of dPIP4K or sktl did not exacerbate the electrical response to light or the retinal degeneration phenotype of rdgB9.

Bottom Line: Loss of dPIP5K causes profound defects in the electrical response to light and light-induced PIP2 dynamics at the photoreceptor membrane.These results provide evidence for the existence of a unique dPIP5K dependent pool of PIP2 required for normal Drosophila phototransduction.Our results define the existence of multiple pools of PIP2 in photoreceptors generated by distinct lipid kinases and supporting specific molecular processes at neuronal membranes.

View Article: PubMed Central - PubMed

Affiliation: Inositide Laboratory, Babraham Institute, Cambridge, United Kingdom.

ABSTRACT
Multiple PIP2 dependent molecular processes including receptor activated phospholipase C activity occur at the neuronal plasma membranes, yet levels of this lipid at the plasma membrane are remarkably stable. Although the existence of unique pools of PIP2 supporting these events has been proposed, the mechanism by which they are generated is unclear. In Drosophila photoreceptors, the hydrolysis of PIP2 by G-protein coupled phospholipase C activity is essential for sensory transduction of photons. We identify dPIP5K as an enzyme essential for PIP2 re-synthesis in photoreceptors. Loss of dPIP5K causes profound defects in the electrical response to light and light-induced PIP2 dynamics at the photoreceptor membrane. Overexpression of dPIP5K was able to accelerate the rate of PIP2 synthesis following light induced PIP2 depletion. Other PIP2 dependent processes such as endocytosis and cytoskeletal function were unaffected in photoreceptors lacking dPIP5K function. These results provide evidence for the existence of a unique dPIP5K dependent pool of PIP2 required for normal Drosophila phototransduction. Our results define the existence of multiple pools of PIP2 in photoreceptors generated by distinct lipid kinases and supporting specific molecular processes at neuronal membranes.

Show MeSH
Related in: MedlinePlus