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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 controls the light response in Drosophila photoreceptors.(A) Western blot of fly head extracts probed with dPIP5K specific antibody. The genotypes of the flies are labeled above each lane. The arrows indicate three bands corresponding to three different forms of dPIP5K detected with this antibody in wild type flies. All three bands are missing in both the knockout lines labeled as dPIP5K18 and dPIP5K30. A protein loading control is shown labeled with a black arrow. (B) Representative ERG traces depicting the response of control and dPIP5K18 photoreceptors to single 2s flash of green light of intensity 3 (cf. X-axis of Fig. 2D). The genotypes corresponding to each trace are indicated on the right side. 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. Genotypes: Control-wild type; Mutant- dPIP5K18; Bac[dPIP5K] represents BAC clone containing the dPIP5K gene. (C) Graphical representation comparing the light response between control and dPIP5K18. The X-axis represents increasing light intensity in log units. The Y-axis represents peak amplitude of each response in mV. Error bars: Mean +/− S.D. (D) Intensity response function of the light response in control and dPIP5K18 flies. Wild type and dPIP5K18 flies with matched eye color are shown. The X-axis represents increasing light intensity in log units and Y-axis the peak response amplitude at each intensity normalized to the response at the maximum intensity. p values were determined using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05). Quantification of the decay time (time taken for the amplitude of the ERG response to reach 50% of its peak amplitude) (E) and the rise time of the ERG response (F) in control, dPIP5K18 and dPIP5K18; Bac[dPIP5K]. (G) Representative light responses from control and dPIP4K29 flies to single 2s flashes of green light. Scale bar at the bottom shows the axes; X-axis represents time in seconds and Y-axis represents amplitude of the response in mV. The duration of the light pulse is indicated. (H) Quantification of the intensity- response to light function from control and dPIP4K29 flies. The X-axis represents light intensity in log units and Y-axis represents the peak amplitude of the response at a given intensity normalized to the response at the maximum intensity. (I) Quantification of the decay time (time taken for the amplitude of the ERG response to reach 50% of its peak amplitude) (J) and the rise time of the ERG response in controls and dPIP4K29.
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pgen.1004948.g002: dPIP5K controls the light response in Drosophila photoreceptors.(A) Western blot of fly head extracts probed with dPIP5K specific antibody. The genotypes of the flies are labeled above each lane. The arrows indicate three bands corresponding to three different forms of dPIP5K detected with this antibody in wild type flies. All three bands are missing in both the knockout lines labeled as dPIP5K18 and dPIP5K30. A protein loading control is shown labeled with a black arrow. (B) Representative ERG traces depicting the response of control and dPIP5K18 photoreceptors to single 2s flash of green light of intensity 3 (cf. X-axis of Fig. 2D). The genotypes corresponding to each trace are indicated on the right side. 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. Genotypes: Control-wild type; Mutant- dPIP5K18; Bac[dPIP5K] represents BAC clone containing the dPIP5K gene. (C) Graphical representation comparing the light response between control and dPIP5K18. The X-axis represents increasing light intensity in log units. The Y-axis represents peak amplitude of each response in mV. Error bars: Mean +/− S.D. (D) Intensity response function of the light response in control and dPIP5K18 flies. Wild type and dPIP5K18 flies with matched eye color are shown. The X-axis represents increasing light intensity in log units and Y-axis the peak response amplitude at each intensity normalized to the response at the maximum intensity. p values were determined using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05). Quantification of the decay time (time taken for the amplitude of the ERG response to reach 50% of its peak amplitude) (E) and the rise time of the ERG response (F) in control, dPIP5K18 and dPIP5K18; Bac[dPIP5K]. (G) Representative light responses from control and dPIP4K29 flies to single 2s flashes of green light. Scale bar at the bottom shows the axes; X-axis represents time in seconds and Y-axis represents amplitude of the response in mV. The duration of the light pulse is indicated. (H) Quantification of the intensity- response to light function from control and dPIP4K29 flies. The X-axis represents light intensity in log units and Y-axis represents the peak amplitude of the response at a given intensity normalized to the response at the maximum intensity. (I) Quantification of the decay time (time taken for the amplitude of the ERG response to reach 50% of its peak amplitude) (J) and the rise time of the ERG response in controls and dPIP4K29.

Mentions: In order to reveal the function of dPIP5K in vivo, a loss-of-function mutant was generated using ends-out homologous recombination [20]. This results in the insertion of a dominant selection marker (Pw+) flanked by multiple stop codons within the gene such that the kinase domain of dPIP5K was disrupted and the mutant allele should produce no protein. A total of eight independent knock-out alleles were isolated by following phenotypic markers, genetic mapping and molecular screening. Two of these namely dPIP5K18 and dPIP5K30 were studied in detail and are described in this study. All eight alleles were semi-lethal; very few homozygous mutant flies emerged as viable adults. Using a polyclonal antibody generated against a relatively unique C-terminal region of dPIP5K, we found that dPIP5K18 and dPIP5K30 were protein alleles of dPIP5K (Fig. 2A).


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 controls the light response in Drosophila photoreceptors.(A) Western blot of fly head extracts probed with dPIP5K specific antibody. The genotypes of the flies are labeled above each lane. The arrows indicate three bands corresponding to three different forms of dPIP5K detected with this antibody in wild type flies. All three bands are missing in both the knockout lines labeled as dPIP5K18 and dPIP5K30. A protein loading control is shown labeled with a black arrow. (B) Representative ERG traces depicting the response of control and dPIP5K18 photoreceptors to single 2s flash of green light of intensity 3 (cf. X-axis of Fig. 2D). The genotypes corresponding to each trace are indicated on the right side. 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. Genotypes: Control-wild type; Mutant- dPIP5K18; Bac[dPIP5K] represents BAC clone containing the dPIP5K gene. (C) Graphical representation comparing the light response between control and dPIP5K18. The X-axis represents increasing light intensity in log units. The Y-axis represents peak amplitude of each response in mV. Error bars: Mean +/− S.D. (D) Intensity response function of the light response in control and dPIP5K18 flies. Wild type and dPIP5K18 flies with matched eye color are shown. The X-axis represents increasing light intensity in log units and Y-axis the peak response amplitude at each intensity normalized to the response at the maximum intensity. p values were determined using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05). Quantification of the decay time (time taken for the amplitude of the ERG response to reach 50% of its peak amplitude) (E) and the rise time of the ERG response (F) in control, dPIP5K18 and dPIP5K18; Bac[dPIP5K]. (G) Representative light responses from control and dPIP4K29 flies to single 2s flashes of green light. Scale bar at the bottom shows the axes; X-axis represents time in seconds and Y-axis represents amplitude of the response in mV. The duration of the light pulse is indicated. (H) Quantification of the intensity- response to light function from control and dPIP4K29 flies. The X-axis represents light intensity in log units and Y-axis represents the peak amplitude of the response at a given intensity normalized to the response at the maximum intensity. (I) Quantification of the decay time (time taken for the amplitude of the ERG response to reach 50% of its peak amplitude) (J) and the rise time of the ERG response in controls and dPIP4K29.
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Related In: Results  -  Collection

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pgen.1004948.g002: dPIP5K controls the light response in Drosophila photoreceptors.(A) Western blot of fly head extracts probed with dPIP5K specific antibody. The genotypes of the flies are labeled above each lane. The arrows indicate three bands corresponding to three different forms of dPIP5K detected with this antibody in wild type flies. All three bands are missing in both the knockout lines labeled as dPIP5K18 and dPIP5K30. A protein loading control is shown labeled with a black arrow. (B) Representative ERG traces depicting the response of control and dPIP5K18 photoreceptors to single 2s flash of green light of intensity 3 (cf. X-axis of Fig. 2D). The genotypes corresponding to each trace are indicated on the right side. 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. Genotypes: Control-wild type; Mutant- dPIP5K18; Bac[dPIP5K] represents BAC clone containing the dPIP5K gene. (C) Graphical representation comparing the light response between control and dPIP5K18. The X-axis represents increasing light intensity in log units. The Y-axis represents peak amplitude of each response in mV. Error bars: Mean +/− S.D. (D) Intensity response function of the light response in control and dPIP5K18 flies. Wild type and dPIP5K18 flies with matched eye color are shown. The X-axis represents increasing light intensity in log units and Y-axis the peak response amplitude at each intensity normalized to the response at the maximum intensity. p values were determined using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05). Quantification of the decay time (time taken for the amplitude of the ERG response to reach 50% of its peak amplitude) (E) and the rise time of the ERG response (F) in control, dPIP5K18 and dPIP5K18; Bac[dPIP5K]. (G) Representative light responses from control and dPIP4K29 flies to single 2s flashes of green light. Scale bar at the bottom shows the axes; X-axis represents time in seconds and Y-axis represents amplitude of the response in mV. The duration of the light pulse is indicated. (H) Quantification of the intensity- response to light function from control and dPIP4K29 flies. The X-axis represents light intensity in log units and Y-axis represents the peak amplitude of the response at a given intensity normalized to the response at the maximum intensity. (I) Quantification of the decay time (time taken for the amplitude of the ERG response to reach 50% of its peak amplitude) (J) and the rise time of the ERG response in controls and dPIP4K29.
Mentions: In order to reveal the function of dPIP5K in vivo, a loss-of-function mutant was generated using ends-out homologous recombination [20]. This results in the insertion of a dominant selection marker (Pw+) flanked by multiple stop codons within the gene such that the kinase domain of dPIP5K was disrupted and the mutant allele should produce no protein. A total of eight independent knock-out alleles were isolated by following phenotypic markers, genetic mapping and molecular screening. Two of these namely dPIP5K18 and dPIP5K30 were studied in detail and are described in this study. All eight alleles were semi-lethal; very few homozygous mutant flies emerged as viable adults. Using a polyclonal antibody generated against a relatively unique C-terminal region of dPIP5K, we found that dPIP5K18 and dPIP5K30 were protein alleles of dPIP5K (Fig. 2A).

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