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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 PIP2 dynamics in Drosophila photoreceptors.(A) Diagrammatic representation of the experimental protocol used to study PIP2 dynamics in the intact eye. The blue symbols indicate the time of image acquisition. The color of the bar indicates the light condition at which the fly was kept during the experiment. Black color indicates total dark and red indicates in red light illumination. The time points are labeled above the bar in minute. The detailed experimental procedure is discussed in material and methods section. (B) Fluorescent deep pseudopupil (dpp) imaging to study PIP2 dynamics using flies expressing the PIP2 biosensor (see main text for details). The time scale of the imaging is indicated on the top of each panel. Arrows indicate the timing of a 90 ms flash of blue light used for imaging the dpp. Images were acquired from control, dPIP5K18 and norpAP24. The genotypes used for the image acquisition are labeled at the left of the image panel. norpAP24, which is a protein  mutant of PLCβ, is used to show the dependence of dpp dynamics on PLCβ activity. (C) Quantitative representation of PIP2 dynamics. X-axis represents time in minutes between the depleting flash of blue light and the next image acquired. During this period eyes were illuminated in red light. Y-axis represents the level of fluorescence represented as a % of the value in the initial image. Error bars represents mean +/− S.D from five flies. p values were calculated using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05) (D) Western blot from head extracts depicting the level of dPIP5K protein expression in wild type flies and those overexpressing dPIP5K. The blot was probed with antibody to dPIP5K. Tubulin was used as loading control. (E) Representative images of dpp imaging in control flies and those overexpressing dPIP5K. (F) Quantification of PIP2 dynamics in flies overexpressing dPIP5K compared to controls. X-axis represents time in minutes and Y-axis represents the level of fluorescence represented as a % of the value in the initial image. Error bars represents mean +/− S.D from five flies. p values were determined using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05).
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pgen.1004948.g004: dPIP5K controls PIP2 dynamics in Drosophila photoreceptors.(A) Diagrammatic representation of the experimental protocol used to study PIP2 dynamics in the intact eye. The blue symbols indicate the time of image acquisition. The color of the bar indicates the light condition at which the fly was kept during the experiment. Black color indicates total dark and red indicates in red light illumination. The time points are labeled above the bar in minute. The detailed experimental procedure is discussed in material and methods section. (B) Fluorescent deep pseudopupil (dpp) imaging to study PIP2 dynamics using flies expressing the PIP2 biosensor (see main text for details). The time scale of the imaging is indicated on the top of each panel. Arrows indicate the timing of a 90 ms flash of blue light used for imaging the dpp. Images were acquired from control, dPIP5K18 and norpAP24. The genotypes used for the image acquisition are labeled at the left of the image panel. norpAP24, which is a protein mutant of PLCβ, is used to show the dependence of dpp dynamics on PLCβ activity. (C) Quantitative representation of PIP2 dynamics. X-axis represents time in minutes between the depleting flash of blue light and the next image acquired. During this period eyes were illuminated in red light. Y-axis represents the level of fluorescence represented as a % of the value in the initial image. Error bars represents mean +/− S.D from five flies. p values were calculated using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05) (D) Western blot from head extracts depicting the level of dPIP5K protein expression in wild type flies and those overexpressing dPIP5K. The blot was probed with antibody to dPIP5K. Tubulin was used as loading control. (E) Representative images of dpp imaging in control flies and those overexpressing dPIP5K. (F) Quantification of PIP2 dynamics in flies overexpressing dPIP5K compared to controls. X-axis represents time in minutes and Y-axis represents the level of fluorescence represented as a % of the value in the initial image. Error bars represents mean +/− S.D from five flies. p values were determined using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05).

Mentions: dPIP5K is a PIP kinase that is predicted to convert PI4P into PIP2. To test its requirement in regulating the dynamics of light induced PIP2 turnover at the rhabdomeral membrane we used a live fly preparation in which a fluorescent biosensor consisting of the PH domain of PLCδ fused to GFP (hereafter called PIP2 biosensor) is expressed in photoreceptors. When eyes are illuminated with bright light (λmax 488 nm) high rates of PLC activation result in the hydrolysis of PIP2 and as a consequence the fluorescent biosensor is detached from the membrane and diffuses out of the microvillar cytoplasm resulting in the loss of the fluorescent pseudopupil signal. Under red light illumination that converts metarhodopsin to rhodopsin thus terminating PLCβ activity, PIP2 levels recover as a consequence of ongoing PIP2 resynthesis and the PIP2 biosensor signal in the pseudopupil recovers (Fig. 4A,B). The kinetics of the fluorescent pseudopupil are blocked in norpAP24 which lacks appreciable PLCβ activity (Fig. 4B). Using this approach we studied the kinetics of light induced PIP2 turnover in dPIP5K18 and compared it to wild type controls. This analysis revealed a clear delay in the kinetics of PIP2 resynthesis in dPIP5K18 compared to wild type controls and suggests that dPIP5K activity is required to support normal PIP2 resynthesis following phototransduction (Fig. 4B).


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 PIP2 dynamics in Drosophila photoreceptors.(A) Diagrammatic representation of the experimental protocol used to study PIP2 dynamics in the intact eye. The blue symbols indicate the time of image acquisition. The color of the bar indicates the light condition at which the fly was kept during the experiment. Black color indicates total dark and red indicates in red light illumination. The time points are labeled above the bar in minute. The detailed experimental procedure is discussed in material and methods section. (B) Fluorescent deep pseudopupil (dpp) imaging to study PIP2 dynamics using flies expressing the PIP2 biosensor (see main text for details). The time scale of the imaging is indicated on the top of each panel. Arrows indicate the timing of a 90 ms flash of blue light used for imaging the dpp. Images were acquired from control, dPIP5K18 and norpAP24. The genotypes used for the image acquisition are labeled at the left of the image panel. norpAP24, which is a protein  mutant of PLCβ, is used to show the dependence of dpp dynamics on PLCβ activity. (C) Quantitative representation of PIP2 dynamics. X-axis represents time in minutes between the depleting flash of blue light and the next image acquired. During this period eyes were illuminated in red light. Y-axis represents the level of fluorescence represented as a % of the value in the initial image. Error bars represents mean +/− S.D from five flies. p values were calculated using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05) (D) Western blot from head extracts depicting the level of dPIP5K protein expression in wild type flies and those overexpressing dPIP5K. The blot was probed with antibody to dPIP5K. Tubulin was used as loading control. (E) Representative images of dpp imaging in control flies and those overexpressing dPIP5K. (F) Quantification of PIP2 dynamics in flies overexpressing dPIP5K compared to controls. X-axis represents time in minutes and Y-axis represents the level of fluorescence represented as a % of the value in the initial image. Error bars represents mean +/− S.D from five flies. p values were determined using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4310717&req=5

pgen.1004948.g004: dPIP5K controls PIP2 dynamics in Drosophila photoreceptors.(A) Diagrammatic representation of the experimental protocol used to study PIP2 dynamics in the intact eye. The blue symbols indicate the time of image acquisition. The color of the bar indicates the light condition at which the fly was kept during the experiment. Black color indicates total dark and red indicates in red light illumination. The time points are labeled above the bar in minute. The detailed experimental procedure is discussed in material and methods section. (B) Fluorescent deep pseudopupil (dpp) imaging to study PIP2 dynamics using flies expressing the PIP2 biosensor (see main text for details). The time scale of the imaging is indicated on the top of each panel. Arrows indicate the timing of a 90 ms flash of blue light used for imaging the dpp. Images were acquired from control, dPIP5K18 and norpAP24. The genotypes used for the image acquisition are labeled at the left of the image panel. norpAP24, which is a protein mutant of PLCβ, is used to show the dependence of dpp dynamics on PLCβ activity. (C) Quantitative representation of PIP2 dynamics. X-axis represents time in minutes between the depleting flash of blue light and the next image acquired. During this period eyes were illuminated in red light. Y-axis represents the level of fluorescence represented as a % of the value in the initial image. Error bars represents mean +/− S.D from five flies. p values were calculated using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05) (D) Western blot from head extracts depicting the level of dPIP5K protein expression in wild type flies and those overexpressing dPIP5K. The blot was probed with antibody to dPIP5K. Tubulin was used as loading control. (E) Representative images of dpp imaging in control flies and those overexpressing dPIP5K. (F) Quantification of PIP2 dynamics in flies overexpressing dPIP5K compared to controls. X-axis represents time in minutes and Y-axis represents the level of fluorescence represented as a % of the value in the initial image. Error bars represents mean +/− S.D from five flies. p values were determined using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05).
Mentions: dPIP5K is a PIP kinase that is predicted to convert PI4P into PIP2. To test its requirement in regulating the dynamics of light induced PIP2 turnover at the rhabdomeral membrane we used a live fly preparation in which a fluorescent biosensor consisting of the PH domain of PLCδ fused to GFP (hereafter called PIP2 biosensor) is expressed in photoreceptors. When eyes are illuminated with bright light (λmax 488 nm) high rates of PLC activation result in the hydrolysis of PIP2 and as a consequence the fluorescent biosensor is detached from the membrane and diffuses out of the microvillar cytoplasm resulting in the loss of the fluorescent pseudopupil signal. Under red light illumination that converts metarhodopsin to rhodopsin thus terminating PLCβ activity, PIP2 levels recover as a consequence of ongoing PIP2 resynthesis and the PIP2 biosensor signal in the pseudopupil recovers (Fig. 4A,B). The kinetics of the fluorescent pseudopupil are blocked in norpAP24 which lacks appreciable PLCβ activity (Fig. 4B). Using this approach we studied the kinetics of light induced PIP2 turnover in dPIP5K18 and compared it to wild type controls. This analysis revealed a clear delay in the kinetics of PIP2 resynthesis in dPIP5K18 compared to wild type controls and suggests that dPIP5K activity is required to support normal PIP2 resynthesis following phototransduction (Fig. 4B).

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