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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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Subcellular localization of different PIPKs in adult photoreceptors.(A) Western blot showing localization of dPIP5K in different sub-cellular fractions prepared from adult Drosophila heads. Fractions shown are: HL-total head lysate; C-cytoplasm; N, nuclear; M-microsomal/membrane. An antibody to INAD is used as a membrane marker. Histone 1 has been used as a nuclear marker and Tubulin as a marker for cytosol. (B) Confocal image showing the distribution of endogenous dPIP5K as detected by a polyclonal antibody from wild type and dPIP5K18 photoreceptors. The enrichment of dPIP5K staining at the rhabdomere membrane (arrow) in wild type is missing in dPIP5K18. (C) Double staining experiments on wild type retinae showing the co-localization of dPIP5K (green) with Rh1 (red). (D) Localization of dPIP5K overexpressed from its endogenous genomic locus. Confocal image from retinae of control flies and those overexpressing dPIP5K using Rh1-GAL4. The protein is shown localized to the rhabdomere membrane. (E) Confocal image showing localization of overexpressed dPIP4K in adult Drosophila photoreceptors. Phalloidin staining marking the rhabdomeres shown in green and dPIP4K localization detected by antibody labeling shown in red. Merged image shows that dPIP4K is excluded from the rhabdomeres.
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pgen.1004948.g006: Subcellular localization of different PIPKs in adult photoreceptors.(A) Western blot showing localization of dPIP5K in different sub-cellular fractions prepared from adult Drosophila heads. Fractions shown are: HL-total head lysate; C-cytoplasm; N, nuclear; M-microsomal/membrane. An antibody to INAD is used as a membrane marker. Histone 1 has been used as a nuclear marker and Tubulin as a marker for cytosol. (B) Confocal image showing the distribution of endogenous dPIP5K as detected by a polyclonal antibody from wild type and dPIP5K18 photoreceptors. The enrichment of dPIP5K staining at the rhabdomere membrane (arrow) in wild type is missing in dPIP5K18. (C) Double staining experiments on wild type retinae showing the co-localization of dPIP5K (green) with Rh1 (red). (D) Localization of dPIP5K overexpressed from its endogenous genomic locus. Confocal image from retinae of control flies and those overexpressing dPIP5K using Rh1-GAL4. The protein is shown localized to the rhabdomere membrane. (E) Confocal image showing localization of overexpressed dPIP4K in adult Drosophila photoreceptors. Phalloidin staining marking the rhabdomeres shown in green and dPIP4K localization detected by antibody labeling shown in red. Merged image shows that dPIP4K is excluded from the rhabdomeres.

Mentions: Given our prediction that dPIP5K generates PIP2 that is used as a substrate for light induced PLCβ activity, it is likely that the enzyme is localized to the microvillar plasma membrane where PLCβ is localized. Initial fractionation experiments showed that almost all of the dPIP5K is localized to a membrane fraction (Fig. 6A) where it co-fractionates with key phototransduction proteins such as INAD. We attempted to establish the localization of dPIP5K expressed at endogenous levels using immunocytochemistry; under these conditions the dPIP5K antibody was able to detect the protein localized at the microvillar membrane (Fig. 6B); this was abolished in retinae from dPIP5K18 photoreceptors that are protein alleles for this gene. Double labeling experiments showed that dPIP5K co-localizes with Rh1 at the microvillar membrane (Fig. 6C). We exploited a genetic tool [34] that allowed us to elevate the expression level of untagged endogenous dPIP5K in photoreceptors, expressed from the endogenous gene locus. Under these conditions too, we found dPIP5K clearly localized to the microvillar membrane (Fig. 6D). By contrast a dPIP4K::GFP transgene was excluded from the microvillar plasma membrane (Fig. 6E).


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)

Subcellular localization of different PIPKs in adult photoreceptors.(A) Western blot showing localization of dPIP5K in different sub-cellular fractions prepared from adult Drosophila heads. Fractions shown are: HL-total head lysate; C-cytoplasm; N, nuclear; M-microsomal/membrane. An antibody to INAD is used as a membrane marker. Histone 1 has been used as a nuclear marker and Tubulin as a marker for cytosol. (B) Confocal image showing the distribution of endogenous dPIP5K as detected by a polyclonal antibody from wild type and dPIP5K18 photoreceptors. The enrichment of dPIP5K staining at the rhabdomere membrane (arrow) in wild type is missing in dPIP5K18. (C) Double staining experiments on wild type retinae showing the co-localization of dPIP5K (green) with Rh1 (red). (D) Localization of dPIP5K overexpressed from its endogenous genomic locus. Confocal image from retinae of control flies and those overexpressing dPIP5K using Rh1-GAL4. The protein is shown localized to the rhabdomere membrane. (E) Confocal image showing localization of overexpressed dPIP4K in adult Drosophila photoreceptors. Phalloidin staining marking the rhabdomeres shown in green and dPIP4K localization detected by antibody labeling shown in red. Merged image shows that dPIP4K is excluded from the rhabdomeres.
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Related In: Results  -  Collection

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pgen.1004948.g006: Subcellular localization of different PIPKs in adult photoreceptors.(A) Western blot showing localization of dPIP5K in different sub-cellular fractions prepared from adult Drosophila heads. Fractions shown are: HL-total head lysate; C-cytoplasm; N, nuclear; M-microsomal/membrane. An antibody to INAD is used as a membrane marker. Histone 1 has been used as a nuclear marker and Tubulin as a marker for cytosol. (B) Confocal image showing the distribution of endogenous dPIP5K as detected by a polyclonal antibody from wild type and dPIP5K18 photoreceptors. The enrichment of dPIP5K staining at the rhabdomere membrane (arrow) in wild type is missing in dPIP5K18. (C) Double staining experiments on wild type retinae showing the co-localization of dPIP5K (green) with Rh1 (red). (D) Localization of dPIP5K overexpressed from its endogenous genomic locus. Confocal image from retinae of control flies and those overexpressing dPIP5K using Rh1-GAL4. The protein is shown localized to the rhabdomere membrane. (E) Confocal image showing localization of overexpressed dPIP4K in adult Drosophila photoreceptors. Phalloidin staining marking the rhabdomeres shown in green and dPIP4K localization detected by antibody labeling shown in red. Merged image shows that dPIP4K is excluded from the rhabdomeres.
Mentions: Given our prediction that dPIP5K generates PIP2 that is used as a substrate for light induced PLCβ activity, it is likely that the enzyme is localized to the microvillar plasma membrane where PLCβ is localized. Initial fractionation experiments showed that almost all of the dPIP5K is localized to a membrane fraction (Fig. 6A) where it co-fractionates with key phototransduction proteins such as INAD. We attempted to establish the localization of dPIP5K expressed at endogenous levels using immunocytochemistry; under these conditions the dPIP5K antibody was able to detect the protein localized at the microvillar membrane (Fig. 6B); this was abolished in retinae from dPIP5K18 photoreceptors that are protein alleles for this gene. Double labeling experiments showed that dPIP5K co-localizes with Rh1 at the microvillar membrane (Fig. 6C). We exploited a genetic tool [34] that allowed us to elevate the expression level of untagged endogenous dPIP5K in photoreceptors, expressed from the endogenous gene locus. Under these conditions too, we found dPIP5K clearly localized to the microvillar membrane (Fig. 6D). By contrast a dPIP4K::GFP transgene was excluded from the microvillar plasma membrane (Fig. 6E).

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