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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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dPIP5K18 photoreceptors have normal ultrastructure and unaltered levels of transduction proteins.(A) TEM images showing the ultrastructure of control (i-ii), dPIP5K30 (iii-iv) and dPIP5K18 (v-vi). The cross sectional view of a single ommatidium (i, iii, v) and a high magnification view of a single rhabdomere (ii, iv, vi) are shown for each genotype. (B) Optical neutralization images of dPIP5K18 retinae showing normal rhabdomere ultrastructure in flies grown in a 12h L/D cycle as well as in 24 hrs constant light. Images shown are from flies aged nine days post-eclosion. (C) Western blot analysis of head extracts from wild type, dPIP5K18, dPIP5K30 probed with antibodies to each of the major phototransduction proteins. The antibodies used are indicated at the right side of each panel. Tubulin is used as loading control for each set of blots. (D) Single optical transverse sections of a control and dPIP5K18 retina probed with antibodies to Rhodopsin (Rh1) (i, ii) and TRP (iii, iv).
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pgen.1004948.g003: dPIP5K18 photoreceptors have normal ultrastructure and unaltered levels of transduction proteins.(A) TEM images showing the ultrastructure of control (i-ii), dPIP5K30 (iii-iv) and dPIP5K18 (v-vi). The cross sectional view of a single ommatidium (i, iii, v) and a high magnification view of a single rhabdomere (ii, iv, vi) are shown for each genotype. (B) Optical neutralization images of dPIP5K18 retinae showing normal rhabdomere ultrastructure in flies grown in a 12h L/D cycle as well as in 24 hrs constant light. Images shown are from flies aged nine days post-eclosion. (C) Western blot analysis of head extracts from wild type, dPIP5K18, dPIP5K30 probed with antibodies to each of the major phototransduction proteins. The antibodies used are indicated at the right side of each panel. Tubulin is used as loading control for each set of blots. (D) Single optical transverse sections of a control and dPIP5K18 retina probed with antibodies to Rhodopsin (Rh1) (i, ii) and TRP (iii, iv).

Mentions: To test this hypothesis, we studied photoreceptor ultrastructure using transmission electron microscopy (TEM). This revealed that photoreceptors R1-R7 from 0 day old flies were normal in dPIP5K18(Fig. 3A). Microvilli were completely intact and showed no vesiculation or blebbing and only minimal defects were seen at the base of the microvilli; however these changes did not increase with age or illumination and rhabdomere structure remained completely intact (Fig. 3B).


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)

dPIP5K18 photoreceptors have normal ultrastructure and unaltered levels of transduction proteins.(A) TEM images showing the ultrastructure of control (i-ii), dPIP5K30 (iii-iv) and dPIP5K18 (v-vi). The cross sectional view of a single ommatidium (i, iii, v) and a high magnification view of a single rhabdomere (ii, iv, vi) are shown for each genotype. (B) Optical neutralization images of dPIP5K18 retinae showing normal rhabdomere ultrastructure in flies grown in a 12h L/D cycle as well as in 24 hrs constant light. Images shown are from flies aged nine days post-eclosion. (C) Western blot analysis of head extracts from wild type, dPIP5K18, dPIP5K30 probed with antibodies to each of the major phototransduction proteins. The antibodies used are indicated at the right side of each panel. Tubulin is used as loading control for each set of blots. (D) Single optical transverse sections of a control and dPIP5K18 retina probed with antibodies to Rhodopsin (Rh1) (i, ii) and TRP (iii, iv).
© Copyright Policy
Related In: Results  -  Collection

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pgen.1004948.g003: dPIP5K18 photoreceptors have normal ultrastructure and unaltered levels of transduction proteins.(A) TEM images showing the ultrastructure of control (i-ii), dPIP5K30 (iii-iv) and dPIP5K18 (v-vi). The cross sectional view of a single ommatidium (i, iii, v) and a high magnification view of a single rhabdomere (ii, iv, vi) are shown for each genotype. (B) Optical neutralization images of dPIP5K18 retinae showing normal rhabdomere ultrastructure in flies grown in a 12h L/D cycle as well as in 24 hrs constant light. Images shown are from flies aged nine days post-eclosion. (C) Western blot analysis of head extracts from wild type, dPIP5K18, dPIP5K30 probed with antibodies to each of the major phototransduction proteins. The antibodies used are indicated at the right side of each panel. Tubulin is used as loading control for each set of blots. (D) Single optical transverse sections of a control and dPIP5K18 retina probed with antibodies to Rhodopsin (Rh1) (i, ii) and TRP (iii, iv).
Mentions: To test this hypothesis, we studied photoreceptor ultrastructure using transmission electron microscopy (TEM). This revealed that photoreceptors R1-R7 from 0 day old flies were normal in dPIP5K18(Fig. 3A). Microvilli were completely intact and showed no vesiculation or blebbing and only minimal defects were seen at the base of the microvilli; however these changes did not increase with age or illumination and rhabdomere structure remained completely intact (Fig. 3B).

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