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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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dPIP5K is not required to support cytoskeleton function and dynamin mediated endocytosis in adult photoreceptors.(A) Confocal images of phalloidin stained retinae from control and dPIP5K18 photoreceptors showing normal staining of the rhabdomeres. (B) Confocal images of retinae stained with a p-moesin specific antibody from control and dPIP5K18. Red pseudo color represents p-moesin staining and green marks the rhabdomeric region stained with phalloidin. (C) Confocal images showing longitudinal sections from retinae stained with an antibody to Rh1. The arrows indicated Rhodopsin Loaded Vesicle (RLV) involved in Rh1 endocytosis and recycling. (D) Rate of photoreceptor degeneration in norpAP24 and norpAP24; dPIP5K18 monitored using optical neutralization. The flies were reared in continuous light at 2300 lux. The X-axis represents age of the flies and the Y-axis represents number of rhabdomere visualized in each ommatidium. Error bars represent mean +/− S.D from 50 ommatidia taken from at least five flies. (E) Representative transmission electron micrographs of retinae from norpAP24 and norpAP24; dPIP5K18 flies showing the degree of preservation of ultrastructure. Images shown are from flies that are three days old gown under the same illumination conditions as for panel D. (F) Quantitative representation of Rhodopsin Loaded Vesicles (RLVs) in control, norpAp24 and norpAp24;dPIP5K18. The genotype of the fly is shown in the X-axis and Y-axis represents the count of RLVs.
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pgen.1004948.g005: dPIP5K is not required to support cytoskeleton function and dynamin mediated endocytosis in adult photoreceptors.(A) Confocal images of phalloidin stained retinae from control and dPIP5K18 photoreceptors showing normal staining of the rhabdomeres. (B) Confocal images of retinae stained with a p-moesin specific antibody from control and dPIP5K18. Red pseudo color represents p-moesin staining and green marks the rhabdomeric region stained with phalloidin. (C) Confocal images showing longitudinal sections from retinae stained with an antibody to Rh1. The arrows indicated Rhodopsin Loaded Vesicle (RLV) involved in Rh1 endocytosis and recycling. (D) Rate of photoreceptor degeneration in norpAP24 and norpAP24; dPIP5K18 monitored using optical neutralization. The flies were reared in continuous light at 2300 lux. The X-axis represents age of the flies and the Y-axis represents number of rhabdomere visualized in each ommatidium. Error bars represent mean +/− S.D from 50 ommatidia taken from at least five flies. (E) Representative transmission electron micrographs of retinae from norpAP24 and norpAP24; dPIP5K18 flies showing the degree of preservation of ultrastructure. Images shown are from flies that are three days old gown under the same illumination conditions as for panel D. (F) Quantitative representation of Rhodopsin Loaded Vesicles (RLVs) in control, norpAp24 and norpAp24;dPIP5K18. The genotype of the fly is shown in the X-axis and Y-axis represents the count of RLVs.

Mentions: PIP5K has been shown to have a role in actin remodeling in both yeast and mammalian systems [25]. In dPIP5K18, photoreceptor ultrastructure was largely normal by TEM analysis and growing flies under conditions of bright light illumination did not result in disruption of microvillar ultrastructure. This suggests that the actin cytoskeleton is unaffected by the absence of dPIP5K activity. Further, phalloidin staining suggested that the actin cytoskeleton was largely unaffected in dPIP5K18 (Fig. 5A). The Ezrin/Radixin/Moesin (ERM) family of proteins are regulated by PIP2 and act as cross-linkers between cortical actin and plasma membrane thus playing a key role in maintaining membrane projections, such as microvilli and filopodia [26][27]. ERM proteins are regulated by PIP2 and in the presence of PIP2 the active, phosphorylated form of the protein is attached to the membrane; on PIP2 hydrolysis the proteins are dephosphorylated and the inactive form is released to cytosol [28]. Drosophila has only a single ERM protein, dMoesin, which is required for morphogenesis and maintenance of microvillar structure in photoreceptors. Moesin localizes to the base of the rhabdomere in wild-type flies in the dark [29], but is dephosphorylated and translocates to the cytosol under bright illumination. Using immunolabelling we studied the distribution of p-Moesin in photoreceptors and found this to be no different between controls and dPIP5K18(Fig. 5B). Collectively these findings suggest that dPIP5K activity is not required to support cytoskeletal function in adult Drosophila photoreceptors.


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 not required to support cytoskeleton function and dynamin mediated endocytosis in adult photoreceptors.(A) Confocal images of phalloidin stained retinae from control and dPIP5K18 photoreceptors showing normal staining of the rhabdomeres. (B) Confocal images of retinae stained with a p-moesin specific antibody from control and dPIP5K18. Red pseudo color represents p-moesin staining and green marks the rhabdomeric region stained with phalloidin. (C) Confocal images showing longitudinal sections from retinae stained with an antibody to Rh1. The arrows indicated Rhodopsin Loaded Vesicle (RLV) involved in Rh1 endocytosis and recycling. (D) Rate of photoreceptor degeneration in norpAP24 and norpAP24; dPIP5K18 monitored using optical neutralization. The flies were reared in continuous light at 2300 lux. The X-axis represents age of the flies and the Y-axis represents number of rhabdomere visualized in each ommatidium. Error bars represent mean +/− S.D from 50 ommatidia taken from at least five flies. (E) Representative transmission electron micrographs of retinae from norpAP24 and norpAP24; dPIP5K18 flies showing the degree of preservation of ultrastructure. Images shown are from flies that are three days old gown under the same illumination conditions as for panel D. (F) Quantitative representation of Rhodopsin Loaded Vesicles (RLVs) in control, norpAp24 and norpAp24;dPIP5K18. The genotype of the fly is shown in the X-axis and Y-axis represents the count of RLVs.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4310717&req=5

pgen.1004948.g005: dPIP5K is not required to support cytoskeleton function and dynamin mediated endocytosis in adult photoreceptors.(A) Confocal images of phalloidin stained retinae from control and dPIP5K18 photoreceptors showing normal staining of the rhabdomeres. (B) Confocal images of retinae stained with a p-moesin specific antibody from control and dPIP5K18. Red pseudo color represents p-moesin staining and green marks the rhabdomeric region stained with phalloidin. (C) Confocal images showing longitudinal sections from retinae stained with an antibody to Rh1. The arrows indicated Rhodopsin Loaded Vesicle (RLV) involved in Rh1 endocytosis and recycling. (D) Rate of photoreceptor degeneration in norpAP24 and norpAP24; dPIP5K18 monitored using optical neutralization. The flies were reared in continuous light at 2300 lux. The X-axis represents age of the flies and the Y-axis represents number of rhabdomere visualized in each ommatidium. Error bars represent mean +/− S.D from 50 ommatidia taken from at least five flies. (E) Representative transmission electron micrographs of retinae from norpAP24 and norpAP24; dPIP5K18 flies showing the degree of preservation of ultrastructure. Images shown are from flies that are three days old gown under the same illumination conditions as for panel D. (F) Quantitative representation of Rhodopsin Loaded Vesicles (RLVs) in control, norpAp24 and norpAp24;dPIP5K18. The genotype of the fly is shown in the X-axis and Y-axis represents the count of RLVs.
Mentions: PIP5K has been shown to have a role in actin remodeling in both yeast and mammalian systems [25]. In dPIP5K18, photoreceptor ultrastructure was largely normal by TEM analysis and growing flies under conditions of bright light illumination did not result in disruption of microvillar ultrastructure. This suggests that the actin cytoskeleton is unaffected by the absence of dPIP5K activity. Further, phalloidin staining suggested that the actin cytoskeleton was largely unaffected in dPIP5K18 (Fig. 5A). The Ezrin/Radixin/Moesin (ERM) family of proteins are regulated by PIP2 and act as cross-linkers between cortical actin and plasma membrane thus playing a key role in maintaining membrane projections, such as microvilli and filopodia [26][27]. ERM proteins are regulated by PIP2 and in the presence of PIP2 the active, phosphorylated form of the protein is attached to the membrane; on PIP2 hydrolysis the proteins are dephosphorylated and the inactive form is released to cytosol [28]. Drosophila has only a single ERM protein, dMoesin, which is required for morphogenesis and maintenance of microvillar structure in photoreceptors. Moesin localizes to the base of the rhabdomere in wild-type flies in the dark [29], but is dephosphorylated and translocates to the cytosol under bright illumination. Using immunolabelling we studied the distribution of p-Moesin in photoreceptors and found this to be no different between controls and dPIP5K18(Fig. 5B). Collectively these findings suggest that dPIP5K activity is not required to support cytoskeletal function in adult Drosophila photoreceptors.

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