PMC

(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.

(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).

(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 norpA^P24 and norpA^P24; 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 norpA^P24 and norpA^P24; 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, norpA^p24 and norpA^p24;dPIP5K¹⁸. The genotype of the fly is shown in the X-axis and Y-axis represents the count of RLVs.

A) Multiple alignment of the protein sequences of PIP4K and PIP5K genes. The sequence around the activation loop is presented with the region indicated by a grey line. Amino acid residues common between PIP5K and PIP4K proteins are marked in blue; residues unique to PIP5K are marked in yellow and unique residues for PIP4K in grey. The red arrow indicates the single residue described as responsible for the unique substrate specificity of PIP4K and PIP5K. Notations used for gene names are; hs-Homo sapiens; a-alpha; b-beta; g-gamma; CG17471-Drosophila PIP4K. B) RNA expression pattern of PIP kinases in various fly tissues: Qualitative RT (reverse transcription) PCR analysis with RNA extracted from various fly tissues. The tissue sources are labeled above the lanes. ‘+’ denotes +RT and ‘−’ denotes −RT. The corresponding gene names are indicated on the left side of the agarose gel picture. C) Comparative real time PCR analysis showing eye enrichment of dPIP5K and dPIP4K; the X-axis indicates gene names and the Y-axis represents transcript level expression in arbitrary units (A.U). White bars represent expression levels from cDNA samples of wild type fly heads and black bars represent samples from heads of so^D (mutants that lack eyes). Values shown are the means ± S.D of three independent samples. p values between wild type and so^D samples were determined using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05)

(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 ). 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.

(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 norpA^P24. The genotypes used for the image acquisition are labeled at the left of the image panel. norpA^P24, which is a protein null 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).

(A) Representative ERG traces depicting the response of control, dPIP5K¹⁸, rdgB⁹ and rdgB⁹; dPIP5K¹⁸. Responses to single 2s flash of green light (intensity 5 cf. X-axis of ) are depicted. The genotypes corresponding to each trace are indicated on the top of the graph. 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. (B) Comparison of maximum amplitude of the light response among control, dPIP5K¹⁸, rdgB⁹ and rdgB⁹; dPIP5K¹⁸. Y-axis represents mean +/− S.D of the peak amplitude from five flies. (C) Representative optical neutralization images from control, rdgB⁹, dPIP5K¹⁸ and rdgB⁹; dPIP5K¹⁸ showing the exacerbation of degeneration in rdgB⁹; dPIP5K¹⁸ compared to rdgB⁹, dPIP5K¹⁸ alone does not show any degeneration. The representative images shown are collected from one-day-old flies maintained in L/D cycle (900 lux). (D) Quantification of the effect of dPIP5K¹⁸ loss of function on photoreceptor structure in rdgB⁹. (E) Schematic diagram showing the light induced PIP₂ cycle in Drosophila photoreceptors. Genes encoding a given enzyme activity where identified are marked in italics. Notations used: PI(4,5)P₂-phosphatidylinositol 4,5 bisphosphate, norpA- no receptor potential A, PLCβ-phospholipase C beta, DAG- diacylglycerol, DGK- diacylglycerol kinase, rdgA- retinal degeneration A, laza-lipid phosphate phosphohydrolase (PA phosphatase), PA-phosphatidic acid, CDP-DAG- Cytidine diphosphate diacylglycerol; CDS CDP-DAG synthase, PI- phosphatidylinositol, rdgB- retinal degeneration B, PITP- phosphatidylinositol transfer protein, PI(4)P- phosphatidylinositol 4-phosphate (F) Schematic representation of the pools of PIP₂ in Drosophila photoreceptor membranes. Representations are only semi-quantitative. The total pool of PIP₂ in the plasma membrane is shown bounded by the solid black line. The PLC sensitive PIP₂ pool sensitive to light induced PLC activity is shown in the rectangle bounded by the broken/dashed line indicating that PLC will likely also use a non-dPIP5K dependent pool. The basal PIP₂ pool is indicated in green. The major function of each pool is indicated. Enzymes responsible for the synthesis of each pool are marked.