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Rhabdomere biogenesis in Drosophila photoreceptors is acutely sensitive to phosphatidic acid levels
Correspondence to Padinjat Raghu: raghu.padinjat{at}bbsrc.ac.uk
Phosphatidic acid (PA) is postulated to have both structural and signaling functions during membrane dynamics in animal cells. In this study, we show that before a critical time period during rhabdomere biogenesis in Drosophila melanogaster photoreceptors, elevated levels of PA disrupt membrane transport to the apical domain. Lipidomic analysis shows that this effect is associated with an increase in the abundance of a single, relatively minor molecular species of PA. These transport defects are dependent on the activation state of Arf1. Transport defects via PA generated by phospholipase D require the activity of type I phosphatidylinositol (PI) 4 phosphate 5 kinase, are phenocopied by knockdown of PI 4 kinase, and are associated with normal endoplasmic reticulum to Golgi transport. We propose that PA levels are critical for apical membrane transport events required for rhabdomere biogenesis.
© 2009 Raghu et al.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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In the simple eukaryote Saccharomyces cerevisiae that recapitulates most basal transport pathways conserved in higher eukaryotes, genetic analysis has implicated several lipids in regulating membrane traffic. Evidence showing that DAG and PA can affect membrane transport comes from yeast through analysis of SEC14, a gene that encodes a PI/PC transfer protein essential for viability and transport from the Golgi (Bankaitis et al., 1990). The sec14 phenotype can be suppressed/bypassed by mutants in several genes that control biosynthesis of PI and PC (Cleves et al., 1991). However, the ability of such mutants to bypass sec14 has an obligate requirement for SPO14 that encodes phospholipase D (PLD; Xie et al., 1998), an enzyme that generates PA from PC. Although Spo14p is not required for vegetative growth (Sreenivas et al., 1998; Xie et al., 1998), it is required to form the prospore membrane (Rudge et al., 1998) and for PA synthesis during sporulation (Rudge et al., 2001); loss of Spo14p leads to accumulation of undocked prospore membrane precursors vesicles on the spindle pole body (Nakanishi et al., 2006). Thus, in yeast, PA generated by Spo14p activity plays a key role in this membrane trafficking event. Although the analysis of spo14 has implicated PA and its downstream lipid metabolites in membrane transport, to date there is little direct evidence to suggest that PA can function as a regulator of membrane traffic in metazoans. The idea that PA can function in a signaling capacity during membrane transport has been fueled by the observations that (a) in vitro ADP ribosylation factor (Arf) proteins, key mediators of membrane transport, can regulate the activity of PLD (Brown et al., 1993; Cockcroft et al., 1994), (b) overexpression of PLD in several different cell types affects processes likely to require exocytosis (Vitale et al., 2001; Choi et al., 2002; Cockcroft et al., 2002; Huang et al., 2005), and (c) overexpression of mammalian PLD1 is reported to promote generation of β–amyloid precursor protein-containing vesicles from the TGN (Cai et al., 2006). However, the role of PA in regulating secretion in these settings remains unclear, and currently, there is little evidence linking demonstrable changes in PA levels with the molecular machinery that regulates membrane traffic in vivo.
In this study, we have used Drosophila melanogaster photoreceptors as a model system to test the effect of altered PA levels on membrane traffic. We show that elevated levels of PA disrupt membrane transport to the apical domain of photoreceptors with defects in the endomembrane system.
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90% of the membrane surface area of photoreceptors (Leonard et al., 1992); its growth is triggered during the last 30% of pupal development (pd) by a process of intense membrane biogenesis and polarized vesicle trafficking.
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Finally, we also tested the effect of overexpressing DGK encoded by the rdgA gene (Masai et al., 1993). This resulted in the accumulation of a large number of tubular and vesicular transport intermediates within the cell body (Fig. 2 H). Such structures were not seen in control photoreceptors (Fig. 2 D). Because increased levels of PA are the immediate common biochemical outcome of all three genetic manipulations, our findings strongly suggest that in vivo, elevated levels of PA are able to perturb endomembrane homeostasis in photoreceptors.
To test whether increased levels of PA are associated with this phenotype, we measured the levels of this lipid in retinal extracts from cds1, Pld, and rdgA overexpressing retinae using liquid chromatography followed by mass spectrometry. These experiments revealed no significant elevation in total retinal PA levels (unpublished data). However, because total PA includes contributions from a larger biosynthetic pool as well as a smaller signaling pool, we examined the abundance of 20 individual species of PA that can be separately quantified by liquid chromatography mass spectrometry. In the case of cds1, we found a significant elevation of a single species of PA (predicted fatty acyl composition 16:0/18:2, also called 34:2) relative to controls (Fig. 3 A). Remarkably, when Pld was overexpressed, a smaller elevation of the same species of PA was seen relative to both wild-type (no overexpression) as well as the overexpression of catalytically dead PLD (Fig. 3 B). Finally, in retinae overexpressing rdgA, an elevation in 16:0/18:2 PA was also seen (Fig. 3 C). The acyl chain composition of this species reflects that of the most abundant species of PI and PC in Drosophila retinal lipid extracts (unpublished data). PI and PC are the substrates from which PA is generated by PLC (in cds1 grown in bright light to trigger PA accumulation) and PLD (Pld overexpression), respectively. The levels of PC, DAG, and PI were not altered in retinae from these genotypes (Fig. 3, D–F). Our findings demonstrate a common biochemical basis, namely elevation in the abundance of a single molecular species of PA, for the endomembrane defects described in this study.
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Enhanced activity of Arf1 but not Arf6 mediates the effects of elevated PA levels
To understand the mechanism by which elevated PA disrupts rhabdomere biogenesis, we modulated the activity of two members of the Arf family of small GTPases that play a key role in the organization of secretory and endocytic membrane traffic (D'Souza-Schorey and Chavrier, 2006; Gillingham and Munro, 2007). Arf1 is thought to regulate the assembly of coat complexes along the early secretory pathway, notably between ER and Golgi, between the Golgi cisternae and at the trans-Golgi, whereas Arf6 is thought to regulate endosomal traffic at the plasma membrane. To alter the activity of Arf1 (class I Arf), we overexpressed the guanine nucleotide exchange factor (GEF) that is expected to enhance levels of active Arf1-GTP (Fig. 5 G). In Drosophila, the garz gene (Kraut et al., 2001) encodes a protein with homology to the mammalian Arf1-GEF Golgi-specific brefeldin resistance factor (Cox et al., 2004). To overexpress garz in photoreceptors, we used an enhancer promoter line inserted upstream of the garz gene that allows overexpression of endogenous garz using the GAL4/upstream activation sequence (UAS) system (Rorth, 1996). We overexpressed garz in developing photoreceptors using GMR-GAL4. TEM analysis revealed that overexpression of garz results in normal rhabdomere development with no abnormal accumulation of endomembranes in the cell body (Fig. 5 C). To test the consequence of enhanced Arf1 activity on photoreceptors with elevated PA levels, we overexpressed garz along with Pld. This resulted in a massive enhancement of the phenotype seen by overexpressing Pld alone (Fig. 5, compare C with E). In any given retina, we could at best detect one or two poorly developed rhabdomeres; apart from this, there was virtually no development of rhabdomeres. To test whether this was a direct consequence of enhanced Arf1 activity, we tested the effects of overexpressing the GTPase-activating protein (GAP) for Arf1. Arf1-GAP is expected to enhance the hydrolysis of GTP on GTP-Arf1 to GDP-Arf1, thereby reducing Arf1 activity (Fig. 5 G). To do this, we generated transgenic flies expressing Drosophila Arf1-GAP (dArf1-GAP), which is encoded by the gene Gap69C (Frolov and Alatortsev, 2001). When overexpressed in wild-type photoreceptors using the GAL4/UAS system, dArf1-GAP results in a mild rough eye phenotype. TEM revealed that rhabdomeres developed normally, and there were at best mild endomembrane defects in the cell body (Fig. 5 D). To examine the effect of reducing Arf1-GTP levels in photoreceptors with elevated PA levels, we coexpressed dArf1-GAP and Pld. This resulted in a substantial but incomplete rescue of rhabdomere size compared with flies overexpressing Pld alone (Fig. 5, compare D with F).
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-adaptin function does not suppress the effects of Pld overexpression-adaptin, a specific subunit of the AP-2 complex. The AP-2 complex is found at the plasma membrane and is not known to have a function at the TGN or endosomes (Robinson, 2004). Because null mutants in the only Drosophila gene-encoding -adaptin (CG4260) are homozygous lethal during embryonic development (Gonzalez-Gaitan and Jackle, 1997), we disrupted its function specifically in the eye by transgenic RNAi knockdown using GMR-GAL4 (Dietzl et al., 2007). Three independent RNAi lines for -adaptin were used with essentially similar results. Data are presented for one of the lines.
When -adaptin function is reduced by GMR-GAL4* UAS-RNAi-adaptin, rhabdomere biogenesis is disrupted. Individual rhabdomeres (Fig. 8, r) are smaller and ill formed (Fig. 8 H), the base of the rhabdomeres is distorted, and the cell bodies show accumulation of membranous structures (Fig. 8, m). We coexpressed Pld and UAS-RNAi-adaptin using GMR-GAL4. TEM analysis revealed that retinae from such animals have virtually no detectable rhabdomeres (Fig. 8 I). The cell bodies of the photoreceptors from such animals continued to show accumulation of membrane transport intermediates (Fig. 8, m). These results strongly suggest that reducing the function of AP-2 complexes cannot suppress the transport defects seen as a result of elevated PA levels.
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The effects of Pld overexpression require the activity of type I PIPkin
The activity of type I PI 4 phosphate 5 kinase (PIPkin), a key enzyme in the synthesis of cellular PI 4,5 bisphosphate (PI(4,5)P2), is known to be regulated by PA (Jenkins et al., 1994). To test whether type I PIPkin activity is required to mediate the effects of PA on transport in photoreceptors, we used a severe hypomorph in sktl (skittles), one of the two type I PIPkin genes encoded in the Drosophila genome (Hassan et al., 1998). Although the sktl gene product is essential for eye development and null mutants in this gene are cell lethal for photoreceptors (Hassan et al., 1998), the sktl
20/sktl1-1 allelic combination (that has <5% sktl RNA by quantitative PCR analysis) develops viable photoreceptors that appear normal (Fig. 9 B; Garcia-Murillas et al., 2006). We overexpressed Pld in sktl20/sktl1-1 photoreceptors; this revealed a dramatic increase in rhabdomere size compared with photoreceptors overexpressing Pld alone (Fig. 9, compare C with D). This finding strongly suggests a requirement for type I PIPkin activity in generating the endomembrane defects observed in photoreceptors with elevated PA levels. An immediate prediction of this observation is that overexpression of sktl in developing photoreceptors might also affect rhabdomere biogenesis. We tested this idea by overexpressing sktl in developing photoreceptors and compared its effect to that of expressing a point mutant shown to abolish the catalytic activity (kinase-dead sktl) of this enzyme (Ishihara et al., 1998). This study revealed that overexpression of wild-type sktl resulted in an almost complete block in rhabdomere biogenesis; in contrast, overexpression of kinase-dead sktl did not result in this phenotype (Fig. 9, compare F with G). The effects of elevating PA levels by Pld overexpression required the enzyme to be present before the onset of rhabdomere biogenesis (Fig. 4). If these effects are mediated by enhanced sktl activity, one might predict that the effects of sktl overexpression on rhabdomere biogenesis might also only work before this critical time window. To test this idea, we overexpressed sktl using Rh1-GAL4 and found that in contrast to overexpression using GMR-GAL4, there was no effect on rhabdomere biogenesis (Fig. 9, compare F with H). These findings suggest that enhanced type I PIPkin activity mediates the effects of elevated PA levels in developing photoreceptors.
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). This analysis revealed that down-regulation of CG2929 using RNAi phenocopied key aspects of the phenotype of Pld overexpression: (a) down-regulation in the levels of Rh1 protein (Fig. 10 C), (b) formation of small and deformed rhabdomere (Fig. 10 A), and (c) accumulation of abnormal endomembranes within the cell body (Fig. 10 B). These findings suggest that the activity of PI4K is important for membrane transport.
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Interestingly, although cds1, Pld, and rdgA overexpression all caused endomembrane defects in photoreceptors, the ultrastructural features of the abnormal transport intermediates were variable. All three genotypes showed variable degrees of defect in rhabdomere biogenesis. In addition, in the case of cds1, the accumulated endomembranes in the cell body resembled ER-like structures (Fig. 2 C); with Pld overexpression, there were concentric and sheetlike tubular membranes (Fig. 2 G), whereas with rdgA overexpression, in addition to tubular membranes, there were several vesicular intermediates that accumulated (Fig. 2 H). It is likely that these differences reflect the distinct subcellular locations at which PA accumulates in each genotype. In cds1, PA probably accumulates in the ER site at which CDP-DAG synthase activity is normally present; PLD localization is limited to a compartment at the base of the rhabdomeres, and when overexpressed, DGK is distributed in punctate fashion throughout the ER (unpublished data). The generation of a suitable probe to visualize PA levels in a spatial dimension will be required to address this issue.
During development, the precursor cells of the Drosophila eye undergo a substantial increase in size with the concomitant requirement for generating new plasma membrane (Longley and Ready, 1995). During the last 30% of pd, photoreceptors show an approximately fourfold increase in plasma membrane surface area (Raghu et al., 2000), a process that requires a massive surge in polarized membrane transport capacity starting at 70% pd. In this study, we have defined a critical time window 70% pd before which elevation of PA levels by overexpressing Pld results in the endomembrane defects. As this window precedes the onset of rapid membrane transport accompanying rhabdomere biogenesis, we postulate that PA regulates the activity of a component of the molecular machinery that mediates polarized membrane transport during this period. Conceptually, in this respect our findings are reminiscent of observations in the yeast Spo14 mutant, in which membrane transport defects are evident only during the generation of the prospore membrane. To our knowledge, these findings are the first report of regulation of polarized membrane transport by PA in metazoans.
PA affects Arf1-dependent transport
During this study, we observed that the effects of elevated PA (through both cds1 and Pld overexpression) were sensitive to the activation state of Arf1. In the cds1 mutant, in which PA is likely to be elevated in the ER, overexpression of the Arf1-GEF garz resulted in significantly less developed apical rhabdomere membrane (Fig. 6 B) but was not associated with enhanced accumulation of membranes in the cell body, which is consistent with the known effects of expressing constitutively active Arf1 in cells (Dascher and Balch, 1994). In contrast, overexpression of dArf1-GAP resulted in an enhancement of defective rhabdomere biogenesis as well as a massive accumulation of ER membrane–like intermediates in the cell body (Fig. 6 D). This observation suggests that the PA accumulating at the ER in cds1 influences the Arf1 cycle in this setting, resulting in the transport defects described. Previous biochemical analysis has shown that the activity of Arf1-GAP proteins can be regulated by at least three different lipids relevant to this study, namely PC, DAG (Antonny et al., 1997), and PA (Yanagisawa et al., 2002). In our lipidomic analysis of cds1 retinae, we found that the levels of 34:2 DAG and 34:2 PC were no different from wild type (Fig. 3 D), whereas levels of 34:2 PA were elevated. On the basis of these findings, it is likely that the 34:2 PA that accumulates in cds1 photoreceptors causes the transport defects we have described by down-regulating the activity of Arf1 via dArf1-GAP.
The development and maintenance of apical membranes in polarized cells requires both sorting at the TGN with exocytic transport as well as endocytosis (Bomsel et al., 1989). Thus, the phenotypes resulting from PLD overexpression could be a result of (a) altered membrane transport along one of the steps in the secretory pathway from the ER to the developing rhabdomere or (b) the consequence of enhanced endocytosis from the rhabdomere into the cell body.
Experimental evidence presented in this study shows that in photoreceptors overexpressing Pld, the defect in rhabdomere biogenesis was dependent on the levels of active Arf1. In contrast, we found that (a) altering the activity of Arf6, (b) down-regulation of -adaptin, and (c) a reduction in the function of dynamin (shi) did not suppress the effects of overexpressing Pld. Collectively, these three observations strongly suggest that excessive clathrin-mediated endocytosis of rhabdomeral plasma membrane does not underlie the endomembrane defects resulting from Pld overexpression. A recent study has suggested a role for Arf1 in regulating a dynamin-independent endocytic pathway in Drosophila cells (Kumari and Mayor, 2008). The role of this pathway in the effects of Pld overexpression remains unknown.
Arf1 also exerts several effects on distinct steps of the exocytic pathway, including bidirectional transport between the ER and Golgi (Lee et al., 2004) between Golgi cisternae and the regulation of exit from the late Golgi (D'Souza-Schorey and Chavrier, 2006). In photoreceptors overexpressing Pld, our analysis suggests that ER to trans-Golgi transport was normal (Fig. S2), implying that the observed phenotypes are likely to involve a transport step between the TGN and plasma membrane, although observed phenotypes do not phenocopy exocyst loss of function (Fig. S3). In Drosophila photoreceptors, PLD localizes to a restricted subcompartment at the base of the rhabdomeres (Fig. S5 A). Although the molecular identity of this compartment has not been established, its subcellular localization is consistent with the ability of PA produced by PLD to regulate transport between the rhabdomeres and cell body. In TEMs of photoreceptors overexpressing Pld, the endomembranes we observed in the cell body showed a tubulovesicular morphology extending throughout the cytoplasm (e.g., Fig. 2 E). These membranes resemble large pleiomorphic carriers (Polishchuk et al., 2000; Luini et al., 2005), transport intermediates that derive from the TGN destined for acceptor compartments like the plasma membrane. Furthermore, vesicles containing proteins destined for and normally restricted to the apical rhabdomere membrane (such as Rh1) are found in the cell body of photoreceptors overexpressing Pld (Fig. 4 F). These observations are particularly interesting in the light of previous studies suggesting that PA generated by PLD can regulate the release of vesicles from the Golgi in an Arf1-dependent manner (Ktistakis et al., 1996; Chen et al., 1997). However, in the absence of a clear identification of the accumulated membranes, the precise definition of the affected transport intermediates we have observed remains elusive.
Arf1 can influence several events at the TGN (for review see De Matteis and Luini, 2008), including the recruitment and activation of phospholipid-metabolizing enzymes. These include the recruitment and activation of PI4KIIIβ (Godi et al., 1999) generating PI(4)P as well a direct role in activating the type I PIPkin on Golgi membranes in vitro (Jones et al., 2000). During this study, we found that (a) down-regulating the levels of a PI4K expressed during photoreceptor development phenocopies key aspects of that seen with Pld overexpression (Fig. 10), and (b) a strong hypomorph of the type I PIPkin (sktl) was able to substantially suppress the effects of Pld overexpression on rhabdomere biogenesis. These observations reflect the importance of tightly regulating type I PIPkin activity by PA for normal transport to the apical domain in polarized cells. They suggest that the regulation of PI(4)P levels is critical for rhabdomere biogenesis. In the context of interpreting the effects of Pld overexpression, it is possible that raised PA levels lead to enhanced activity of type I PIPkin consuming PI(4)P at the TGN, resulting in consequent transport defects to the apical membrane. Although we have not been able to demonstrate reduced PI(4)P or increased PI(4,5)P2 levels at the Golgi in photoreceptors overexpressing Pld, our observation that overexpression of sktl in developing photoreceptors before the critical time window (but not a kinase-dead version) results in a massive defect in rhabdomere biogenesis underscores the importance of tight regulation of type I PIPkin activity during this process. Thus, a tight regulation of the balance of PI(4)P and PI(4,5)P2 levels through Arf1 activity may underlie the effects of PA in this system.
Given the large number of effectors that can be regulated by PA (Stace and Ktistakis, 2006), in the future, it will be important to identify and understand the functions of those that play a role in the biogenesis of rhabdomeres during photoreceptor development.
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EM
Eyes were prepared for histology by dissecting in ice-cold fixative (2% formaldehyde and 2.5% glutaraldehyde in 0.1 M Na cacodylate buffer, pH 7.3). After 4 h fixation at 4°C, eyes were buffer washed, postfixed in 1% OsO4 for 1 h, and stained en bloc in uranyl acetate for 1 h. Eyes were dehydrated in an alcohol series and embedded in Spurr's. 70-nm ultrathin sections were stained with uranyl acetate and lead citrate and viewed on a transmission electron microscope (CM100; Phillips).
Generation of Pld overexpression flies
The Pld cDNA used in this study originated from the clone pOT2a-GH07346 generated by the Berkeley Drosophila Genome Project. This clone has been fully sequenced by the Berkeley Drosophila Genome Project, and its sequence is available in GenBank (accession no. AF145640). Overexpression in the retina was perfomed using the modular GAL4-UAS system. To generate transgenic flies, the Pld open reading frame was subcloned into the EcoRI and NotI sites of pUAST (Brand and Perrimon, 1993). To generate a catalytically dead Pld (the K1086R mutation; Fig. S2), the wild-type gene was subcloned into pBluescript using EcoRI and NotI, and the mutation was introduced by standard site-directed mutagenesis. After sequencing, the K/R Pld was subcloned into the germline transformation vector pUAST using EcoRI/NotI. Germline transformation was performed using established protocols. w1118 embryos were injected, and several independent transgenic lines were obtained. They were mapped using standard genetic crosses to obtain stocks carrying single mapped insertions. Such stocks were used in the experiments described in this study.
Immunohistochemistry
Whole-mount immunohistochemistry was performed using a modification of previously published methods (Karagiosis and Ready, 2004). Retinae were dissected in ice-cold PBS and fixed with 4% paraformaldehyde in PBS for 1 h on ice. Fixed eyes were given three 10-min washes in PBST (PBS with 0.25% Triton X-100). Blocking was performed using 10% FBS for 1 h at room temperature. Incubations with primary antibodies (diluted in PBST with 5% FBS) were performed at 4°C overnight. After washes in PBST, samples were incubated in secondary antibodies diluted in PBST for 2 h at room temperature. Samples were washed in PBST and mounted in Citifluor (Agar Scientific). Samples were viewed using a confocal microscope (LSM 510 META; Carl Zeiss, Inc.) using either a Plan-Neofluar 40x NA 1.30 or a Plan-Apochromat 63x NA 1.40 oil objective (Carl Zeiss, Inc.).
Isolation of pure retinal tissue
Pure preparations of retinal tissue were collected using previously described methods (Garcia-Murillas et al., 2006). In brief, flies were snap frozen in liquid nitrogen and dehydrated in acetone at –20°C for 48 h. The acetone was drained off, and the retinae dried at room temperature. They were cleanly separated from the head at the level of the basement membrane using a flattened insect pin. This allows the preparation of a largely pure collection of retinae.
Heat shock expression experiments
Wandering third instar larvae were collected at pupariation (0% pd) and aged at 25°C on a moist Whatman paper in a Petri dish in a cooled incubator. Under these conditions, in our hands, adult flies eclosed at 100 h. Pupae from this collection were removed and subjected to a 1-h heat shock at 37°C in a water bath. After this, they were transferred into a vial with normal culture medium and allowed to complete development in the vial until adult flies emerged. 0–12-h-old flies were separated into nonexpressing controls and Pld-expressing animals (flies with curly or straight wings, respectively). They were decapitated using a sharp razor blade, and protein extracts were prepared from the heads as described in Western blot analysis.
Western blot analysis
Protein analysis was performed with flies aged 0–12 h posteclosion. Heads were prepared by decapitating flies cooled on ice. Where specified, the samples used were dissected freeze-dried retinae. Samples were homogenized in 2x SDS-PAGE sample buffer followed by boiling at 100°C for 1 min. Samples were separated using SDS-PAGE and electroblotted onto supported nitrocellulose membrane (Hybond-C extra; GE Healthcare) using wet transfer. The uniformity of transfer onto membranes was checked by staining with Ponceau S. After blocking in 5% nonfat milk (Marvel), blots were incubated for 1 h at room temperature in appropriate dilutions of primary antibody. Immunoreactive protein was visualized after incubation in a 1:10,000 dilution of donkey –rabbit IgG coupled to horseradish peroxidase (Jackson ImmunoResearch Laboratories) for 1 h at room temperature, and the blots were developed with ECL (GE Healthcare).
Antibodies
The following antibodies were used in this study: antirhodopsin mouse monoclonal 4C5 (Developmental Studies Hybridoma Bank), anti-Golgi mouse monoclonal 7H6D7C2 (EMD), anti-Bip rat monoclonal antibody clone 143 (Babraham Bioscience Technologies), antiwind rabbit polyclonal (provided by D. Ferrari, Max Planck Institute, Gottingen, Germany), antisyntaxin16 (provided by W. Trimble, University of Toronto, Toronto, Ontario, Canada), anti-PLC (provided by B. Hwa-Shieh, Vanderbilt University, Nashville, TN), anti-TRP (provided by C. Montell, John Hopkins University, Baltimore, MD), and anti-KDEL (provided by G. Butcher, The Babraham Institute, Cambridge, England, UK). For immunofluorescence, anti–mouse Alexa Fluor 488 and anti–rat Alexa Fluor 633 (Invitrogen) were used.
Generation of anti-Pld antibody
A fragment of Pld encompassing amino acids 607–789 (start, RWDDHHHRLT; end, GQEVAITTSS) was subcloned into the pGEX vector (GE Healthcare) and expressed as a GST fusion protein in Escherichia coli BL-21 cells. After purification of this fragment using standard procedures, 3 mg total protein was injected into rabbits to produce polyclonal antibodies. Highest titers were obtained from the final bleed, and most experiments shown have used this serum. In some experiments, antibodies were affinity purified as follows: 2 ml total serum was incubated with beads coupled to GST, and nonbound material was collected. This material was incubated with beads coupled to GST-PLD, and bound material was eluted with glycine, pH 2.5. After neutralization, the purified antibodies were aliquoted and stored at –20°C until use.
Analysis of retinal lipids
150 freeze-dried retinae (dissected as described in Isolation of pure retinal tissue) were homogenized in 0.5 ml methanol (containing 500 ng each of 12:0/12:0 species of DAG, PA, PC, phosphatidylethanol, PG, and PS as internal standards) using a 1-ml glass homogenizer until completely disrupted (>100 strokes). The methanolic homogenate was transferred into a glass screw-capped tube. Further methanol (0.5 ml) was used to wash the homogenizer and was combined in the glass tube. 2 ml chloroform was added and left to stand for 10 min. 0.88% KCl (1 ml) was added to split the phases. After removal of the upper phase and interfacial material, the lower organic phase containing the lipids was dried, resuspended in 15 µl chloroform, and finally transferred into a silanized autosampler vial ready for analysis.
Phospholipids (1-µl injection) were separated on a 1.0 x 150–mm silica column (3 µm, Luna; Phenomenex) using 100% chloroform/methanol/water (90:9.5:0.5) containing 7.5 mM ethylamine changing to 100% acetonitrile/chloroform/methanol/water (30:30:35:5) containing 10 mM ethylamine over 20 min at 100 µl/min. Detection was performed by electrospray ionization in both positive (PC) and negative modes (PA, phosphatidylethanol, PG, PI, and PS) on a single quadruple mass spectrometer (probe voltage, ±4 kV; nebulizer gas, 4 liters/min N2; desolvation line temperature, 300°C; QP8000; Shimadzu). Subsequent lipid analysis adopted a similar chromatographic separation but analyzed the lipid structures in a semiquantitative manner using a mass spectrometer (LCMS-IT-TOF; Shimadzu). The detection methodology of this machine has a considerably greater sensitivity and mass accuracy; consequently, the data in Fig. 3 (compare A and B with C) are qualitatively similar but slightly different quantitatively. In all graphs, the control and experimental data presented have been gathered on the same machine.
Generation of stable Pld-expressing S2 cell lines
The gene for Pld was subcloned using EcoRI/NotI into vector pCMV3-myc, which introduces a myc tag at the N terminus. The myc-Pld construct was subsequently subcloned into pRmHa-3 using Kpn1. The pRmHa-3-Pld vector was transfected into S2 cells using Fugene (Roche), and stable cell lines were selected after growth of resistant cells in 0.5 mg/ml of G418. In these cells, expression of Pld is inducible using CuSO4 (usually 500–700 µM overnight). A similar strategy was used to derive S2 cells expressing the Pld-K1086R catalytically inactive mutant.
Biochemical analysis of PLD activity
Measurement of PLD activity was done in stable S2 cells expressing Pld or in COS-7 cells transfected with myc-tagged Pld or the K1086R mutant. In all cases, cells in 6-well plates were labeled overnight in 1% dialysed serum containing 300 µCi per well of 3H palmitic acid. The following morning, the medium was made to contain 1% butanol (as indicated) for 30 min, and the cells were collected and extracted for lipid analysis as described previously (Manifava et al., 1999).
Online supplemental material
Fig. S1 shows the biochemical characterization of Drosophila Pld expressed in S2 cells. Fig. S2 shows data from transport assays that ER to Golgi transport is normal in photoreceptors with elevated PA levels. Fig. S3 shows the photoreceptor phenotypes of sec6 and sec15 mutants in Drosophila. Fig. S4 shows the generation of the Arf6 mutant. Fig. S5 shows the subcellular localization of Pld and dARf1-GAP when expressed in Drosophila photoreceptors. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200807027/DC1.
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Acknowledgments |
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This work was funded by the Biotechnology and Biological Sciences Research Council UK and The Wellcome Trust.
Submitted: 7 July 2008
Accepted: 26 February 2009
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