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Dictyostelium uses ether‐linked inositol phospholipids for intracellular signalling

2014; Springer Nature; Volume: 33; Issue: 19 Linguagem: Inglês

10.15252/embj.201488677

1460-2075

Jonathan Clark, Robert R. Kay, Anna Kielkowska, Izabella Niewczas, Louise Fets, David Oxley, Len R. Stephens, Phillip T. Hawkins,

Zebrafish Biomedical Research Applications

Article1 September 2014free access Dictyostelium uses ether-linked inositol phospholipids for intracellular signalling Jonathan Clark Jonathan Clark Babraham Biosciences Technology, Babraham Research Campus, Cambridge, UK Search for more papers by this author Robert R Kay Corresponding Author Robert R Kay MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK Search for more papers by this author Anna Kielkowska Anna Kielkowska Babraham Biosciences Technology, Babraham Research Campus, Cambridge, UK Search for more papers by this author Izabella Niewczas Izabella Niewczas Babraham Biosciences Technology, Babraham Research Campus, Cambridge, UK Search for more papers by this author Louise Fets Louise Fets MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK Search for more papers by this author David Oxley David Oxley Signalling Programme, Babraham Research Campus, Cambridge, UK Search for more papers by this author Len R Stephens Corresponding Author Len R Stephens Signalling Programme, Babraham Research Campus, Cambridge, UK Search for more papers by this author Phillip T Hawkins Corresponding Author Phillip T Hawkins Signalling Programme, Babraham Research Campus, Cambridge, UK Search for more papers by this author Jonathan Clark Jonathan Clark Babraham Biosciences Technology, Babraham Research Campus, Cambridge, UK Search for more papers by this author Robert R Kay Corresponding Author Robert R Kay MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK Search for more papers by this author Anna Kielkowska Anna Kielkowska Babraham Biosciences Technology, Babraham Research Campus, Cambridge, UK Search for more papers by this author Izabella Niewczas Izabella Niewczas Babraham Biosciences Technology, Babraham Research Campus, Cambridge, UK Search for more papers by this author Louise Fets Louise Fets MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK Search for more papers by this author David Oxley David Oxley Signalling Programme, Babraham Research Campus, Cambridge, UK Search for more papers by this author Len R Stephens Corresponding Author Len R Stephens Signalling Programme, Babraham Research Campus, Cambridge, UK Search for more papers by this author Phillip T Hawkins Corresponding Author Phillip T Hawkins Signalling Programme, Babraham Research Campus, Cambridge, UK Search for more papers by this author Author Information Jonathan Clark1,‡, Robert R Kay 2,‡, Anna Kielkowska1, Izabella Niewczas1, Louise Fets2,4, David Oxley3, Len R Stephens 3,‡ and Phillip T Hawkins 3,‡ 1Babraham Biosciences Technology, Babraham Research Campus, Cambridge, UK 2MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK 3Signalling Programme, Babraham Research Campus, Cambridge, UK 4Present address: MRC National Institute for Medical Research, The Ridgeway, London, UK ‡These authors contributed equally ‡These authors contributed equally *Corresponding author. Tel: +44 1223 267039; Fax: +44 1223 268306; E-mail: [email protected] *Corresponding author. Tel: +44 1223 496615; Fax: +44 1223 496043; E-mail: [email protected] *Corresponding author. Tel: +44 1223 496615; Fax: +44 1223 496043; E-mail: [email protected] The EMBO Journal (2014)33:2188-2200https://doi.org/10.15252/embj.201488677 See also: GRV Hammond & T Balla (October 2014) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Inositol phospholipids are critical regulators of membrane biology throughout eukaryotes. The general principle by which they perform these roles is conserved across species and involves binding of differentially phosphorylated inositol head groups to specific protein domains. This interaction serves to both recruit and regulate the activity of several different classes of protein which act on membrane surfaces. In mammalian cells, these phosphorylated inositol head groups are predominantly borne by a C38:4 diacylglycerol backbone. We show here that the inositol phospholipids of Dictyostelium are different, being highly enriched in an unusual C34:1e lipid backbone, 1-hexadecyl-2-(11Z-octadecenoyl)-sn-glycero-3-phospho-(1'-myo-inositol), in which the sn-1 position contains an ether-linked C16:0 chain; they are thus plasmanylinositols. These plasmanylinositols respond acutely to stimulation of cells with chemoattractants, and their levels are regulated by PIPKs, PI3Ks and PTEN. In mammals and now in Dictyostelium, the hydrocarbon chains of inositol phospholipids are a highly selected subset of those available to other phospholipids, suggesting that different molecular selectors are at play in these organisms but serve a common, evolutionarily conserved purpose. Synopsis A new mass spectrometry technique reveals that inositol phospholipids in Dictyostelium are based on a novel backbone. These plasmanyl inositides are functionally identical to the canonical diacyl glycerol configurations found in mammalian cells. Most inositol lipids in Dictyostelium are plasmanylinositols with a C16:0-ether, C18:1-acyl glycerol backbone. Dictyostelium uses these unusually structured lipids as signalling molecules in an analogous fashion to their phosphatidylinositol counterparts in mammalian cells. Other major phospholipid classes in Dictyostelium are comprised of a more heterogeneous collection of diacyl and acyl/ether species, suggesting evolutionary pressure to create a molecularly homogenous pool of plasmanylinositols in this organism. Introduction Inositol phospholipids are believed to be ubiquitous amongst the eukaryotes, where they play crucial roles in organising a wide variety of cellular functions, particularly vesicular trafficking and signal transduction (Balla, 2013; Di Paolo & De Camilli, 2006; Michell, 2008). Eight of these lipids are commonly described, the most abundant of which is phosphatidylinositol (PtdIns). PtdIns is distributed throughout the intracellular membrane systems of eukaryotes and usually represents approximately 10% of total cellular phospholipids. The other inositol phospholipids, where present, are much less abundant and carry one or more phosphate groups on their inositol ring, namely PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3. These more highly phosphorylated inositol lipids are metabolically interconverted by a series of reactions catalysed by specific kinases and phosphatases, and characteristically exhibit a much more restricted cellular distribution relating to their function. Phylogenetic surveys indicate that genes encoding type-III PI3K and PI4K inositol lipid kinases (making PtdIns(3)P and PtdIns(4)P, respectively) and PI(4)P5K (making PtdIns(4,5)P2) are likely to be present across the eukaryotes. In contrast, Class I PI3Ks, producing PtdIns(3,4,5)P3 at the plasma membrane, are absent in plants and fungi, but are found in the Amoebozoa, such as Dictyostelium discoideum (Brown & Auger, 2011; Engelman et al, 2006; Zhou et al, 1995). In mammalian cells, PtdIns(3)P and PtdIns(3,5)P2 play important roles in endosomal/lysosommal trafficking and the induction of autophagy (Burman & Ktistakis, 2010; Raiborg et al, 2013). PtdIns(4)P and PtdIns(4,5)P2 play roles in plasma membrane identity, cytoskeletal organisation and secretion (Di Paolo & De Camilli, 2006; Saarikangas et al, 2010). PtdIns(4,5)P2 also plays a major role as the substrate for two major signal transduction pathways; PLC-catalysed conversion of PtdIns(4,5)P2 to the messenger molecules diacylglycerol and inositol 1,4,5-trisphosphate (Kadamur & Ross, 2013) and Class I PI3K-catalysed conversion of PtdIns(4,5)P2 to the messenger PtdIns(3,4,5)P3 (Hawkins et al, 2006). To a greater or lesser extent, all of the phosphorylated inositol lipids signal via the binding of their specific inositol phosphate head groups to several defined protein domains, the best studied of which are the binding of PtdIns(3,4,5)P3 to a subfamily of PH-domain containing proteins at the inner leaflet of the plasma membrane and the binding of PtdIns(3)P to FYVE or PX domains at the cytoplasmic face of endosomes and autophagosomes (Lemmon, 2008). These lipids are thus envisaged to act as regulatable membrane scaffolds dictating the localisation and function of proteins at the surface in which they reside. Thus far, the composition of the hydrocarbon chains in inositol phospholipids has received very little attention. In part, this is because they are envisaged to merely anchor the inositol phospholipid in the bilayer, with minimal influence on the functional interaction of the head group with specific protein domains. However, it is also because most of the widely used techniques to measure these lipids in cell extracts do not yield the composition of the hydrocarbon chains and, indeed, often rely on anion-exchange chromatography of their deacylated derivatives (Guillou et al, 2007). Mass spectrometry-based approaches have the potential to define the full structures of lipids, but they have generally lacked sufficient sensitivity to accurately measure the more highly phosphorylated inositol lipids (Pettitt et al, 2006). We have recently solved some of these problems through chemical derivatisation of the phosphate groups on the inositol ring, allowing sensitive detection of PI, PIP, PIP2 and PIP3 species (the abbreviation PI(P)n is used here to refer to inositol phospholipids without implying the nature of the hydrocarbon linkage to the glycerol unit; PtdIns(P)n is used where the linkages are known to be esters, that is defining a ‘phosphatidyl’ unit) (Clark et al, 2011; Kielkowska et al, 2014). However, these methods do not distinguish between regioisomers of these lipids, that is, PI3P/PI4P/PI5P and PI(3,4)P2/PI(3,5)P2/PI(4,5)P2; PI(3,4,5,)P3 is the only known isomer of PIP3 found in cells. The mass spectrometric analysis of the fatty acyl/alkyl composition of highly phosphorylated inositol lipids is still in its infancy and has focused thus far on lipid extracts of mammalian origin, confirming that these lipids possess the characteristic diacylglycerol backbone of their precursor, PtdIns (Holub, 1986). The acyl groups that make up this backbone are highly selected: typically stearoyl (C18:0; 18 carbons: 0 double bonds) at the sn-1 position and arachidonoyl (C20:4) at the sn-2 position (C38:4 in total), particularly in primary tissue samples, with less abundant species containing palmitoyl (C16:0), oleoyl (C18:1) and linoloyl (C18:2) groups (Anderson et al, 2013; Lee et al, 2012; Milne et al, 2005; Rouzer et al, 2006). This raises the questions of whether the same C38:4 backbone is used in other organisms and whether there is the same strong selection for this (or another) backbone in the inositol lipids compared to the heterogeneity of backbone available in the other phospholipids. The social amoeba Dictyostelium discoideum has genetic and cell biological features commending it as a model organism for investigating biological organisation across species (King & Insall, 2009; Muller-Taubenberger et al, 2013). Dictyostelium cells possess 7 recognisable PI3K genes, 5 of which have Ras-binding domains indicative of regulation through Ras (Hoeller & Kay, 2007; Zhou et al, 1995), a clear PTEN homologue (Iijima & Devreotes, 2002), and a number of PIP3-binding effector proteins, including a homologue of the protein kinase PKB/AKT (Meili et al, 2000; Zhang et al, 2010). Genetic studies show that PI3K signalling is required for efficient macropinocytosis (Buczynski et al, 1997; Hoeller et al, 2013; Zhou et al, 1998) and for the relay of cyclic-AMP signals during aggregation (Insall et al, 1994; Loovers et al, 2006). Its role in chemotaxis to cyclic-AMP and folic acid is much more controversial (Kay et al, 2008). Although PIP3 is made in response to both chemoattractants (see later) and can be polarised towards the leading edge of chemotaxing cells (Parent et al, 1998), genetic elimination of PIP3 signalling does not prevent efficient chemotaxis to either chemical and indeed improves chemotaxis to folic acid (Hoeller et al, 2013; Hoeller & Kay, 2007; Takeda et al, 2007; Veltman et al, 2014). There is also evidence that PIP3 is involved in bleb-driven cell movement (Zatulovskiy et al, 2014), and as PIP3 is produced at phagosomes (Dormann et al, 2004), it is likely to be involved in phagocytosis. Despite this intense interest in phosphoinositide signalling, very few measurements have been published quantifying changes in the inositol lipids themselves in this organism. We applied our recent mass spectrometry techniques to analyse inositol phospholipids from Dictyostelium. To our surprise, we discovered that the vast majority of the inositol phospholipids in this organism are ether lipids, with a novel plasmanyl-C34:1e structure (the ‘e’ is used here to indicate that one of the hydrocarbon chains is linked to the glycerol via an ether linkage). This C34:1e species is much more enriched in the inositol phospholipids than the other major phospholipid classes, suggesting it has been selected to convey specific properties to this pool. We also show through the use of appropriate mutants that these plasmanylinositols are used by the PI3K signalling pathway and define the kinetics of C34:1e PIP3 production in response to chemoattractants. Results Inositol phospholipids in Dictyostelium are C34:1e plasmanylinositols We recently described a new HPLC-ESI mass spectrometry method to analyse phosphorylated inositol lipids in cellular lipid extracts (Kielkowska et al, 2014). This method uses methylation of acidic phosphate groups with TMS-diazomethane, reverse phase chromatography on a C4 column, fragmentation of the lipids at the phosphodiester phosphate and measuring the charged molecular species derived from the neutral loss of a specific methylated head group. We applied this methodology to measure inositol phospholipids in lipid extracts from Dictyostelium discoideum grown in axenic medium. Neutral loss scans corresponding to the loss of the inositide head groups (inositol, methylated inositol phosphate or methylated inositol bisphosphate) indicated that the most abundant inositide species had unexpected and unusual masses (m/z 837.6, 945.6 and 1053.6 respectively; Fig 1). The corresponding glycerol fragment had an m/z of 563.6, which suggested either it was derived from a diacylglycerol containing a fatty acyl group with an odd number of carbons or, that one chain was attached to the glycerol via an ether linkage. Both of these possibilities would appear to have the same mass at the resolution of the mass spectrometer used, although fatty acids with an odd carbon chain length are rarely found in eukaryotes. Figure 1. The major molecular species of inositol phospholipids present in lipid extracts of Dictyostelium discoideum possess a C34:1e backboneLipid extracts were prepared from D. discoideum grown in axenic medium, then methylated with TMS-diazomethane and analysed by HPLC-ESI mass spectrometry. Neutral loss scans are shown which describe the major species of PI, PIP and PIP2 present (the mass of the individual neutral fragments corresponding to the mass of methylated inositol phosphate ‘head groups’ are listed in parentheses and differ by multiples of 108, the mass of a methylated phosphate). The most abundant species detected in each case corresponded to the generation of a glycerol fragment with an m/z of 563.6, suggesting the presence of either one ether-linked hydrocarbon chain plus one acyl chain (C34:1e) or two acyl chains with an odd number of total carbon atoms (C33:1). Further high-resolution mass analysis (Supplementary Fig S1A) and fragmentation (Supplementary Fig S1B) confirmed the presence of C16:0 alkyl and C18:1 acyl chains. Similar results were obtained when lipid extracts were prepared from D. discoideum grown on bacteria (Supplementary Fig S1C) or in a fully defined medium containing no added fatty acids (Supplementary Fig S1D). Download figure Download PowerPoint To resolve this ambiguity, a sample of methylated PIP2 was isolated by HPLC and an accurate mass obtained on an Orbitrap mass spectrometer capable of working at a higher mass resolution. A value of m/z for MH+ of 1053.5809 was obtained (Supplementary Fig S1A). The theoretical m/z for a C34:1e ether/acyl PIP2 is 1053.5804, whereas the theoretical m/z for the alternative C33:1 diacyl compound would be 1053.5440 (which was not seen). Further fragmentation studies of the PIP2 species revealed an ion with a m/z of 265.4 (Supplementary Fig S1B), suggestive of a C18:1 acyl cation. If this was indeed a C18:1 acyl cation, then the other chain would most likely be a C16:0 ether-linked chain, which was consistent with the other ions observed (see Supplementary Fig S1B). Previous work with other organisms has shown that ether-containing phospholipids are synthesised by a metabolic pathway that first exchanges an acyl chain in the sn-1 position of the glycerol for an alcohol, in a reaction catalysed by alkyl-DHAP synthase (Nenci et al, 2012). We therefore attempted to confirm the structure and pathway for synthesis of a putative C34:1e lipid by feeding Dictyostelium cells a C16:0 alcohol (hexadecan-1-ol or palmitol) which contained two deuterium nuclei at the C1 position. Both deuteriums were efficiently incorporated into the C34:1e structure, indicating that the palmityl ether was present (Fig 2). The high proportion of deuterium incorporation (80% of PIP2 molecules had incorporated two deuteriums by 490 min; Supplementary Fig S2), and the lack of any significant incorporation of a single deuterium nucleus, ruled out significant metabolism at the C1 of D2-palmitol and hence more indirect routes of deuterium incorporation. Importantly, the lack of incorporation of a single deuterium also ruled out the presence of a vinyl ether linkage, a characteristic feature of a group of ether lipids called plasmalogens (Brites et al, 2004). Figure 2. Determination of the alkyl chain structure in Dictyostelium discoideum inositol lipidsMethylated lipid extracts were prepared from D. discoideum grown in axenic medium in the absence (left panel) or presence (right panel) of D2-hexadecan-1-ol for 120 min and then analysed by HPLC-ESI mass spectrometry. The mass data are shown with a centroid presentation to allow differences of one mass unit to be more easily discerned. Both the unlabelled and labelled signals show the typical pattern obtained in mass spectra of compounds predominantly made up of carbon, hydrogen and oxygen atoms, which is the result of the natural abundance of 13C. D2-hexadecan-1-ol was synthesised with both deuterium nuclei in the C1 position, and the mass data indicate both deuteriums were efficiently incorporated into PIP2; that is, m/z peaks were shifted by precisely two mass units. There was no detectable increase in the m/z 1054.6 signal, indicating no significant incorporation of a single deuterium nucleus, and hence the absence of a vinyl ether linkage to the C16 (palmityl) chain (illustrated by the coloured structures). A more detailed description of the rate and extent of D2-hexadecan-1-ol incorporation is shown in Supplementary Fig S2. Download figure Download PowerPoint Dictyostelium C34:1e lipids were sensitive to hydrolysis by phospholipase A2 (Scott et al, 1990), indicating that the C18:1 acyl chain is in the sn-2 position (Fig 3A). Finally, the position of the C=C double bond in the C18:1 chain was identified by ozonolysis to be delta-11, identifying the acyl chain as the 11-ocatadecenoyl group (Fig 3B). It is most likely that the double bond is the cis isomer because this is the isomer which is most commonly found in biologically important fatty acids and because Z11-ocatadecenoic acid has recently been shown to be an abundant fatty acid in Dictyostelium discoideum (Blacklock et al, 2008). Thus, the structures of the most abundant species of inositol lipids in Dictyostelium discoideum are defined as 1-hexadecyl-2-(11Z-octadecenoyl)-sn-glycero-3-phospho-(1’-myo-inositol) and phosphates thereof. Figure 3. Determination of the acyl chain structure in Dictyostelium discoideum inositol lipids The PI in D. discoideum is susceptible to hydrolysis by PLA2. Lipid extracts prepared from D. discoideum were mixed with phospholipase A2 (PLA2; from bee venom), and the levels of PI and lyso-PI measured over time by HPLC-ESI mass spectrometry. The quantitative conversion of PI to lyso-PI demonstrates the presence of an acyl chain in the sn-2 position. The PIP2 in D. discoideum contains an 11-octadecenoyl acyl chain. Methylated lipid extracts prepared from D. discoideum were subjected to ozonolysis. The mass of the fragment generated indicates the presence of an 11-octadecenoyl chain. Download figure Download PowerPoint We also analysed the major species of inositol phospholipids in Dictyostelium discoideum grown on bacteria or in a fully defined medium with no added fatty acids, SIH medium (Han et al, 2004) and, in each case, found a similarly high proportion of the C34:1e species (Supplementary Fig S1C and D), indicating that this structure is independent of the availability of particular fatty acyl chains in the medium. Further, we additionally examined three other species of social amoebae—Dictyostelium purpureum, Dictyostelium mucoroides and Polysphondylium violaceum—and in each case found that the major species of PIP2 is the same mass as that from D. discoideum (m/z 1053.6), suggesting that the use of plasmanylinositols is common amongst these amoebae (unpublished observations). The C34:1e plasmanyl motif is enriched in inositol lipids compared to other phospholipid classes The striking selection for a particular combination of acyl tails in mammalian phosphoinositides raises the speculation that this selectivity could be functionally important; but equally with only this single example, it could be happenstance. We therefore asked whether a similar molecular selectivity exists in the Dictyostelium phosphoinositides. The relative abundances of molecular species of PC, PS, PE and PA in Dictyostelium were assessed by analogous neutral loss scans to those described above for the inositol phospholipids (Fig 4). It is immediately apparent that the C34:1e species is the most abundant form of PA, but is a smaller component of the other major phospholipid pools, which have the greater proportion comprised of a mixture of diacyl- and acyl/ether-species (relative comparison of these molecular species is given in Supplementary Fig S3). Figure 4. The molecular species of the major phospholipid classes in Dictyostelium discoideum are highly heterogeneousNeutral loss (PA, PS, PE) or precursor ion (PC) scans are shown describing the major molecular species of abundant phospholipids present in methylated lipid extracts of D. discoideum grown under axenic conditions (see 4 for the MRM transitions monitored). The C34:1e species highlighted in red is analogous to the major species of inositol lipids found in D. discoideum. Relative quantification of some of these species is given in Supplementary Fig S3. Download figure Download PowerPoint The most abundant species of PI, PIP, PIP2 and PA were targeted for more careful comparison by multiple reaction monitoring (MRM), which indicated that the vast majority of each of these lipid pools was comprised of the C34:1e species under both axenic (Fig 5) and bacterially fed conditions (unpublished observations). The accepted pathway for de novo synthesis of PI in eukaryotes is via the formation of CDP-DG from PA and CTP, catalysed by CDP-DG synthase (Saito et al, 1997), followed by the formation of PI from CDP-DG and inositol, catalysed by PI synthase (Paulus & Kennedy, 1960). Thus, the enrichment of C34:1e species of PI/PIP/PIP2 is naturally explained by the relative abundance of C34:1e PA. However, the other major phospholipids are derived from diacylglycerol by base activation (the Kennedy Pathway; Vance & Vance, 2004). Analogous neutral loss scans of DG were not possible, so we quantified selected DG species based on the major species detected for PI and PC, described above. The results indicated that the DG pool appears to be comprised mostly of diacyl-species, with only small quantities of C34:1e (Supplementary Fig S4). This suggests that the major pools of PA and DG are maintained with very different molecular compositions, consistent with their differential roles as the sources of PI and other phospholipids, respectively. Figure 5. Measurement of the relative abundance of the major molecular species of inositol phospholipids and PA in Dictyostelium discoideumMethylated lipid extracts prepared from D. discoideum were grown under axenic conditions and analysed by HPLC-ESI mass spectrometry. MRM traces were integrated to provide relative abundances of the major species of PA, PI, PIP and PIP2 present. Download figure Download PowerPoint C34:1e plasmanylinositols are used in Dictyostelium signalling pathways Previous work has characterised important roles for the PI3K signalling pathway in the response of Dictyostelium amoebae to chemoattractants. Thus far, however, measurements of the inositol phospholipids themselves in this organism have rarely been attempted, with most work using in vivo reporters instead. Although the levels of PIP3 were too low to obtain good neutral loss spectra (see above), specifically targeting the C34:1e species produced a very good signal/noise ratio and accurate measurement was readily achievable (see Supplementary Fig S5). We first measured changes in the levels of C34:1e-PI, -PIP, -PIP2 and -PIP3 in response to added cyclic-AMP using an adenylyl cyclase mutant to reduce background levels of cyclic-AMP and thus sharpen the response (acaA−; Fig 6). Addition of cyclic-AMP produced a very fast and transient rise in PIP3, with a minimal associated drop in PIP2. The level of PIP3 peaked at approximately 5 s. A very similar pattern of changes was seen in the response of the Ax2 strain to a different chemoattractant, folic acid (Fig 7). Figure 6. Changes in inositol phospholipids in response to cAMP in Dictyostelium discoideum (acaA−)The acaA− strain of D. discoideum was starved and rendered competent to respond to cAMP. Individual samples of cells were then stimulated with 10 μM cAMP for the times shown. Methylated lipid extracts were prepared and analysed by HPLC-ESI mass spectrometry. Integrated MRM values (mean ± SD, n = 3 individual cell incubations) for the C34:1e species of PI, PIP, PIP2 and PIP3 are shown; for example, traces from which the integrations were performed are shown in Supplementary Fig S5. This experiment has been repeated three times with qualitatively very similar results. These data are uncorrected for differences in extraction and ionisation of different lipid classes, and so, no significance can be placed on differences between the signal intensity of PI, PIP, PIP2 or PIP3. Download figure Download PowerPoint Figure 7. Changes in inositol phospholipids in response to folic acid in Dictyostelium discoideum (Ax2)The Ax2 strain of D. discoideum was grown on bacteria and stimulated with 100 μM folic acid for the times shown. Methylated lipid extracts were prepared and analysed by HPLC-ESI mass spectrometry. Integrated MRM values (mean ± SD, n = 3 individual cell incubations) for the C34:1e species of PI, PIP, PIP2 and PIP3 are shown. This experiment has been repeated three times with qualitatively very similar results. These data are uncorrected for differences in extraction and ionisation of different lipid classes, and so, no significance can be placed on differences between the signal intensity of PI, PIP, PIP2 or PIP3. Download figure Download PowerPoint To examine the genetic dependence of the response to cyclic-AMP, we used mutants lacking key enzymes in inositol phospholipid signalling pathways. C34:1e inositol lipids were measured in these mutants before and after a 5-s stimulation with cyclic-AMP (Fig 8; note that because these mutants were created in the Ax2 strain, endogenous production of cyclic-AMP likely led to a somewhat de-sensitised response compared to the acaA− strain described above). Loss of all of the five PI3Ks with a Ras-binding domain (Hoeller & Kay, 2007) prevented any PIP3 response to cyclic-AMP, though a basal level of PIP3 was still detectable; this indicates that these PI3Ks are the cyclic-AMP-sensitive enzymes responsible for PIP3 synthesis, but another minor pathway of PIP3 synthesis must also exist in this organism. Loss of the PIP3 phosphatase PTEN (Iijima & Devreotes, 2002) resulted in a hugely elevated basal and stimulated level of PIP3, indicating it is a major PIP3 phosphatase in this organism. Loss of PI4P5K dramatically reduced the levels of PIP2 as previously reported (Fets et al, 2014), but remarkably, this diminished level could still support substantial cyclic-AMP-stimulated PIP3 synthesis, conflicting with previous measurements made using an ELISA. Figure 8. Changes in inositol phospholipids in response to cAMP in mutants of Dictyostelium discoideumThe parental Ax2 strain of D. discoideum, or the indicated mutant strains derived from it (PI3K1-5-, pikA−, pikB−, pikC−, pikF−, pikG−; PI4P5K-, pikI−; PTEN-, ptenA−) were grown on bacteria and then rendered compet