Dictyostelium uses ether-linked inositol phospholipids for intracellular signalling

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.

Keywords: Dictyostelium; PI3K; ether lipids; phosphoinositides; plasmanylinositol.

Figure 1. The major molecular species of inositol phospholipids present in lipid extracts of Dictyostelium discoideum possess a C34:1e backbone

Lipid 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).

Figure 2. Determination of the alkyl chain structure in Dictyostelium discoideum inositol lipids

Methylated 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.

Figure 3. Determination of the acyl chain structure in Dictyostelium discoideum inositol lipids

1. 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.

2. 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.

Figure 4. The molecular species of the major phospholipid classes in Dictyostelium discoideum are highly heterogeneous

Neutral 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 Materials and Methods 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.

Figure 5. Measurement of the relative abundance of the major molecular species of inositol phospholipids and PA in Dictyostelium discoideum

Methylated 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.

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.

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.

Figure 8. Changes in inositol phospholipids in response to cAMP in mutants of Dictyostelium discoideum

The 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 competent to respond to cAMP. Individual samples of cells were then stimulated with 10 μM cAMP or vehicle for 5 s. 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 in differences between the signal intensity of PI, PIP, PIP2 or PIP3.