The mammalian phospholipid exchange protein PITPα (phosphatidylinositol transfer protein alpha), found in both extranuclear and endonuclear compartments, is thought in part to facilitate nuclear import of the PtdIns (phosphatidylinositol) consumed in the generation of proliferation-associated endonuclear diacylglycerol accumulations. Unlike phosphatidylcholine, endonuclear PtdIns is not synthesized in situ. However, despite progressive postnatal lethality of PITPα ablation in mice, PITPα−/− MEF (mouse embryonic fibroblasts) lack an obviously impaired proliferative capacity. We used ESI-MS (tandem electrospray ionization-MS) to monitor incorporation of the deuterated phospholipid precursors, choline-d9 and inositol-d6, into molecular species of whole cell and endonuclear phosphatidylcholine and PtdIns over 24 h to assess the contribution of PITPα to the nuclear import of PtdIns into MEF cells. In cells labelled for 1, 3, 6, 12 and 24 h fractional inositol-d6 incorporation into whole-cell PtdIns species was consistently higher in PITPα−/− MEF implying greater flux through its biosynthetic pathway. Moreover, endonuclear accumulation of PtdIns-d6 was apparent in the PITPα−/− cells and mirrored that in PITPα+/+ cells. Together, these results suggest that the essential endonuclear PtdIns import via PITPα can be accommodated by other mechanisms.

Introduction

PITPα (phosphatidylinositol transfer protein α) in mammalian cells is distributed between extranuclear and endonuclear compartments [1], implying a functional requirement to transport PtdIns/PtdCho (phosphatidylinositol/phosphatidylcholine) within or between these compartments. Endonuclear PtdIns and PtdCho act as sources of the periodic nuclear accumulations of diacylglycerol, which accompany cell proliferation [2], although endogenous capacity for highly saturated endonuclear PtdCho synthesis [3] is not matched by an equivalent capacity for endonuclear PtdIns synthesis de novo [4,5]. Accordingly, PtdIns must be transported into the nuclear matrix before conversion into polyphosphoinositides and evidence points to the involvement of PITPα [5]. Notwithstanding such an assertion, genetic ablation of PITPα from the whole mouse, although it is ultimately terminal post-natally [6], does not appear to compromise the ability of altered cells to proliferate and derived MEF (mouse embryonic fibroblasts) cells grow equally well with or without PITPα.

Use of tandem ESI-MS (electrospray ionization-MS) and the deuterated phospholipid precursors choline-d9 and inositol-d6 permits us to evaluate both the endogenous content and pattern of synthesis of PtdCho and PtdIns in whole cells and endonuclear compartments [3,4]. We have used this technology to probe the acquisition of endonuclear PtdIns and PtdCho over 24 h in PITPα+/+ and PITPα−/− MEF cells to ascertain whether PITPα is essential for nuclear PtdIns accretion.

Materials and methods

myo-d6-Inositol was obtained from C/D/N Isotopes Inc (Pointe-Claire, Quebec, Canada), choline-d9 and other biochemicals were acquired from Sigma–Aldrich (Poole, Dorset, U.K.), organic solvents were from Fisher Scientific (Fair Lawn, NJ, U.S.A.). The PITPα−/− mice, as described by Alb et al. [6], were used alongside control mice to generate MEF cells from PITPα+/+ and PITPα−/− mouse embryos (E16). Only third or fourth passage MEFs were employed in these experiments. MEFs were cultured on 150 mm charged tissue culture dishes in Dulbecco's modified Eagle's medium, supplemented with fetal bovine serum (10%, v/v) and glucose (4.5 g/l). Sub-confluent cells were labelled with inositol-d6 and choline-d9 for up to 24 h as described elsewhere [3,4]. Trypsinized cells were washed and prepared as either whole cells or nuclear envelope-stripped nuclei [2], and Triton X-100-mediated removal of nuclear envelopes was confirmed by TEM. Following subsequent cell/nuclei lipid extraction [2], ESI-MS/MS of endogenous and newly synthesized PtdCho and PtdIns in the total extract was undertaken on a Micromass Ultima Quatro electrospray ionization tandem mass spectrometer either by direct injection for the cell extracts or by nanoflow capillary infusion of endonuclear extracts and employing precursor scans of m/z 184+ and m/z 193+ under positive ionization for PtdCho [3] and m/z 241 and m/z 247 under negative ionization for PtdIns [4] as described. Among many other lipid scans, we also scanned for diacyl PtdEtn (phosphatidylethanolamine) molecular species using neutral loss of a m/z 141 fragment [3].

Results and discussion

Composition of whole cell and endonuclear MEF phospholipids

Analyses and comparison of whole cell and endonuclear phospholipids compositions between PITPα+/+ and PITPα−/− MEF cells revealed no significant variation attributable to genotypic loss of PITPα (results not shown). However, comparisons between whole cell and endonuclear phospholipids (Figure 1) showed that endonuclear PtdCho (Figure 1b) and PtdEtn (Figure 1d) were considerably more saturated and depleted in polyunsaturated species than the corresponding lipids of whole cells (Figures 1a and 1c) in agreement with our previous reports of mammalian PtdCho [3,4,7], although not recorded in the case of PtdEtn. Intriguingly, activity of the endonuclear CDP:choline sn-1,2 diacylglycerol cholinephosphotransferase in IMR-32 cells [2] was accompanied by an unreported ethanolaminephosphotransferase activity present in identical proportions (∼16% of whole cell activity), which may indicate a parallel endonuclear capacity for synthesis of saturated PtdEtn. The precursors of m/z 184+ scans of the endonuclear compartments (Figure 1b) also revealed a characteristic endonuclear enrichment of SM (sphingomyelin) species, principally 16:0 SM and 20:1 SM [4].

ESI-MS analyses of selected whole cell and endonuclear MEF phospholipids

Figure 1
ESI-MS analyses of selected whole cell and endonuclear MEF phospholipids

The total chloroform–methanol extract of whole cell or endonuclear fractions from PITPα+/+ MEF were subjected to ESI-MS analyses as described in the Materials and methods section. (a) Precursor scan of m/z 184+ fragment over the mass range 700–840 showing endogenous whole cell PtdCho and SM species. (b) Precursor scan of m/z 184+ fragment over the mass range 700–840 showing endogenous endonuclear PtdCho and SM species. (c) Neutral loss scan of m/z 141 over the mass range 710–790 showing endogenous whole cell PtsEtn species (but not alkenylacyl species). (d) Neutral loss scan of m/z 141 fragment over the mass range 710–790 showing endogenous endonuclear diacyl PtdEtn species (but not alkenylacyl species). (e) Precursor scan of m/z 241 over the mass range 825–920 showing endogenous whole cell PtdIns species. (f) Precursor scan of m/z 241 over the mass range 825–920 showing endogenous endonuclear PtdIns species.

Figure 1
ESI-MS analyses of selected whole cell and endonuclear MEF phospholipids

The total chloroform–methanol extract of whole cell or endonuclear fractions from PITPα+/+ MEF were subjected to ESI-MS analyses as described in the Materials and methods section. (a) Precursor scan of m/z 184+ fragment over the mass range 700–840 showing endogenous whole cell PtdCho and SM species. (b) Precursor scan of m/z 184+ fragment over the mass range 700–840 showing endogenous endonuclear PtdCho and SM species. (c) Neutral loss scan of m/z 141 over the mass range 710–790 showing endogenous whole cell PtsEtn species (but not alkenylacyl species). (d) Neutral loss scan of m/z 141 fragment over the mass range 710–790 showing endogenous endonuclear diacyl PtdEtn species (but not alkenylacyl species). (e) Precursor scan of m/z 241 over the mass range 825–920 showing endogenous whole cell PtdIns species. (f) Precursor scan of m/z 241 over the mass range 825–920 showing endogenous endonuclear PtdIns species.

PtdIns molecular species, in contrast, were essentially identical in both endonuclear compartments and whole cells (Figures 1e and 1f) and this was also true for PITPα−/− (results not shown). In all cases, PtdIns was dominated by the polyunsaturated stearoylarachidonyl species, 18:0/20:4, suggesting maintenance of an adequate endonuclear PtdIns pool. Accordingly, PITPα ablation does not appear to compromise endonuclear PtdIns content.

Pattern of incorporation of myo-d6-inositol into whole cell and endonuclear PtdIns

At each time point assessed, the fractional incorporation of myo-d6-inositol into whole cell PtdIns was higher in PITPα−/− MEF cells than in PITPα+/+ (results not shown) which, together with a lack of cellular accumulation of PtdIns and no apparent derangement in proliferation rate, would be consistent with a higher PtdIns turnover in PITPα−/− cells. It is unclear from these preliminary results whether or how PITPα ablation may be altering flux through the de novo synthesis pathway, although the possibility that PITPα-bound lipid may ‘report’ either cellular PtdIns status, or the balance between PtdIns and PtdCho content to the nucleus poses intriguing possibilities.

In molecular species terms, newly synthesized PtdIns (represented by precursors of m/z 247 in Figure 2) from whole cells (Figures 2b and 2d) showed higher representation of 18:1/18:1 species when compared with whole endogenous cell contents (Figure 1e). This presumably reflects an ongoing remodelling of PtdIns synthesized de novo towards the endogenous composition. In contrast, newly acquired endonuclear PtdIns (Figures 2a and 2c) at all time points (full time-course results not shown) reflects endogenous endonuclear composition (Figure 1f) without 18:1/18:1 enrichment. Imported endonuclear PtdIns, therefore, appears fully remodelled.

ESI-MS of incorporation of myo-d6-inositol into whole MEF cell and endonuclear PtdIns species after 24 h

Figure 2
ESI-MS of incorporation of myo-d6-inositol into whole MEF cell and endonuclear PtdIns species after 24 h

The total chloroform–methanol extract of whole cell or endonuclear fractions from PITPα+/+ and PITPα−/− MEF were subjected to ESI-MS analyses as described in the Materials and methods section, 24 h after incubation with myo-d6-inositol. (a) Precursor scan of m/z 247 fragment over the mass range 825–920 showing the newly acquired endonuclear PtdIns of PITPα+/+ MEF. (b) Precursor scan of m/z 247 fragment over the mass range 825–920 showing newly synthesized whole cell PtdIns of PITPα+/+ MEF. (c) Precursor scan of m/z 247 fragment over the mass range 825–920 showing the newly acquired endonuclear PtdIns of PITPα−/− MEF. (d) Precursor scan of m/z 247 fragment over the mass range 825–920 showing newly synthesized whole cell PtdIns of PITPα−/− MEF.

Figure 2
ESI-MS of incorporation of myo-d6-inositol into whole MEF cell and endonuclear PtdIns species after 24 h

The total chloroform–methanol extract of whole cell or endonuclear fractions from PITPα+/+ and PITPα−/− MEF were subjected to ESI-MS analyses as described in the Materials and methods section, 24 h after incubation with myo-d6-inositol. (a) Precursor scan of m/z 247 fragment over the mass range 825–920 showing the newly acquired endonuclear PtdIns of PITPα+/+ MEF. (b) Precursor scan of m/z 247 fragment over the mass range 825–920 showing newly synthesized whole cell PtdIns of PITPα+/+ MEF. (c) Precursor scan of m/z 247 fragment over the mass range 825–920 showing the newly acquired endonuclear PtdIns of PITPα−/− MEF. (d) Precursor scan of m/z 247 fragment over the mass range 825–920 showing newly synthesized whole cell PtdIns of PITPα−/− MEF.

Accordingly, from these results, we conclude that

  1. PITPα ablation does not compromise the capacity of cells to maintain an endonuclear PtdIns content that is sufficient to permit normal rates of cell growth and division.

  2. In PITPα−/− MEF cells, a PITPα-independent mechanism facilitates endonuclear accretion of newly synthesized PtdIns which is not subsequently remodelled.

  3. PITPα may directly or indirectly alter flux through the de novo pathway of PtdIns biosynthesis.

Research Colloquia: Research Colloquia at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by M. Bouvier (Montreal, Canada), G. Milligan (Glasgow, U.K.), V. O'Donnell (Cardiff, U.K.), M. Brand (MRC-Dunn Human Nutrition Unit, Cambridge, U.K.), M. Schweizer (Heriot-Watt University, Edinburgh, U.K.), R. Insall (Birmingham, U.K.), A. Ridley (Ludwig Institute for Cancer Research, London, U.K.) and M. Sutcliffe (Leicester, U.K.). The first eight papers featured in this Section were presented as a part of the GPCR Regulation and Signalling Research Colloquium, incorporating the GPCR–Ion Channel Interactions Pfizer-Sponsored Research Colloquium.

Abbreviations

     
  • ESI-MS

    electrospray ionization-MS

  •  
  • MEF

    mouse embryonic fibroblasts

  •  
  • PITPα

    phosphatidylinositol transfer protein α

  •  
  • PtdCho

    phosphatidylcholine

  •  
  • PtdEtn

    phosphatidylethanolamine

  •  
  • PtdIns

    phosphatidylinositol

  •  
  • SM

    sphingomyelin

This collaborative venture was undertaken under a BBSRC International Scientific Interchange Scheme award to A.N.H.

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