Highly Saturated Endonuclear Phosphatidylcholine Is Synthesizedin Situ and Colocated with CDP-choline Pathway Enzymes

Chromatin-associated phospholipids are well recognized. A report that catalytically active endonuclear CTP:choline-phosphate cytidylyltransferase α is necessary for cell survival questions whether endonuclear, CDP-choline pathway phosphatidylcholine synthesis may occur in situ. We report that chromatin from human IMR-32 neuroblastoma cells possesses such a biosynthetic pathway. First, membrane-free nuclei retain all three CDP-choline pathway enzymes in proportions comparable with the content of chromatin-associated phosphatidylcholine. Second, following supplementation of cells with deuterated choline and using electrospray ionization mass spectrometry, both the time course and molecular species labeling pattern of newly synthesized endonuclear and whole cell phosphatidylcholine revealed the operation of spatially separate, compositionally distinct biosynthetic routes. Specifically, endogenous and newly synthesized endonuclear phosphatidylcholine species are both characterized by a high degree of diacyl/alkylacyl chain saturation. This unusual species content and synthetic pattern (evident within 10 min of supplementation) are maintained through cell growth arrest by serum depletion and when proliferation is restored, suggesting that endonuclear disaturated phosphatidylcholine enrichment is essential and closely regulated. We propose that endonuclear phosphatidylcholine synthesis may regulate periodic nuclear accumulations of phosphatidylcholine-derived lipid second messengers. Furthermore, our estimates of saturated phosphatidylcholine nuclear volume occupancy of around 10% may imply a significant additional role in regulating chromatin structure. Chromatin-associated phospholipids are well recognized. A report that catalytically active endonuclear CTP:choline-phosphate cytidylyltransferase α is necessary for cell survival questions whether endonuclear, CDP-choline pathway phosphatidylcholine synthesis may occur in situ. We report that chromatin from human IMR-32 neuroblastoma cells possesses such a biosynthetic pathway. First, membrane-free nuclei retain all three CDP-choline pathway enzymes in proportions comparable with the content of chromatin-associated phosphatidylcholine. Second, following supplementation of cells with deuterated choline and using electrospray ionization mass spectrometry, both the time course and molecular species labeling pattern of newly synthesized endonuclear and whole cell phosphatidylcholine revealed the operation of spatially separate, compositionally distinct biosynthetic routes. Specifically, endogenous and newly synthesized endonuclear phosphatidylcholine species are both characterized by a high degree of diacyl/alkylacyl chain saturation. This unusual species content and synthetic pattern (evident within 10 min of supplementation) are maintained through cell growth arrest by serum depletion and when proliferation is restored, suggesting that endonuclear disaturated phosphatidylcholine enrichment is essential and closely regulated. We propose that endonuclear phosphatidylcholine synthesis may regulate periodic nuclear accumulations of phosphatidylcholine-derived lipid second messengers. Furthermore, our estimates of saturated phosphatidylcholine nuclear volume occupancy of around 10% may imply a significant additional role in regulating chromatin structure. phosphatidylcholine phosphatidylethanolamine phosphatidylinositol CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) diacylglycerol mass spectrometry electrospray ionization MS Hanks' balanced salt solution phosphatidic acid 9-deuterated methylcholine fetal calf serum Considerable evidence supports the existence of an endonuclear pool of phospholipid, in association with the nuclear matrix and distinct from the nuclear envelope (1Rose H.G. Frenster J.H. Biochim. Biophys. Acta. 1965; 106: 577-591Crossref PubMed Scopus (85) Google Scholar, 2Vann L.R. Wooding F.B.P. Irvine R.F. Divecha N. Biochem. J. 1997; 327: 569-576Crossref PubMed Scopus (108) Google Scholar, 3Albi E. Mersel M. LeRay C. Tomassoni M.L. ViolaMagni M.P. Lipids. 1994; 29: 715-719Crossref PubMed Scopus (59) Google Scholar, 4Maraldi N.M. Santi S. Zini N. Ognibene A. Rizzoli R. Mazzotti G. Di Primo R. Baregg R. Bertagnolo V. Pagliarini C. Capitani S. J. Cell Sci. 1993; 104: 853-859PubMed Google Scholar). This endonuclear phospholipid is remarkable for several reasons. Although all the major membrane phospholipids, predominantly phosphatidylcholine (PtdCho),1phosphatidylethanolamine (PtdEtn), phosphatidylserine, and phosphatidylinositol (PtdIns), may be present typically at 4–10% of total cell content (2Vann L.R. Wooding F.B.P. Irvine R.F. Divecha N. Biochem. J. 1997; 327: 569-576Crossref PubMed Scopus (108) Google Scholar), transmission electron microscopy has failed to reveal endonuclear membranous systems (5Lewis J.D. Tllervey D. Science. 2000; 288: 1385-1389Crossref PubMed Scopus (180) Google Scholar). Hence the molecular organization of endonuclear phospholipids in eukaryotic cells is still unclear. Extensive histochemical and cytochemical studies suggest that their spatial distributions overlap that of decondensed chromatin domains (3Albi E. Mersel M. LeRay C. Tomassoni M.L. ViolaMagni M.P. Lipids. 1994; 29: 715-719Crossref PubMed Scopus (59) Google Scholar, 4Maraldi N.M. Santi S. Zini N. Ognibene A. Rizzoli R. Mazzotti G. Di Primo R. Baregg R. Bertagnolo V. Pagliarini C. Capitani S. J. Cell Sci. 1993; 104: 853-859PubMed Google Scholar). A number of in vitro studies suggest a functional relationship between various endonuclear phospholipids and gene expression/transcription (6Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (349) Google Scholar, 7Manzoli F.A. Capitani S. Mazzotti G. Barnabei O. Maraldi N.M. Adv. Enzyme Regul. 1982; 20: 247-262Crossref PubMed Scopus (60) Google Scholar, 8Boronenkov I.V. Loijens J.C. Umeda M. Anderson R.A. Mol. Biol. Cell. 1998; 9: 3547-3560Crossref PubMed Scopus (282) Google Scholar, 9Fraschini A. Biggiogera M. Bottone M.G. Martin T.E. Eur. J. Cell Biol. 1999; 78: 416-423Crossref PubMed Scopus (17) Google Scholar, 10Hirai H. Natori S. Sekimizu K. Biochim. Biophys Acta. 1991; 1088: 191-196Crossref PubMed Scopus (12) Google Scholar). Moreover, cell studies have shown that the amounts of endonuclear phospholipids change during progression through the cell cycle (4Maraldi N.M. Santi S. Zini N. Ognibene A. Rizzoli R. Mazzotti G. Di Primo R. Baregg R. Bertagnolo V. Pagliarini C. Capitani S. J. Cell Sci. 1993; 104: 853-859PubMed Google Scholar). The potential physiological importance of intranuclear phospholipid has recently been highlighted by the recognition that the α isoform (CCTα) of CTP:choline-phosphate cytidylyltransferase, the principal regulatory enzyme of PtdCho biosynthesis (11Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (192) Google Scholar), is confined to the nucleus throughout the cell cycle (12DeLong C.J. Qin L. Cui Z. J. Biol. Chem. 2000; 275: 32325-32330Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Furthermore, modified Chinese hamster ovary MT 58 cells (13Esko J.D. Raetz C.R. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 5192-5196Crossref PubMed Scopus (54) Google Scholar) expressing a temperature-sensitive mutation of CCTα are not viable when grown at the restrictive temperature (12DeLong C.J. Qin L. Cui Z. J. Biol. Chem. 2000; 275: 32325-32330Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), suggesting that endonuclear synthesis of PtdCho is essential for cell survival. This hypothesis is supported by the observation that extranuclear PtdCho biosynthesis, which occurs at the endoplasmic reticulum (14Vance D.E. Choy P.C. Farren S.B. Lim P.H. Schneider W.J. Nature. 1977; 270: 268-269Crossref PubMed Scopus (82) Google Scholar) and involves one or both of the β isoform(s) of cytidylyltransferase (CCTβ-1, CCTβ-2) (15Lykidis A. Baburina I. Jackowski S. J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), cannot rescue mutant cells grown at the restrictive temperature (12DeLong C.J. Qin L. Cui Z. J. Biol. Chem. 2000; 275: 32325-32330Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). However, the mechanism whereby endonuclear PtdCho biosynthesis regulates cell proliferation is not clear, and as yet there has been no direct demonstration of the intact pathway within the nucleus. One possible role for endonuclear PtdCho biosynthesis might be to regulate the periodic accumulations of diacylglycerol (DAG) within the nucleus that are functionally linked to cell proliferation (16Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (255) Google Scholar). Mobilization of DAG in the nucleus is bipartite (17D'Santos C. Clarke J.H. Irvine R.F. Divecha N. Curr. Biol. 1999; 9: 437-440Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) and involves two pools: one derived from predominantly unsaturated PtdIns and one derived from largely saturated PtdCho. Accordingly, a nuclear CDP-choline pathway for endonuclear PtdCho biosynthesis could regulate the nuclear DAG content by recycling endonuclear PtdCho-derived DAG together with choline and or phosphocholine. Consequently, we sought to determine whether expression of CCT activity within the nucleus does indeed represent part of an intact compartmentalized CDP-choline pathway for intranuclear PtdCho biosynthesis. First, we measured activities of individual CDP-choline pathway enzymes to establish the potential capacity for PtdCho synthesis within the nucleus. Subsequently, we employed electrospray ionization mass spectrometry (ESI-MS) of PtdCho isolated from cells cultured with deuterated choline to quantify the rate of endonuclear PtdCho synthesis. ESI-MS also permitted a detailed analysis of the molecular species compositions of endogenous and newly synthesized molecular species of PtdCho within the nucleus. Stock IMR-32 human neuroblastoma cells, obtained from Imperial Laboratories (Andover, Hampshire, UK), were maintained in monolayer culture in RPMI 1640 containing 10% fetal bovine serum and penicillin/streptomycin/amphotericin (Life Technologies, Inc.) at 37 °C in 5% (v/v) CO2. Sterile Hanks' balanced salt solution (HBSS) and HBSS without Ca2+/Mg2+ was also obtained from Life Technologies, Inc. Electrophoresis chemicals were from Promega Hampshire, PtdCho were obtained from chemicals for electron microscopy were from UK), and chemicals were of from or from and were by and choline was from IMR-32 cells were with 10 of HBSS without Ca2+/Mg2+ at with for cell enzyme and of HBSS the cell was in of without Ca2+/Mg2+ containing at min of cells were and of in The nuclear from through the at for min was in of HBSS without Ca2+/Mg2+ at °C and at for were in of HBSS without and was with and a were for or enzyme IMR-32 cell or nuclei in in were in in for in were in for by a in and a using in at and and three of were with (Life Hampshire, for for and and for The were using an in by for at microscopy were using a and were and with of was a transmission electron The of for of the nuclear matrix from cells, from with nuclear was by a This is the observation that the of nuclei containing nuclear envelope is D. M. D. D. A at Scholar). Consequently, such nuclei are from the not through the D. M. D. D. A at Scholar). that is for the nuclear envelope is D. M. D. D. A at Scholar). has been as a of both endoplasmic reticulum and nuclear envelope of nuclear matrix in and of is an to of nuclear matrix (16Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (255) Google Scholar, S. L. L. J. A. Biochem. Biophys. 1997; PubMed Scopus Google Scholar, L. A. S. G. L. Google Scholar). from of cell 10 and isolated nuclear from the were by 10% to analysis by was using with using and were using a from whole cells or isolated nuclei were to and W.J. J. Biochem. PubMed Scopus Google Scholar), and the PtdCho was in the of as Biochem. J. PubMed Scopus Google Scholar). a to an phospholipid containing phosphatidylserine, and PtdIns was with acid containing choline using was to choline mass of PtdCho and were at the of the to quantify individual molecular endogenous synthesized PtdCho IMR-32 cells were in RPMI 1640 with choline containing in at enrichment in the for to ESI-MS of PtdCho from whole cells or from nuclei was a mass with an electrospray ionization were in and the mass by or by with endogenous PtdCho a with C.J. Cui Z. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), to the synthesized which an C.J. Cui Z. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). of the and permitted the of endogenous and newly synthesized PtdCho species C.J. Cui Z. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). for the PtdCho species for the two time were to were and using to to and for and for with the PtdCho species were as a of the total present in the The for with was for PtdCho as a a to a of for PtdCho disaturated species at the following the were mass were by of by ESI-MS and of from whole cell or nuclei was in with with of a of to the J. PubMed Scopus Google Scholar). permitted of species present within the activity was by the of and K. PubMed Scopus Google Scholar). CCT were as Biochim. Biophys. Acta. PubMed Scopus Google Scholar), with with were to and in were to all were at activity was measured in the of as by and PubMed Scopus Google Scholar). and additional were to activity PubMed Scopus Google Scholar), and all activities were using in of PubMed Scopus Google Scholar). (EC activities were as Biochem. J. 1994; PubMed Scopus Google Scholar) additional lipid for nuclear activity to for high endogenous activities were in to a number of cells or isolated nuclei as during nuclei of both endoplasmic reticulum and nuclear envelope from nuclear matrix is an essential for of chromatin-associated phospholipid compositions and biosynthetic have been by of nuclear and be to retain D. G. Biochem. PubMed Scopus Google Scholar) or (2Vann L.R. Wooding F.B.P. Irvine R.F. Divecha N. Biochem. J. 1997; 327: 569-576Crossref PubMed Scopus (108) Google Scholar, M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (255) Google Scholar, Biochem. 1997; PubMed Scopus Google Scholar) the nuclear membrane from isolated was for the present a report that the nuclear envelope from isolated nuclei of (2Vann L.R. Wooding F.B.P. Irvine R.F. Divecha N. Biochem. J. 1997; 327: 569-576Crossref PubMed Scopus (108) Google Scholar). and high electron microscopy a membrane in whole IMR-32 cell nuclei a and that was from nuclei isolated in the of and evidence for of endoplasmic reticulum and evidence of nuclear envelope membrane were also by the of to of isolated nuclei of has been as of nuclear matrix in studies (16Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (255) Google S. L. L. J. A. Biochem. Biophys. 1997; PubMed Scopus Google Scholar, L. A. S. G. L. Google Scholar), nuclear envelope content the of cannot be by such This in a high of membrane-free nuclei was of Ca2+/Mg2+ from the the of a of nuclear matrix The capacity of the nuclear matrix of IMR-32 cells to a compartmentalized synthesis of PtdCho was by the and the of of the three enzymes of the CDP-choline choline (EC and choline activity was that of the whole endonuclear CCT without was of whole cell This endonuclear CCT activity was not by with in to the of the extranuclear enzyme by The of an of lipid nuclear CCT may have been to an of the to the nuclear or to which may lipid Chromatin-associated activity of whole cell is that and of DAG to the nuclear may have activity in the that choline activity may also have been by was to be CDP-choline pathway enzymes in IMR-32 cells and without with activities of choline and were in 10 of cells and of a of nuclei of their nuclear from the as and the activity for in a The activities of choline and were in 10 of cells and of a of nuclei of their nuclear from the as and the activity for the enzyme activities we the capacity vitro endonuclear of of the PtdCho to their biosynthetic not the existence of functionally compartmentalized in intact we as a in with ESI-MS to PtdCho biosynthesis C.J. Cui Z. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The of endogenous to newly synthesized PtdCho molecular species was by IMR-32 cells with and the PtdCho from isolated nuclei and from whole of the of of and were to quantify endogenous and newly synthesized PtdCho species C.J. Cui Z. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) of PtdCho from whole cells was by 10 min and for to which time of whole cell PtdCho the The time for the of the endonuclear PtdCho was the of the species were within 10 min However, a rate of choline was the of supplementation the time for of the the endonuclear choline and nuclear PtdCho synthesis in and IMR-32 a of endogenous PtdCho synthesized in cell and following of with in cells for cells and for and 10% during cells IMR-32 cells were with for or synthesized PtdCho was in whole cells and in isolated nuclei and is shown as the for and and as the for the ESI-MS analysis permitted the analysis of the molecular species of both endogenous and newly synthesized PtdCho a and using PtdCho as an we were to determine an endonuclear PtdCho of nuclei to This of the PtdCho present in the of the species of the endonuclear and whole cell PtdCho obtained from the of revealed that endonuclear PtdCho was in species both were The of such disaturated PtdCho to total PtdCho was for whole cells in was for endonuclear PtdCho PtdCho species with acid or of the total PtdCho from whole cells were the of in the endonuclear PtdCho pool of the This observation an additional of the of the nuclear matrix PtdCho species containing are a major of the nuclear envelope Lipids. PubMed Scopus Google Scholar, J. 2000; Full Text Full Text PDF PubMed Google Scholar). of the of the and that both nuclear and whole cell synthesized PtdCho molecular species with a to of their endogenous PtdCho However, of the revealed a whole cell PtdCho synthesis was indeed to the endogenous PtdCho with of within that time of newly synthesized endonuclear PtdCho and the endogenous pool that endonuclear Specifically, of newly synthesized endonuclear PtdCho was disaturated with of endogenous and the between saturated species and species were in newly synthesized endonuclear PtdCho. Moreover, the pattern of endonuclear PtdCho synthesis 10 min of supplementation of newly synthesized PtdCho to saturated with the pool with chromatin is to be saturated the of the whole cell M. A. S. D. J. K. J. 2000; Full Text Full Text PDF PubMed Google Scholar). activity with the nuclear matrix has the for a for of acid the nucleus A. Mol. Cell. Biochem. Google Scholar). Consequently, the necessary for of endonuclear PtdCho to saturated species are present within the nuclear PtdCho species containing or of whole cell PtdCho synthesis were from nuclear PtdCho synthesis at 10 min This that are synthesized within the nucleus are to saturated of newly synthesized whole cell and endonuclear PtdCho 10 min of a ESI-MS of the of the with of a endonuclear PtdCho following whole cell supplementation with for 10 to were in nuclear time ESI-MS of the of the of the whole cell PtdCho from a of cells from the The enrichment of in newly synthesized endonuclear PtdCho 10 min with whole cell PtdCho synthesis and the of synthesis are evidence for the existence of a compartmentalized PtdCho synthesis pathway within the nucleus. is possible that PtdCho synthesized the endoplasmic reticulum could be the of newly synthesized endonuclear PtdCho 10 min of with However, of saturated PtdCho species from the endoplasmic an as yet saturated PtdCho the nuclear envelope and the nuclear such have been for PtdCho that are both and and endonuclear PtdCho synthesis is the for our The together with CDP-choline pathway enzyme activities and the for active CCTα (12DeLong C.J. Qin L. Cui Z. J. Biol. Chem. 2000; 275: 32325-32330Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), for the existence of a functional pathway for the synthesis of endonuclear PtdCho in the intact ESI-MS studies also the of all endonuclear of and permitted the of the as well as the species of endonuclear and of and were and to the between PtdCho compositions between the whole cell and the compositions of and were between whole cell and endonuclear isolated from both was largely of species not was unsaturated both whole cell and endonuclear species containing or of the with between the of individual unsaturated the saturated of endonuclear PtdCho might be to in a the of the unsaturated is to endonuclear lipid a and active at physiological The of in the endonuclear phospholipid with a high degree of chain is also necessary for CCT A that CCTα activity in vitro was by the of in a membrane Jackowski S. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar), which in was by the lipid of that CCTα was in of saturated of PtdCho with unsaturated or of species to an are as have our ESI-MS we that of the endonuclear phospholipids were a with for CCTα of endonuclear and from the ESI-MS studies were to the volume that in the nucleus D. of Scholar). The molecular volume of a be from the volume of a or or of the and the number of or in chain The following for phospholipids in an were in the The amounts of endonuclear species as by ESI-MS were to the volume by species PtdCho which a total volume of This volume is the volume that be by the were a for the total volume be by a of to The volume of a IMR-32 nucleus was to be by an with transmission electron microscopy with and in that are such to a volume between and which to of the nuclear volume for IMR-32 nuclei the in human a volume of the total mass of in chromatin is to the mass of the volume by the is to be of the of of the nuclear The of to endonuclear by the and lipid from the nuclear volume no species or are is between and using a molecular of for the we that at the the endonuclear phospholipids we were present at an of 10% of of The high phospholipid to the nucleus suggest that are to be present as and as the synthesis of extranuclear PtdCho with the cell cycle S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), we sought to establish whether the synthesis of endonuclear PtdCho and species were or to IMR-32 cells were to a by in for This proliferation and of was cells were to the cell cycle by supplementation with 10% by growth cell and proliferation the growth arrest by serum the whole cell PtdCho during of with to of supplementation with 10% the whole cell PtdCho synthesis to of PtdCho synthesis during the cell the of the endonuclear the synthesis following growth arrest was of the cell cycle to an in synthesis to of in whole cell PtdCho and endonuclear PtdCho species were following serum and were by the species of the newly synthesized PtdCho. However, the of disaturated species in both endogenous and newly synthesized endonuclear PtdCho that unusual pattern was and J.C. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) that of cells by serum supplementation was by a of nuclear CCTα to the endoplasmic demonstration of nuclear of CCTα throughout the cell cycle (12DeLong C.J. Qin L. Cui Z. J. Biol. Chem. 2000; 275: 32325-32330Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) that of the have been to of CCT or cell change (12DeLong C.J. Qin L. Cui Z. J. Biol. Chem. 2000; 275: 32325-32330Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). not of CDP-choline pathway enzyme activities during serum and cannot that as we that both nuclear and whole cell PtdCho synthesis in IMR-32 cells following serum that the nuclear as a for CCT for the endoplasmic reticulum of IMR-32 cells at a time when endonuclear PtdCho synthetic The for endonuclear PtdCho synthesis is not is that pathway for PtdCho synthesis to membrane phospholipid within the the evidence that is to the synthesis of nuclear envelope as part of cell not of the unsaturated of Lipids. PubMed Scopus Google Scholar, J. 2000; Full Text Full Text PDF PubMed Google Scholar). are First, as the principal regulatory enzyme of PtdCho biosynthesis (1Rose H.G. Frenster J.H. Biochim. Biophys. Acta. 1965; 106: 577-591Crossref PubMed Scopus (85) Google Scholar), CDP-choline for the enzyme of the pathway The of is Accordingly, our a role for an endonuclear CDP-choline pathway in regulating endonuclear disaturated DAG distinct periodic nuclear accumulations of unsaturated DAG from PtdIns (16Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (255) Google Scholar, Clarke J.H. Divecha N. Biochim. Biophys. Acta. 1998; PubMed Scopus Google Scholar) and saturated DAG from PtdCho (17D'Santos C. Clarke J.H. Irvine R.F. Divecha N. Curr. Biol. 1999; 9: 437-440Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) are recognized. CCTα activity is essential for cell survival (12DeLong C.J. Qin L. Cui Z. J. Biol. Chem. 2000; 275: 32325-32330Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), of a DAG nuclear DAG accumulations by to and cell growth (16Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (255) Google Scholar). of through endonuclear PtdCho synthesis at in the cell cycle may to regulate potential endonuclear lipid second by active Martin A. C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Second, the saturated of endonuclear together with the of volume occupancy that we have may a role for endogenous PtdCho within the nuclear the the of a role for saturated endogenous the of the of within in to the is Moreover, the of significant amounts of in the nuclear envelope and as part of the endonuclear and PtdIns Clarke J.H. Divecha N. Biochim. Biophys. Acta. 1998; PubMed Scopus Google Scholar) within the nuclear matrix and in association with significant of of PtdCho in the nuclei may a of endogenous The of endonuclear CCTα to be in a of cultured cell (12DeLong C.J. Qin L. Cui Z. J. Biol. 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