Quantitative profiling of the endonuclear glycerophospholipidome of murine embryonic fibroblasts
2016; Elsevier BV; Volume: 57; Issue: 8 Linguagem: Inglês
10.1194/jlr.m068734
ISSN1539-7262
AutoresEmily K. Tribble, Pavlina T. Ivanova, Aby Grabon, James G. Alb, Irene Faenza, Lucio Cocco, Heather A. Brown, Vytas A. Bankaitis,
Tópico(s)Nuclear Structure and Function
ResumoA reliable method for purifying envelope-stripped nuclei from immortalized murine embryonic fibroblasts (iMEFs) was established. Quantitative profiling of the glycerophospholipids (GPLs) in envelope-free iMEF nuclei yields several conclusions. First, we find the endonuclear glycerophospholipidome differs from that of bulk membranes, and phosphatidylcholine (PtdCho) and phosphatidylethanolamine species are the most abundant endonuclear GPLs by mass. By contrast, phosphatidylinositol (PtdIns) represents a minor species. We also find only a slight enrichment of saturated versus unsaturated GPL species in iMEF endonuclear fractions. Moreover, much lower values for GPL mass were measured in the iMEF nuclear matrix than those reported for envelope-stripped IMF-32 nuclei. The collective results indicate that the nuclear matrix in these cells is a GPL-poor environment where GPL occupies only approximately 0.1% of the total nuclear matrix volume. This value suggests GPL accommodation in this compartment can be satisfied by binding to resident proteins. Finally, we find no significant role for the PtdIns/PtdCho-transfer protein, PITPα, in shuttling PtdIns into the iMEF nuclear matrix. A reliable method for purifying envelope-stripped nuclei from immortalized murine embryonic fibroblasts (iMEFs) was established. Quantitative profiling of the glycerophospholipids (GPLs) in envelope-free iMEF nuclei yields several conclusions. First, we find the endonuclear glycerophospholipidome differs from that of bulk membranes, and phosphatidylcholine (PtdCho) and phosphatidylethanolamine species are the most abundant endonuclear GPLs by mass. By contrast, phosphatidylinositol (PtdIns) represents a minor species. We also find only a slight enrichment of saturated versus unsaturated GPL species in iMEF endonuclear fractions. Moreover, much lower values for GPL mass were measured in the iMEF nuclear matrix than those reported for envelope-stripped IMF-32 nuclei. The collective results indicate that the nuclear matrix in these cells is a GPL-poor environment where GPL occupies only approximately 0.1% of the total nuclear matrix volume. This value suggests GPL accommodation in this compartment can be satisfied by binding to resident proteins. Finally, we find no significant role for the PtdIns/PtdCho-transfer protein, PITPα, in shuttling PtdIns into the iMEF nuclear matrix. The mammalian nuclear matrix is now recognized as a site of lipid biosynthesis and signaling in both normal and pathological states (reviewed in 1Irvine R.F. Nuclear inositide signalling–expansion, structures and clarification.Biochim. Biophys. Acta. 2006; 1761: 505-508Crossref PubMed Scopus (54) Google Scholar, 2Clarke J.H. Letcher A.J. D'Santos C.S. Halstead J.R. Irvine R.F. Divecha N. Inositol lipids are regulated during cell cycle progression in the nuclei of murine erythroleukaemia cells.Biochem. J. 2001; 357: 905-910Crossref PubMed Scopus (125) Google Scholar, 3Hunt A.N. Dynamic lipidomics of the nucleus.Biochim. Biophys. Acta. 2006; 1761: 577-587Crossref PubMed Scopus (19) Google Scholar, 4Monserrate J.P. York J.D. Inositol phosphate synthesis and the nuclear processes they affect.Curr. Opin. Cell Biol. 2010; 22: 365-373Crossref PubMed Scopus (83) Google Scholar, 5Barlow C.A. Laishram R.S. Anderson R.A. Nuclear phosphoinositides: a signaling enigma wrapped in a compartmental conundrum.Trends Cell Biol. 2010; 20: 25-35Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 6Albi E. Viola-Magni M.P. The role of intranuclear lipids.Biol. Cell. 2004; 96: 657-667Crossref PubMed Scopus (118) Google Scholar, 7Fiume R. Stijf-Bultsma Y. Shah Z.H. Keune W.J. Jones D.R. Jude J.G. Divecha N. PIP4K and the role of nuclear phosphoinositides in tumour suppression.Biochim. Biophys. Acta. 2015; 1851: 898-910Crossref PubMed Scopus (39) Google Scholar, 8Divecha N. Phosphoinositides in the nucleus and myogenic differentiation: how a nuclear turtle with a PHD builds muscle.Biochem. Soc. Trans. 2016; 44: 299-306Crossref PubMed Scopus (3) Google Scholar). Radioisotope and stable isotope tracer studies consistently identify glycerophospholipids (GPLs), and the enzymatic activities that metabolize them, within purified nuclear fractions purportedly devoid of nuclear envelope and other cellular membrane contaminants (9Viola-Magni M.P. Gahan P.B. Pacy J. Phospholipids in plant and animal chromatin.Cell Biochem. Funct. 1985; 3: 71-78Crossref PubMed Scopus (42) Google Scholar, 10Cocco L. Gilmour R.S. Ognibene A. Letcher A.J. Manzoli F.A. Irvine R.F. Synthesis of polyphosphoinositides in nuclei of Friend cells. Evidence for polyphosphoinositide metabolism inside the nucleus which changes with cell differentiation.Biochem. J. 1987; 248: 765-770Crossref PubMed Scopus (280) Google Scholar, 11Martelli A.M. Gilmour R.S. Bertagnolo V. Neri L.M. Manzoli L. Cocco L. Nuclear localization and signalling activity of phosphoinositidase C beta in Swiss 3T3 cells.Nature. 1992; 358: 242-245Crossref PubMed Scopus (309) Google Scholar, 12Albi E. Magni M.V. Sphingomyelin synthase in rat liver nuclear membrane and chromatin.FEBS Lett. 1999; 460: 369-372Crossref PubMed Scopus (53) Google Scholar, 13Divecha N. Letcher A.J. Banfic H.H. Rhee S.G. Irvine R.F. Changes in the components of a nuclear inositide cycle during differentiation in murine erythroleukaemia cells.Biochem. J. 1995; 312: 63-67Crossref PubMed Scopus (65) Google Scholar, 14Hunt A.N. Clarke G.T. Attard G.S. Postle A.D. Highly saturated endonuclear phosphatidylcholine is synthesized in situ and colocated with CDP-choline pathway enzymes.J. Biol. Chem. 2001; 276: 8492-8499Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 15Jones D.R. D'Santos C.S. Merida I. Divecha N. T lymphocyte nuclear diacylglycerol is derived from both de novo synthesis and phosphoinositide hydrolysis.Int. J. Biochem. Cell Biol. 2002; 34: 158-168Crossref PubMed Scopus (9) Google Scholar). These findings suggest the existence of an independently-regulated GPL pool within the nuclear matrix, one distinct from the nuclear envelope and from bulk cellular membranes. In support of this interpretation, nuclear isoforms of a number of GPL metabolic enzymes have been described (10Cocco L. Gilmour R.S. Ognibene A. Letcher A.J. Manzoli F.A. Irvine R.F. Synthesis of polyphosphoinositides in nuclei of Friend cells. Evidence for polyphosphoinositide metabolism inside the nucleus which changes with cell differentiation.Biochem. J. 1987; 248: 765-770Crossref PubMed Scopus (280) Google Scholar, 11Martelli A.M. Gilmour R.S. Bertagnolo V. Neri L.M. Manzoli L. Cocco L. Nuclear localization and signalling activity of phosphoinositidase C beta in Swiss 3T3 cells.Nature. 1992; 358: 242-245Crossref PubMed Scopus (309) Google Scholar, 16Smith C.D. Wells W.W. Phosphorylation of rat liver nuclear envelopes. I. Characterization of in vitro lipid phosphorylation.J. Biol. Chem. 1983; 258: 9360-9367Abstract Full Text PDF PubMed Google Scholar, 17Smith C.D. Wells W.W. Phosphorylation of rat liver nuclear envelopes. II. Characterization of in vitro lipid phosphorylation.J. Biol. Chem. 1983; 258: 9368-9373Abstract Full Text PDF PubMed Google Scholar, 18Wang Y. Sweitzer T.D. Weinhold P.A. Kent C. Nuclear localization of soluble CTP:phosphocholine cytidylyltransferase.J. Biol. Chem. 1993; 268: 5899-5904Abstract Full Text PDF PubMed Google Scholar, 19Freyberg Z. Sweeney D. Siddhanta A. Bourgoin S. Frohman M. Shields D. Intracellular localization of phospholipase D1 in mammalian cells.Mol. Biol. Cell. 2001; 12: 943-955Crossref PubMed Scopus (156) Google Scholar, 20Boronenkov I.V. Loijens J.C. Umeda M. Anderson R.A. Phosphoinositide signaling pathways in nuclei are associated with nuclear speckles containing pre-mRNA processing factors.Mol. Biol. Cell. 1998; 9: 3547-3560Crossref PubMed Scopus (279) Google Scholar, 21Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Protein kinase C regulates the nuclear localization of diacylglycerol kinase-zeta.Nature. 1998; 394: 697-700Crossref PubMed Scopus (252) Google Scholar, 22Divecha N. Rhee S.G. Lechter A.J. Irvine R.F. Phosphoinositide signalling enzymes in rat liver nuclei: phosphoinositidase C isoform beta 1 is specifically, but not predominantly, located in the nucleus.Biochem. J. 1993; 289: 617-620Crossref PubMed Scopus (166) Google Scholar). The STAR-PAP RNA poly-A polymerase, the direct modification of a phosphoinositide headgroup presented to an inositide kinase by a nuclear receptor, and nuclear phosphoinositide control of the basal transcription machinery all provide compelling examples of GPL-regulated activities that discharge their functions within the nuclear matrix (23Mellman D.L. Gonzales M.L. Song C. Barlow C.A. Wang P. Kendziorski C. Anderson R.A. A PtdIns4,5P2-regulated nuclear poly(A) polymerase controls expression of select mRNAs.Nature. 2008; 451: 1013-1017Crossref PubMed Scopus (187) Google Scholar, 24Gonzales M.L. Mellman D.L. Anderson R.A. CKIalpha is associated with and phosphorylates star-PAP and is also required for expression of select star-PAP target messenger RNAs.J. Biol. Chem. 2008; 283: 12665-12673Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 25Blind R.D. Miyuki Suzawa M. Ingraham H.A. Direct modification and regulation of a nuclear receptor-phosphoinositide complex by the inositol-lipid kinase IPMK and PTEN.Sci. Signal. 2012; 5: ra44Crossref PubMed Scopus (75) Google Scholar, 26Sablin E.P. Blind R.D. Uthayaruban R. Chiu H.J. Deacon A.M. Das D. Ingraham H.A. Fletterick R.J. Structure of liver receptor homolog-1 (NR5A2) with PIP3 hormone bound in the ligand binding pocket.J. Struct. Biol. 2015; : 342-348Crossref PubMed Scopus (35) Google Scholar, 27Stijf-Bultsma Y. Sommer L. Tauber M. Baalbaki M. Giardoglou P. Jones D.R. Gelato K.A. van Pelt J. Shah Z. Rahnamoun H. et al.The basal transcription complex component TAF3 transduces changes in nuclear phosphoinositides into transcriptional output.Mol. Cell. 2015; 58: 453-467Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). At issue, however, is the scale of nuclear GPL metabolism and nuclear GPL load. This difficult question can now be addressed using quantitative mass spectrometric methods. The single major study on this topic estimates that phosphatidylcholine (PtdCho) alone occupies 10–16% of the nuclear matrix of IRB-32 cells by volume (14Hunt A.N. Clarke G.T. Attard G.S. Postle A.D. Highly saturated endonuclear phosphatidylcholine is synthesized in situ and colocated with CDP-choline pathway enzymes.J. Biol. Chem. 2001; 276: 8492-8499Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). This is a startling conclusion considering that the genome itself is estimated to occupy some 39% of the nuclear volume in the IMF-32 cell line. Furthermore, the endonuclear GPL pool is reported to be unusual in that it is dominated by saturated PtdCho molecular species (14Hunt A.N. Clarke G.T. Attard G.S. Postle A.D. Highly saturated endonuclear phosphatidylcholine is synthesized in situ and colocated with CDP-choline pathway enzymes.J. Biol. Chem. 2001; 276: 8492-8499Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The abundance of endonuclear GPLs, when coupled with their predominantly saturated nature, motivates speculation that phospholipids (PLs) profoundly influence the chemical properties of the nuclear matrix, i.e., by contributing to the formation of gel-like regions within the nuclear matrix (1Irvine R.F. Nuclear inositide signalling–expansion, structures and clarification.Biochim. Biophys. Acta. 2006; 1761: 505-508Crossref PubMed Scopus (54) Google Scholar, 14Hunt A.N. Clarke G.T. Attard G.S. Postle A.D. Highly saturated endonuclear phosphatidylcholine is synthesized in situ and colocated with CDP-choline pathway enzymes.J. Biol. Chem. 2001; 276: 8492-8499Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). This concept raises a fundamental question of how does the nuclear matrix accommodate such a large PL load? That is, how are lipids organized within the nuclear matrix? Given the mounting evidence for endonuclear lipid signaling and the lingering questions regarding how lipids are organized in nuclear matrix, we reinvestigated the problem of endonuclear lipidomics. To this end, we developed a reliable and reproducible method for purification of envelope-free nuclei from immortalized murine embryonic fibroblasts (iMEFs). This method adheres to a stringent quality-control regime for assessing purity of isolated endonuclear compartments. Quantitative GPL profiling of these highly purified fractions describes an endonuclear GPL composition that is indeed distinct from that of bulk cellular membrane. Contrary to previous reports, however, the profile shows no particular enrichment of saturated GPL molecular species. Moreover, the mass measurements record drastically lower GPL contents in endonuclear compartments than those previously reported, at least for a neuroblastoma cell line. While the collective data confirm the nuclear matrix harbors a GPL pool of distinct composition from that of bulk membrane, the data also identify the iMEF nuclear matrix as a PL-poor environment that requires no unusual provisions for PL accommodation other than binding to resident proteins. Chemicals and reagents were purchased from Fisher Scientific (Pittsburg, PA) or from Sigma-Aldrich (St. Louis, MO), unless otherwise stated. All lipid standards were purchased from Avanti Polar Lipids (Alabaster, AL). Organic solvents and supplies used to prepare samples for electron microscopy (EM) were obtained from Electron Microscopy Sciences (Hatfield, PA). The mass labels, myo-inositol-d6 and choline-d9, were obtained from C/D/N Isotopes (Pont-Claire, Quebec, Canada) and Sigma-Aldrich, respectively. DMEM and antibiotics were obtained from Gibco/Invitrogen (Carlsbad, CA). FBS was obtained from Gemini Bio-products (Sacramento, CA). A mouse monoclonal antibody directed toward β-tubulin (product number T5293) and rabbit polyclonal antibody directed toward lamin A (product number L1293) were obtained from Sigma-Aldrich. A mouse monoclonal antibody toward α-tubulin was obtained from Neomarkers Inc. (Fremont, CA; product number MS-581-P). Other rabbit polyclonal antibodies utilized in this study include: an anti-calnexin antibody (Stressgen Assay Designs, Ann Arbor, MI; product number SPA-860), an anti-histone H3 antibody (generous gift of Brian Strahl, University of North Carolina-Chapel Hill), an anti-NURIM antibody (Santa Cruz Biotechnology, Santa Cruz, CA; product number sc-133260), and an anti-fibrillarin antibody (Abcam Inc., Cambridge, MA; product number ab5821). Goat-anti-mouse or goat-anti-rabbit HRP-conjugated secondary antibodies (Bio-Rad, Hercules, CA) were used for development in ECL assays. Donkey-anti-rabbit secondary antibody conjugated to IR Dye 800 was purchased from Rockland Immunochemicals Inc. (Gilbertsville, PA; product number 611-731-127) for use in Odyssey immunoblotting experiments. iMEFs were derived from E14-E16 embryos, and immortalized iMEF lines were generated using the SV40 large T-antigen method (28Shay J.W. Wright W.E. Quantitation of the frequency of immortalization of normal human diploid fibroblasts by SV40 large T-antigen.Exp. Cell Res. 1989; 184: 109-118Crossref PubMed Scopus (237) Google Scholar). Unless otherwise specified, all primary and immortalized cell lines were cultured in complete DMEM containing 4.5 g/l glucose and supplemented with 10% FBS, 1 U/ml penicillin G, and 100 μg/ml streptomycin (complete DMEM). All cell culture was performed at 37°C in a 10% CO2 incubator. Envelope-stripped nuclei were prepared using the method of Martelli et al. (11Martelli A.M. Gilmour R.S. Bertagnolo V. Neri L.M. Manzoli L. Cocco L. Nuclear localization and signalling activity of phosphoinositidase C beta in Swiss 3T3 cells.Nature. 1992; 358: 242-245Crossref PubMed Scopus (309) Google Scholar) with essential modifications: iMEF cells were seeded to 150 mm tissue culture dishes and grown for 24–48 h. Approximately 107 iMEFs were pelleted (for a 1× preparation) and washed three times in Dulbecco's PBS solution. After complete removal of PBS, the cell pellet was resuspended thoroughly in 500 μl of chilled buffer A [10 mM Tris-HCl (pH 7.4), 1% NP-40, 10 mM β-mercaptoethanol, 0.5 mM PMSF] supplemented with Complete Protease Inhibitor cocktail (Roche Biopharmaceuticals). Cells were incubated on ice for 8 min with occasional agitation. An equal volume of ice-cold ddH2O was added to swell the cells. Following a 3 min incubation, swollen cells were subsequently subjected to three passages through a 22 gauge needle. Removal of cellular debris was monitored by examination of several microliters of triturate by phase contrast microscopy. In sufficiently sheared samples, minimally 40 of 50 nuclei examined lacked significant cytosolic or membranous debris. After addition of 0.5 ml of chilled buffer B [10 mM Tris-HCl (pH 7.4), 2 mM MgCl2] supplemented with protease inhibitors, the nuclei were gently triturated and again examined by microscopy. Nuclei were returned to ice for 1 min in preparation for centrifugation, during which a portion of lysate (representing the whole cell fraction) was saved for immunoblot analysis of cellular markers and total protein quantification. Nuclei were sedimented at 86 g for 10 min at 4°C. The supernatant (cytosolic fraction) was either discarded or saved for quality control analysis as needed. The crude pellets were washed once with an excess of buffer B and sedimented again at 86 g for 10 min at 4°C. During an unscaled preparation, the purified nuclei were resuspended in buffer B and distributed as necessary for quality control protein analyses. When generating three times purified nuclear pellets for analysis of PLs by ESI LC-MS, the purified pellet was instead resuspended in 1 ml total of buffer B. Of this suspension, 85% of the final material was pelleted at 500 g for 2 min at 4°C. Following complete removal of the supernatant, the pellet was snap-frozen in liquid nitrogen and stored at −80°C in preparation for PL analysis by MS. Aliquots (150 μl) were collected for protein measurements and quality controls before the final pelleting step. In preparation for immunoblot analysis, whole cell lysates, wash fractions, and nuclear pellets were homogenized in M-Per lysis buffer (Thermo Scientific) supplemented with Complete Protease Inhibitor Cocktail (Roche Biopharmaceuticals). Samples were triturated vigorously through a 25 gauge needle until complete sample disruption was achieved (as confirmed by phase contrast microscopy). Samples were clarified by centrifugation, and the supernatant was separated to a fresh tube for protein precipitation using the SDS-PAGE Clean-up kit (GE Life Sciences, Piscataway, NJ) according to the manufacturer's directions. Precipitates were resuspended in CHAPS buffer (8 M urea, 2% CHAPS, and 50 mM DTT). These samples were solubilized in Laemmli sample buffer resolved by SDS-PAGE (10% gels), and nuclear and membrane markers were visualized by immunoblotting. Sample loading was normalized by "cell equivalents." For Odyssey Westerns, the range of signal linearity was determined for each fraction with each antibody. Resolved proteins were transferred to nitrocellulose membranes by standard methods. Membranes were blocked in the appropriate blocking reagent (as recommended by the manufacturer) for 1 h at room temperature and probed with primary antibody overnight at 4°C. Decorated membranes were washed three times for 10 min in TTBS and incubated for an additional 1–2 h with the corresponding HRP-conjugated secondary antibody diluted in 2% BSA in TTBS. Blots were again washed three times in TTBS and once in PBS before development using the ECL method (Amersham Biosciences). We define the threshold for acceptable purity as lack of detectable calnexin immunoreactivity in a nuclear preparation of 2.4 × 105 cell equivalents. For detection of blotted proteins using the Odyssey platform, transferred membranes were blocked for 1 h at room temperature in Odyssey blocking buffer (LI-COR Biotechnology, Lincoln, NE). The appropriate primary and secondary antibodies were diluted in a 1:1 solution of Odyssey blocking buffer and PBS. Secondary incubations and terminal wash steps were performed in the dark. Decorated membranes were analyzed on the Odyssey® infrared imaging system using Odyssey® 2.0 software (LI-COR Biotechnology). Scan settings were high image quality, resolution was set to 169 μm, and the intensity of the scan was 5.0. Antibody signals were quantified as integrated intensities of the areas above and below the bands of interest. Prior to protein quantification, cellular fractions were reconstituted in 1% SDS and incubated at 95°C for 10 min. Cooled samples were homogenized by at least 30 passages through a 25 gauge needle. Satisfactory sample disruption was confirmed by phase microscopy and samples were subsequently clarified by centrifugation. In preparation for BCA analysis (Thermo Scientific, Rockland, IL), samples were diluted 1:10 or 1:20. Diluted fractions were analyzed in triplicate according to the protocol for BCA assay for microplate reader. Purified nuclei were pelleted and fixed in 2% glutaraldehyde, 1% tannic acid in 0.1 M sodium cacodylate, 2 mM CaCl2, and postfixed in 2% OsO4 in 0.1 M sodium cacodylate, 2 mM CaCl2. Samples were stained in 4% uranyl acetate in 50% ethanol, dehydrated in a 50–100% ethanol/water series, cleared with propylene oxide, and embedded in Embed-812 resin (Electron Microscopy Sciences). Ultrathin sections (50 μm) were prepared using a Leica UCT ultramicrotome, and images were acquired with a Tecnai 12 transmission electron microscope (FEI, Hillsboro, OR), equipped with a Gatan model 794 multiscan digital camera. To examine nuclei at different stages of purification, samples were fixed during extraction, shearing, and "unveiling" stages (see Results) of separate 1× preparations. Nuclei undergoing the initial extraction in buffer A (500 μl total volume) were diluted to 10 ml with an excess of fixative [2% glutaraldehyde, 1% tannic acid, 2 mM CaCl2 in 0.1 M sodium cacodylate (pH 7.4)]. Nuclei were fixed at the 6 min time point. Sheared nuclei (1 ml) and "unveiled" nuclei (1.5 ml) were also diluted to the same final volume in fixative. One milliliter of each dilution was pelleted at 100 g for 10 min at 4°C. Fixed samples were washed two times for 10 min in 1 ml fixative. After washing thoroughly in buffer [2 mM CaCl2, 0.1 M sodium cacodylate(pH 7.4)], samples were processed for EM as described above. To generate iMEF pellets for bulk cellular GPL analysis, 1.2 × 106 cells were seeded onto 150 mm tissue culture dishes and grown for 24 h to 60–70% confluence. Some 107 cells were harvested by trypsinization, pelleted, and washed two times in HBSS without Ca2+ or Mg2+. Cells were then transferred to microfuge tubes in 1 ml of the same buffer and pelleted at 1,000 g for 5 min. Following complete removal of the supernatant, pellets were frozen in liquid nitrogen and stored at −80°C prior to lipid extraction and global PL quantification by MS. GPLs from whole cells or nuclear pellets were extracted using a modified Bligh and Dyer procedure (29Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42694) Google Scholar). Approximately 1 × 107 iMEF cells or 3 × 107 nuclei per pellet in cold 1.5 ml microfuge tubes (Laboratory Product Sales, Rochester, NY) were vortexed with 800 μl of cold 0.1 N HCl:CH3OH (1:1) and 400 μl of cold CHCl3 was added. The extraction proceeded with vortexing (1 min) and centrifugation (5 min, 4°C, 18,000 g). Quantification of GPLs was achieved by the use of ESI LC-MS employing synthetic (non-naturally occurring) diacyl and lysophospholipid standards as communicated elsewhere (30Ivanova P.T. Milne S.B. Byrne M.O. Xiang Y. Brown H.A. Glycerophospholipid identification and quantitation by electrospray ionization mass spectrometry.Methods Enzymol. 2007; 432: 21-57Crossref PubMed Scopus (136) Google Scholar). Typically, 200 ng of each odd-carbon standard was added to each sample. Identification of the individual PLs was accomplished by LC-MS/MS using an MDS SCIEX 4000 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer and a Shimadzu HPLC system with a normal phase Luna Silica column (2 × 250 mm, 5 μm) using a gradient elution (30Ivanova P.T. Milne S.B. Byrne M.O. Xiang Y. Brown H.A. Glycerophospholipid identification and quantitation by electrospray ionization mass spectrometry.Methods Enzymol. 2007; 432: 21-57Crossref PubMed Scopus (136) Google Scholar). Identification of the individual species was based on their chromatographic and mass spectral characteristics and comparison to these of chemically defined standards (30Ivanova P.T. Milne S.B. Byrne M.O. Xiang Y. Brown H.A. Glycerophospholipid identification and quantitation by electrospray ionization mass spectrometry.Methods Enzymol. 2007; 432: 21-57Crossref PubMed Scopus (136) Google Scholar, 31Milne S. Ivanova P. Forrester J. Brown H.A. Lipidomics: an analysis of cellular lipids by ESI-MS.Methods. 2006; 39: 92-103Crossref PubMed Scopus (152) Google Scholar). This analysis allows identification of both fatty acid moieties, but does not determine position on the glycerol backbone (sn-1 vs. sn-2). Data are presented as means plus standard errors. Differences between percentages of the total GPL pool represented by different classes for whole cells versus nuclei were determined by Student's t-test. Rat PITPα cDNA was amplified by PCR and subcloned as a 0.85 kb HindIII-BamHI fragment into pEGFP-N1 (Clontech, Palo Alto, CA). A HA-tagged rat PITPα cDNA (C-terminal tag) was generated by amplification of the rat PITPα cDNA and insertion of the 0.85 kb product into the unique BamHI-NotI sites of pEF3HA, a derivative of pEF4 (Invitrogen, Carlsbad, CA). This construct contains a HA epitope that was incorporated into that construct as an XbaI-PmeI cassette (DNA sequence 5′-TATCCTTACGAC GTTCCAGACTATGCA-3′). Site-directed mutagenesis primers used in this study were from Fisher Scientific, and rat PITPα cDNAs were mutagenized according to QuickChange mutagenesis kit specifications (Stratagene, La Jolla, CA). All mutant constructs were confirmed by nucleotide sequence analysis. Immortalized cell lines were transfected using the FuGene transfection reagent (Roche, Indianapolis, IN). Briefly, Cos7 cells or HeLa lines were seeded onto plastic dishes in complete DMEM 24 h prior to transfection. Once cells settled on the plastic surface, the medium was exchanged for antibiotic-free DMEM. One microgram aliquots of PITPα-EGFP constructs were incubated in 100 μl Opti-MEM (Invitrogen) premixed with 3 μl transfection reagent according to the manufacturer's instructions. The complete transfection cocktail was incubated at room temperature for 1 h before distribution to the medium. At 12 h posttransfection, cells were split onto coverslips and cultured in complete DMEM. HeLa or Cos7 cells, transiently transfected with appropriate PITPα-EGFP or PITPα-HA expression plasmids, were fixed 16–20 h posttransfection in 4% PFA in PBS. After permeabilization in 0.2% Triton X-100, cells expressing PITPα-EGFP constructs were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Molecular Probes) for 1 min. Cells transfected with PITPα-HA constructs were permeabilized similarly, then incubated for 1 h at room temperature in blocking buffer (2% BSA in PBS). Primary anti-HA antibodies (1:2,000 dilution in blocking buffer) were applied to blocked coverslips. After a 12–15 h incubation of coverslips with primary antibody at 4°C, cells were serially washed three times for 10 min in 1% BSA in PBS. Secondary antibodies (1:8,000 in blocking buffer) were then applied onto coverslips and fixed cells were incubated at 4°C for 5–8 h. After several washes (1% BSA in PBS), cells were counterstained with DAPI. Coverslips were mounted on glass slides, imaged on a Zeiss 510 META scanning laser confocal microscope, and images were processed using Adobe Photoshop 6.0. PITPα+/+ or pitpα0/0 iMEFs were seeded in complete DMEM and grown to a subconfluent (∼60%) density. Cells were washed in PBS and labeled for the desired time period in antibiotic-free DMEM containing 10% FBS, 80 μg/ml choline-d9, and 50 μg/ml myo-inositol-d6. For whole cell measurements, PITPα+/+ or pitpα0/0 iMEFs were collected in 800 μl HBSS for GPL extraction and analysis by LC-MS/MS. In experiments involving purified nuclei stripped of membranes, 3 × 107 cells were pelleted and washed in PBS prior to detergent-mediated removal of the nuclear envelope. Extracts pooled from three samples (9 × 107 nuclei) were isolated and analyzed by LC-MS. Purification of quality envelope-free nuclei requires comprehensive removal of contaminating cellular membranes and nuclear envelope with minimal compromise of nuclear integrity or of the biochemical character of the nuclear matrix. Because of the experimental advantages offered by the genetically tractable murine embryonic fibroblast (MEF) system for addressing questions related to nuclear signaling, we developed a method for preparing highly purified envelope-free nuclei from these cells. The method generated purified endonuclear compartments suitable for quantitative lipidomic analyses and yielded reproducible data. The procedure employed serial stripping manipulations in the presence of detergent (1% NP-40), and hypotonic swelling and mechanical shearing in progressively more dilute detergent environments (1% → 0.5% → 0.33% → 0% NP-40) to arrive at envelope-free nuclear preparations. As a general comment, our protocol for purification of envelope-free nuclei wo
Referência(s)