Highly Saturated Endonuclear Phosphatidylcholine Is Synthesized
in Situ and Colocated with CDP-choline Pathway Enzymes*
Alan N.
Hunt
§,
Graeme T.
Clark
,
George S.
Attard¶, and
Anthony D.
Postle
From the
Department of Child Health, University of
Southampton, Southampton General Hospital, Southampton SO16 6YD,
United Kingdom and the ¶ Department of Chemistry, University of
Southampton, Southampton SO17 1BJ, United Kingdom
Received for publication, October 30, 2000, and in revised form, December 15, 2000
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ABSTRACT |
Chromatin-associated phospholipids are well
recognized. A report that catalytically active endonuclear
CTP:choline-phosphate cytidylyltransferase
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.
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INTRODUCTION |
Considerable evidence supports the existence of an endonuclear
pool of phospholipid, in association with the nuclear matrix and
distinct from the nuclear envelope (1-4). This endonuclear phospholipid is remarkable for several reasons. Although all the major
membrane phospholipids, predominantly phosphatidylcholine (PtdCho),1
phosphatidylethanolamine (PtdEtn), phosphatidylserine, and
phosphatidylinositol (PtdIns), may be present typically at 4-10% of
total cell content (2), transmission electron microscopy has failed to
reveal endonuclear membranous systems (5). 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 (3, 4). A number of in vitro studies suggest a
functional relationship between various endonuclear phospholipids and
gene expression/transcription (6-10). Moreover, cell studies have
shown that the amounts of endonuclear phospholipids change during
progression through the cell cycle (4).
The potential physiological importance of intranuclear phospholipid has
recently been highlighted by the recognition that the
isoform
(CCT
) of CTP:choline-phosphate cytidylyltransferase, the
principal regulatory enzyme of PtdCho biosynthesis (11), is confined to
the nucleus throughout the cell cycle (12). Furthermore, modified
Chinese hamster ovary MT 58 cells (13) expressing a temperature-sensitive mutation of CCT
are not viable when grown at
the restrictive temperature (12), 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 (14) and involves one or both of the
isoform(s) of cytidylyltransferase (CCT
-1, CCT
-2) (15), cannot
rescue mutant cells grown at the restrictive temperature (12). 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 (16). Mobilization of DAG
in the nucleus is bipartite (17) 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.
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MATERIALS AND METHODS |
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 (Southampton, Hampshire, UK). PtdCho standards were
obtained from Sigma. Specialist chemicals for electron microscopy were
from Agar Scientific (Stansted, Essex, UK), and other chemicals were of
AnalaR grade from Merck or from Sigma-Aldrich unless otherwise stated.
Phospho[14C]methylcholine and
CDP-[14C]methylcholine were supplied by Amersham
Pharmacia Biotech, and [14C]methyl choline was
from ICN Biomedicals (Thame, Oxfordshire, UK).
Preparation of Membrane-depleted Nuclei--
Trypsinized,
subconfluent IMR-32 cells (2-6 × 107
cells/experiment) were washed twice with 10 ml of HBSS without
Ca2+/Mg2+ at 4 °C, with fractions removed
for cell counting, enzyme assay, and electrophoresis. After removal of
excess HBSS the cell pellet was resuspended in 500 µl of extraction
buffer (HBSS without Ca2+/Mg2+ containing 0.5%
Triton X-100, 4 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, pH 7.4) at 4 °C. After 15 min of
incubation on ice, cells were resuspended and layered over 400 µl of
30% sucrose in extraction buffer. The nuclear pellet recovered from
centrifugation through the sucrose cushion at 4,500 × g for 3 min was resuspended in 1 ml of HBSS without
Ca2+/Mg2+ at 4 °C and centrifuged again at
4,500 × g for 3 min. Nuclear pellets were then
resuspended in 1 ml of HBSS without Ca2+/Mg2+,
and 50 µl was removed, stained with propidium iodide (25 µg/ml), and counted on a hemocytometer. Aliquots (200 µl) were separated for
electrophoresis or enzyme activities.
Electron Microscopy of Membrane-depleted Nuclei--
Whole
IMR-32 cell pellets or membrane-depleted nuclei pellets, washed in 3%
glutaraldehyde in 0.1 M sodium cacodylate, 2 mM CaCl2 buffer, pH 7.4, were incubated in 2%
OsO4 in cacodylate buffer for 1 h. After rinsing in
distilled water they were then incubated in 2% aqueous uranyl acetate
for 30 min, followed by a further rinse in distilled water and a
sequential dehydration protocol using 10-min incubations in ethanol at
70 and 90% and finally three changes of 100% ethanol. Pellets were
then incubated with Histosol (Life Sciences International,
Basingstoke, Hampshire, UK) for 30 min, Histosol/Spurr's resin (50/50)
for 1 h, and finally Spurr's resin and vinyl cyclohexene
dioxide:polypropylene glycol diglycidyl ether:nonenyl succinic
anhydride:dimethylamino ethanol (23:16.3:60.5:1, w/w) for 24 h.
The processed pellets were embedded using an 8-mm flat polythene
TAAB capsule (TAAB Laboratory Equipment Ltd., Aldermaston, Berkshire,
UK) in fresh Spurr's resin followed by polymerization for 16 h at
60 °C. Electron microscopy silver sections were cut using a
Reichert-Jung Ultracut E ultramicrotome and diamond knife. Sections
were then placed on TAAB 100-µm copper grids and counterstained with
Reynold's lead citrate, 8 mM
Pb(NO3)2, 0.12 M sodium citrate,
0.16 M NaOH. Examination of sections was then performed on
a Hitachi H7000 transmission electron microscope.
Nuclear Envelope Marker Analysis--
The method of choice for
purification of the nuclear matrix from cells, free from contamination
with nuclear envelope, was detergent extraction followed by separation
over a sucrose gradient. This procedure is based on the observation
that the buoyant density of nuclei containing residual nuclear envelope
membranes is decreased (18). Consequently, such contaminated nuclei are
effectively removed from the preparation, because they will not
sediment through the sucrose cushion (18). No unambiguous protein
marker that is specific for the nuclear envelope is currently
recognized (18).
-tubulin has been used as a marker of both
endoplasmic reticulum and nuclear envelope contamination of nuclear
matrix preparations in other studies, and absence of immunoreactivity
upon Western blotting is an accepted criterion used to assess purity of
nuclear matrix preparations (16, 19, 20). Paired protein fractions from
post-mitochondrial supernatants of cell homogenates (105
cells; 10,000 × g; 10 min) and isolated nuclear
matrices (105 nuclei) from the same preparation were
separated by 10% discontinuous SDS-polyacrylamide gel electrophoresis.
Following transfer to nitrocellulose, analysis by Western blotting was
performed using monoclonal anti-
-tubulin (1/500, Sigma T5293) with
detection using horseradish peroxidase-conjugated anti-mouse IgG
(Dako, Ely, Cambridge, UK) and
3,3',5,5'-tetramethyl-benzidine-stabilized substrate (Promega).
Molecular weights were determined using a mid-range protein mix
(Promega).
Phospholipid Purification--
Phospholipids from whole cells or
isolated nuclei were extracted according to Bligh and Dyer (21), and
the PtdCho component was separated in the chloroform:methanol (60:40; 1 ml) wash of 100-mg Bond Elut aminopropyl solid phase extraction
cartridges (Varian) as previously described (22). Following a further
methanol wash (1 ml) to elute PtdEtn, an acidic phospholipid fraction
containing PtdOH, phosphatidylglycerol, phosphatidylserine, and PtdIns
was eluted with methanol:water:phosphoric acid (96:4:1; 3 × 1 ml) containing 40 mM choline chloride. Back-extraction, using
chloroform methanol, was performed to remove choline chloride before
mass spectrometry. Internal standards of dimyristoyl PtdCho (PtdCho 14:0/14:0; 15 nmol/107 cells; 0.5 nmol/107
nuclei), dimyristoyl PtdEtn (PtdEtn 14:0/14:0; 4 nmol/107
cells; 0.133 nmol/107 nuclei) and dimyristoyl PtdOH (PtdOH
14:0/14:0; 0.5 nmol/107 cells; 0.0167 nmol/107
nuclei) were added at the start of the extraction to quantify individual molecular species.
ESI-MS of Phospholipids--
For endogenous versus
newly synthesized PtdCho composition analysis, IMR-32 cells were
incubated in RPMI 1640 medium supplemented with choline containing nine
deuterons in methyl groups (d9; C/D/N Isotopes Inc.,
Pointe-Claire, Quebec, Canada) at 8 mg/100 ml (96% enrichment in the
medium) for up to 3 h. ESI-MS of PtdCho extracted from whole cells
or from nuclei was performed on a Micromass Quatro Ultima triple
quadrupole mass spectrometer (Micromass, Wythenshaw, UK) equipped with
an electrospray ionization interface. Samples were dissolved in
methanol:chloroform:water (7:2:1, v/v) and introduced into the mass
spectrometer either by syringe pump or by nanoflow infusion. Following
fragmentation with argon gas, endogenous PtdCho molecules produced a
fragment with m/z = 184 (23), corresponding
to the protonated phosphocholine headgroup. Newly synthesized PtdCho,
which contained nine deuterons, produced an analogous fragment at
m/z = 193 (23). Sequential parent scans of
the m/z 184 and 193 moieties permitted the
determination of endogenous and newly synthesized PtdCho species (23).
Total ion counts for the PtdCho species collected sequentially for the two fragments over identical time scales were ratioed to give percentages synthesized. Data were acquired and processed using MassLynx NT software. After conversion to centroid format according to
area and correction for 13C isotope effects and for reduced
response with increasing m/z values, the PtdCho
species were expressed as a percentage of the total present in the
sample. The formula for reduced response with increased
m/z was determined experimentally for PtdCho as follows: a = 2 × 1013 · b
3.9873, where a = reduced
response factor relative to a value of 1.00 for PtdCho 14:0/14:0, and
b = m/z value. In the
m/z = 184 parent scans, disaturated species
occurred at the following m/z values: 16:0
alkyl/14:0, m/z = 693; 16:0/14:0,
m/z = 707; 16:0 alkyl/16:0, m/z = 721; 16:0/16:0,
m/z = 735; 16:0 alkyl/18:0,
m/z = 749; 16:0/18:0, m/z = 763; 18:0/18:0, m/z = 791. In the
m/z = 193 parent scans the corresponding
peaks were 9 m/z mass units higher. Peak identifications were confirmed by product ion scanning of fatty acyl groups by tandem
MS/MS. ESI-MS and ESI-MS/MS of PtdEtn from whole cell or nuclei was
performed in methanol:chloroform:water (7:2:1 with 0.5% w/v NaI).
Following fragmentation with argon gas, sodium adducts of PtdEtn
lost a neutral fragment of m/z = 141, corresponding to the protonated phosphoethanolamine headgroup (24).
Sequential constant neutral loss scans of
m/z = 141 permitted determination of PtdEtn
species present within the samples.
CDP-choline Pathway Enzyme Determinations--
CK activity was
determined by the method of Ishidate and Nakazawa (25). CCT assays were
performed as previously described (26), with some modifications.
Charcoal washes with water were increased to five, and elution washes
in ethanol/ammonia were increased to three; all were performed at room
temperature. Lipid-stimulated activity was measured in the presence of
PtdCho/oleate vesicles prepared as described by Weinhold and
Feldman (27). ADP (6 mM) and additional MgCl2
(3 mM) were included to block intrinsic nucleotidase activity (27), and all determined activities were corrected using
CDP-[methyl-14C]-choline recovery
standards in each batch of assays (27). CDP-choline:1,2-diacylglycerol
cholinephosphotransferase (EC 2.7.8.2) activities were
determined as reported previously (28) but included additional lipid
blanks for nuclear activity determinations to correct for high
endogenous DAG. All activities were expressed in relation to a unit
number of cells or isolated nuclei (1 × 106) as
determined during nuclei preparation.
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RESULTS AND DISCUSSION |
Exclusion of both endoplasmic reticulum and nuclear envelope
membranes from experimental nuclear matrix preparations is an essential
requirement for quantification of chromatin-associated phospholipid
compositions and biosynthetic capacities. Detergent-based extraction
protocols have been successfully exploited by other groups studying
aspects of nuclear phosphoinositide metabolism and can be designed
either to retain (29) or remove (2, 16, 30) the nuclear membrane from
isolated nuclei. Triton X-100 was selected for the present study based
upon a report that concentrations above 0.04% (w/v) completely removed
the nuclear envelope from isolated nuclei of rat liver (2). Low and
high power electron microscopy clearly showed a typical limiting double
bilayer membrane in whole IMR-32 cell nuclei (Fig.
1, a and b) that
was absent from nuclei isolated in the presence of 0.5% Triton
X-100 (Fig. 1, c and d). Strong evidence for
removal of endoplasmic reticulum and weaker evidence of nuclear
envelope membrane removal were also provided by the lack of reactivity
to anti-
-tubulin upon Western blotting of isolated nuclei (Fig.
1e). Absence of
-tubulin has been widely accepted as
indicative of pure nuclear matrix preparations in previous studies (16,
19, 20), although nuclear envelope content below the level of
immunoreactivity cannot be excluded by such methodology. This procedure
resulted in a reproducibly high recovery of membrane-free nuclei
(67.3 ± 6.7% (mean ± S.D.); n = 18). It
was highly dependent upon exclusion of
Ca2+/Mg2+ from the extraction medium, because
the presence of these ions induced a denaturing aggregation of nuclear
matrix preparations.

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Fig. 1.
Purity of nuclei preparations.
Electron micrographs of whole IMR-32 human neuroblastoma cells
(a, scale bar 10 µm) show at higher magnification
(c, scale bar 0.5 µm) the classical double
layer nuclear envelope. Purified, washed nuclei pellets (b,
scale bar 10 µm) showed no nuclear envelope at the corresponding
higher magnification (d, scale bar 0.5 µm). Western blots
of nuclei (N) and whole cell (C) fractions probed
with monoclonal anti- -tubulin (e) at equivalent cell
loading displayed no immunoreactivity in the nuclear fraction,
consistent with removal of endoplasmic reticulum and minimal nuclear
envelope contamination (21, 22).
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The capacity of the nuclear matrix of IMR-32 cells to sustain a
compartmentalized synthesis of PtdCho was determined by evaluating the
presence, and assaying the activities, of each of the three enzymes of
the CDP-choline pathway, namely choline kinase (EC 2.7.1.32), CCT, and
CDP-choline:1,2-diacylglycerol cholinephosphotransferase (Table
I). Endonuclear choline kinase activity
was 10.3% that of the whole cell, whereas endonuclear CCT without
PtdCho/oleate stimulation was 25.5% of whole cell activity.
This endonuclear CCT activity was not stimulated by incubation with
PtdCho/oleate, in contrast to the 15-fold stimulation of the
extranuclear enzyme by lipid. The absence of an effect of exogenous
lipid on nuclear CCT may have been due either to an inability of the
PtdCho/oleate vesicles to penetrate the nuclear matrices or to residual
Triton X-100, which may compromise lipid stimulation.
Chromatin-associated CDP-choline:1,2-diacylglycerol
cholinephosphotransferase activity comprised 12.8% of whole cell
activity. Likewise, it is probable that Triton X-100 residue and
inability of exogenous DAG vesicles to penetrate the nuclear matrices
may have diminished CDP-choline:1,2-diacylglycerol cholinephosphotransferase activity determinations in this compartment. Notwithstanding the possibility that choline kinase activity may also
have been affected by Triton X-100 exposure, substrate accessibility was less likely to be problematic.
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Table I
Chromatin-associated CDP-choline pathway enzymes in IMR-32 cells and
nuclei
The activities of choline kinase (CK), CCT, and
CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT) were
determined in post-mitochondrial (10,000 × g; 10 min)
supernatants of cells and of a proportion of nuclei stripped of their
nuclear membranes from the same flask as described under "Materials
and Methods." Each value represents the mean activity corrected for
cell/nuclei count (n = 5) ± S.D.
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Whereas the enzyme activities we observed demonstrate the capacity for
in vitro endonuclear transformation of each of the PtdCho intermediates to their proximal biosynthetic products, they do not prove the existence of functionally compartmentalized pathways in intact cells. Consequently we used deuterium-enriched choline-d9 as a labeled substrate in conjunction
with ESI-MS to probe PtdCho biosynthesis (23). The ratio of endogenous to newly synthesized PtdCho molecular species was determined by incubating IMR-32 cells with choline-d9 and then
extracting the PtdCho from isolated nuclei and from whole cells.
Sequential scans of the parents of fragments with
m/z of 184 and 193 were used to quantify
endogenous and newly synthesized PtdCho species (23) (Fig.
2, a-d). Incorporation of
choline-d9 into PtdCho from whole cells was readily
detectable by 10 min and linear for up to 3 h (Fig.
3b), after which time 8.6% of
whole cell PtdCho contained the d9 headgroup (Fig.
3a). The time scale for the incorporation of
choline-d9 into the endonuclear PtdCho was equally
rapid; the parents of the m/z 193 species were detectable within 10 min (Fig. 3b). However, a constant rate
of labeled choline incorporation was only achieved after the first hour
of supplementation (Fig. 3b), presumably representing the time required for equilibration of the d9 substrate
into the endonuclear choline pool.

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Fig. 2.
ESI-MS of PtdCho from whole IMR-32 cells and
nuclear matrices after 3 h of incubation with
choline-d9. a,
scan of parents of fragment with m/z = 184 (choline headgroup); endogenous whole cell PtdCho. Selected
disaturated species are indicated by arrows. b,
scan of parents of fragment with m/z = 193 (choline-d9 headgroup); newly synthesized
whole cell PtdCho. c, scan of parents of fragment with
m/z = 184; endogenous nuclear matrix PtdCho.
Disaturated molecular species are indicated by arrows.
d, scan of parents of fragment with
m/z = 193; newly synthesized nuclear matrix
PtdCho.
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Fig. 3.
Cell and nuclear PtdCho synthesis in normal
and serum-starved IMR-32 cells. a, fraction
of endogenous PtdCho synthesized in cell and nuclei, following 3 h
of incubation with choline-d9, in normal
(control) cells (mean ± S.D., n = 8), starved
(0.5% FCS for 72 h) cells (mean ± S.D., n = 4), and supplemented (suppl) (0.5% FCS for 72 h and then 10% FCS during choline-d9
supplementation) cells (mean ± S.D., n = 5 cells). b, IMR-32 cells were incubated with
choline-d9 for 10, 30, 60, or 180 min. Newly
synthesized PtdCho was linear in whole cells and biphasic in isolated
nuclei and is shown as the mean (n = 3) for 10-, 30-, and 60-min experiments and as the mean ± S.D. (n = 8) for the 180-min experiment.
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ESI-MS analysis permitted the analysis of the molecular species
composition of both endogenous and newly synthesized PtdCho (Fig.
4, a and b). By
using PtdCho 14:0/14:0 as an internal standard, we were able to
determine an endonuclear PtdCho concentration of 3.55 ± 1.48 nmol/106 nuclei (corresponding to 3.55 ± 1.48 × 10
15 mol/nucleus). This represented 5.95 ± 0.69% (n = 8; mean ± S.D.) of the PtdCho
present in the entire cell. Analysis of the species composition of the
endonuclear and whole cell PtdCho obtained from the parents of the
m/z 184 fragment (Fig. 4a) revealed
that endonuclear PtdCho was enriched in species where both fatty acids were saturated. The proportion of such disaturated PtdCho to total PtdCho was 16.8% for whole cells (Fig. 4a) but in contrast
was 60.3% for endonuclear PtdCho (Fig. 4a). PtdCho species
with polyunsaturated fatty acid chains, particularly 20:4 or 22:6,
comprised 27.9% of the total PtdCho from whole cells (Fig.
4a) but were below the limits of detection in the
endonuclear PtdCho pool (<0.5% of the largest peak). This
observation provided an unexpected additional proof of the purity of
the nuclear matrix preparations, because PtdCho species containing 20:4
fatty acids are a major component of the nuclear envelope (31, 32).
Examination of the parents of the m/z 193 fragment (Fig. 2, b and d) suggested that after
3 h both nuclear and whole cell pathways synthesized PtdCho
molecular species with a similar composition to those of their
respective endogenous PtdCho pools. However, closer examination of the
data revealed subtle differences (Fig. 4, a and
b). Whereas whole cell PtdCho synthesis was indeed similar
to the endogenous PtdCho after 3 h, consistent with completion of
any remodeling within that time scale, comparison of newly synthesized
endonuclear PtdCho and the corresponding endogenous pool suggested that
endonuclear remodeling mechanisms probably continue after 3 h.
Specifically, 53.6% of newly synthesized endonuclear PtdCho after
3 h was disaturated (Fig. 4b) compared with 60.3% of
endogenous composition (Fig. 4a), and the distribution
between saturated species differed. Both 16:0/16:0 and 16:0/18:1
species were more abundant in newly synthesized endonuclear PtdCho.
Moreover, the pattern of endonuclear PtdCho synthesis seen after 10 min
of choline-d9 supplementation (Fig.
5a) implies progressive
remodeling of newly synthesized PtdCho to more saturated species. In
agreement with this suggestion, the acyl-CoA pool associated with
chromatin is known to be more saturated than the corresponding acyl-CoA
of the whole cell (33). Additionally, acyl-CoA synthetase activity
associated with the nuclear matrix has the lowest apparent
Km for 16:0, indicating a preference for acylation
of this acid inside the nucleus (34). Consequently, the substrates
necessary for CoA-dependant remodeling of endonuclear PtdCho to more
saturated species are probably present within the nuclear matrix.
PtdCho species containing 20:4 or 22:6 comprised 33.6% of whole cell
PtdCho synthesis after 3 h but were essentially absent from
nuclear PtdCho synthesis (Fig. 4b) even at 10 min (Fig.
5a). This suggests that if they are synthesized within the
nucleus de novo, then they are rapidly remodeled to more
saturated species.

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Fig. 4.
Molecular species compositions of endogenous
and newly synthesized whole IMR-32 cell PtdCho and endonuclear
PtdCho. a, the fractional compositions of
whole cell and endonuclear PtdCho molecular species as determined by
ESI-MS scans of the parents of fragments with
m/z = 184 are shown (n = 5, mean ± S.D.). b, the fractional compositions of newly
synthesized whole cell and endonuclear PtdCho species as determined by
ESI-MS scans of the parents of fragments with
m/z = 193 from the same cells/nuclei.
Identified PtdCho species, which represented >95% of all species
present, are grouped according to degree of saturation. Endogenous and
newly synthesized endonuclear PtdCho were enriched in saturated PtdCho
species, whereas species with polyunsaturated chains were not
detected in these fractions.
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Fig. 5.
ESI-MS of newly synthesized whole cell and
endonuclear PtdCho after 10 min of
choline-d9 exposure.
a, ESI-MS scan of the parents of the fragments
with m/z = 193 of a representative
endonuclear PtdCho fraction following whole cell supplementation with
choline-d9 for 10 min. Signal to noise ratios were
low in nuclear samples, precluding shorter time scale analyses.
b, ESI-MS scan of the parents of the fragments with
m/z = 193 of the whole cell PtdCho fraction
from a proportion of cells from the same flask.
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The enrichment of disaturates in newly synthesized endonuclear PtdCho
after 10 min (Fig. 5a) compared with whole cell PtdCho synthesis (Fig. 5b) and the rapidity of synthesis are
themselves very good evidence for the existence of a compartmentalized
PtdCho synthesis pathway within the nucleus. It is possible that PtdCho synthesized on the endoplasmic reticulum could be the source of newly
synthesized endonuclear PtdCho after 10 min of incubation with
choline-d9 (Fig. 5a). However, this would
require very rapid selective transport of saturated PtdCho species from the endoplasmic reticulum, possibly involving an as yet unrecognized saturated PtdCho transfer protein, across the nuclear envelope and into
the nuclear matrix. No such mechanisms have been demonstrated for
PtdCho transport that are both sufficiently selective and rapid, and
consequently endonuclear PtdCho synthesis is the simplest explanation
for our results. The data presented here, together with CDP-choline
pathway enzyme activities and the recognized requirement for
enzymatically active (endonuclear) CCT
(12), argue strongly
for the existence of a functional pathway for the synthesis of
endonuclear PtdCho in the intact cell.
Our ESI-MS studies also enabled the analyses of all other endonuclear
phospholipids. Simultaneous use of PtdEtn and PtdOH internal standards
permitted the determination of the concentrations as well as the
species of endonuclear PtdEtn and PtdOH pools. Endonuclear
concentrations of PtdEtn and PtdOH were 0.54 ± 0.34 and 0.15 ± 0.003 nmol/106 nuclei, respectively. In contrast to the
dramatic difference between PtdCho compositions between the whole cell
and nucleus, the compositions of PtdOH and PtdEtn were essentially
identical between whole cell and endonuclear pools. PtdOH isolated from both sources was composed largely of monounsaturated species (results not shown), whereas PtdEtn was highly unsaturated (Fig.
6). For both whole cell (Fig.
6a) and endonuclear (Fig. 6b) pools, PtdEtn species containing 20:4 or 22:6 polyunsaturated chains contributed >80% of the total, with relatively minor variation seen between the
fractional contents of individual unsaturated species. Whereas the
highly saturated nature of endonuclear PtdCho might be expected to
exist in a rigid gel phase, the presence of the unsaturated PtdEtn is
likely to render endonuclear lipid a more fluid and metabolically
active phase at physiological temperatures. The presence of PtdEtn in
the endonuclear phospholipid pool, with a high degree of chain
unsaturation, is also probably necessary for CCT activity. A recent
study showed that CCT
activity in vitro was modulated by
the amount of elastic energy stored in a bilayer membrane (35), which
in turn was determined by the lipid composition of that membrane.
Whereas CCT
was essentially inactive in membranes of saturated
PtdCho, introduction either of PtdCho with unsaturated chains or of
PtdEtn species led to an increased activity. These lipids are known as
Type II lipids because they have negative spontaneous curvatures. From
our ESI-MS data we estimate that some 23 mol % of the endonuclear
phospholipids were Type II lipids, a value consistent with support
for CCT
activity.

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Fig. 6.
ESI-MS of endogenous whole cell and
endonuclear PtdEtn. a, ESI-MS constant
neutral loss scan of the fragment with m/z = 141 (ethanolamine headgroup) of a representative whole cell PtdEtn
fraction. b, ESI-MS constant neutral loss scan of the
fragment with m/z = 141 of the endonuclear
PtdEtn fraction from nuclei prepared from a proportion of cells from
the same flask.
|
|
Concentrations of endonuclear PtdCho, PtdEtn, and PtdOH determined from
the ESI-MS studies were used to estimate the volume that these lipids
would occupy in the nucleus (36). The molecular volume (v)
of a phosholipid can be estimated from
|
(Eq. 1)
|
where vj represents the volume of a
CH3, CH2, or CH unit or of the polar headgroup,
and n
represents the number of
CH2 or CH units in each chain (i = 1,2). The following values, appropriate for phospholipids in an
L2 phase, were used in the calculation:
vCH3 = 51.0 Å3; vCH2 = 25.5 Å3; vCH = 20.5 Å3;
vpolar(PtdCho) = 260 Å3;
vpolar(PtdEtn) = 246 Å3; and
vpolar(PtdOH) = 135 Å3. The
amounts of each endonuclear species as determined by ESI-MS were used
to estimate the volume occupied by each species
(VPtdCho = 2.74 × 10
18 m3;
VPtdEtn = 4.40 × 10
19 m3;
VPtdOH = 9.95 × 10
21 m3), which gives a total
volume of 3.15 × 10
18 m3.
This volume is the minimal volume that would be occupied by the lipids
if they were packed into a single bilayer. To allow for hydration the
total volume should be increased by a factor of 1.5 to 2. The volume of
a typical IMR-32 nucleus was estimated to be 3.9 × 10
17 m3 by assuming an oblate
shape, consistent with transmission electron microscopy
observations, with semi-axes 1.5, 1.25, and 5 µm in length.
Assuming that these lipids are fully hydrated, such calculation leads
to a volume between 4.7 × 10
18 and
6.3 × 10
18 m3, which
corresponds to 12-16% of the nuclear volume for IMR-32 nuclei
(3.9 × 10
17 m3). For
comparison, the 6 × 109 nucleotide pairs in human DNA
would occupy a minimum theoretical volume of 6.86 × 10
18 m3. Because the total mass
of histones in chromatin is roughly equal to the mass of DNA, the
volume occupied by the 48 chromosomes is estimated to be of the order
of 1.5 × 10
17 m3 (~38.5%
of the nuclear volume). The maximum amount of water available to
hydrate endonuclear lipids, estimated by subtracting the combined chromosome and lipid volumes from the nuclear volume (and assuming no
other organic species or ions are present), is between 1.7 × 10
11 and 1.9 × 10
11 g. By using a conservative average
molecular weight of 700 for the lipid, we estimate that at the
very least the endonuclear phospholipids we studied were present at an
effective concentration of about 10% (weight of phospholipid/weight
of free water). The relatively high phospholipid to water ratios
inside the nucleus suggest that these lipids are likely to be present
as large complex aggregates and possibly even as liquid crystalline phases.
Because the synthesis of extranuclear PtdCho varies with the cell cycle
(37), we sought to establish whether the synthesis of endonuclear
PtdCho and species composition were constitutive or subject to temporal
regulation. Proliferating IMR-32 cells were induced to enter a
pseudo-differentiated state, G0, by culturing them in
serum-depleted medium (0.5% FCS) for 72 h. This halted proliferation and extension of neurite-like structures was observed. Quiescent cells were susceptible to mitogenic reentry into the cell
cycle by supplementation with 10% FCS (as determined by growth cone
collapse, cell rounding, and proliferation assays). Following the
growth arrest mediated by serum depletion, the basal whole cell PtdCho
synthesis, assayed during 3 h of incubation with
choline-d9, fell to 28% of control values (Fig.
3a). Upon supplementation with 10% FCS, the whole cell
PtdCho synthesis rose to 47% of control (Fig. 3a),
presumably reflecting increased PtdCho synthesis during reentry into
the cell cycle. In the case of the endonuclear PtdCho, the basal
synthesis following growth arrest was 20% of control, whereas reentry
into the cell cycle led to an increase in synthesis to 40% of control.
Minor changes in whole cell PtdCho and endonuclear PtdCho species were
observed following serum depletion, and these were reflected by the
altered species composition of the newly synthesized PtdCho. However,
the proportion of disaturated species in both endogenous and newly
synthesized endonuclear PtdCho pools remained remarkably constant,
indicating that this unusual acylation/alkylation pattern was under
tight homeostatic control. Cornell and co-workers (38) reported that
mitogenic stimulation of serum-starved IIC9 cells by serum
supplementation was accompanied by a translocation of nuclear CCT
to
the endoplasmic reticulum, although demonstration of nuclear
confinement of CCT
throughout the cell cycle (12) apparently
contradicts that result. Explanations of the discrepancy have been
attributed either to insufficient specificity of CCT antibodies or cell
type-specific change (12). Our data did not include measures of
CDP-choline pathway enzyme activities during extracellular serum
manipulation and cannot inform that debate except insofar as we show
that both nuclear and whole cell PtdCho synthesis increased in
serum-starved IMR-32 cells following serum resupplementation. It would
seem unlikely, therefore, that the nuclear matrices would act solely as
a reservoir for exportable CCT destined for the endoplasmic reticulum
of IMR-32 cells at a time when endonuclear PtdCho synthetic flux doubles.
The fundamental function for endonuclear PtdCho synthesis is not clear.
It is very unlikely that this quantitatively minor pathway for PtdCho
synthesis contributes significantly to bulk membrane phospholipid
generation within the cell. Equally, the evidence suggests that it is
unrelated to the synthesis of new nuclear envelope generated as part of
cell division, not least because of the highly unsaturated nature of
these membranes (31, 32). Three possibilities, however, are advanced.
First, CCT, as the principal regulatory enzyme of PtdCho biosynthesis
(1), produces CDP-choline for the proximal enzyme of the pathway,
CDP-choline:1,2-diacylglycerol cholinephosphotransferase,
thereby controlling pathway flux. The other substrate of
CDP-choline:1,2-diacylglycerol cholinephosphotransferase is DAG.
Accordingly, our data would support a role for an endonuclear CDP-choline pathway in regulating endonuclear disaturated DAG content.
Temporally distinct periodic nuclear accumulations of unsaturated DAG
from PtdIns (16, 39) and saturated DAG from PtdCho (17) are recognized.
CCT
activity is clearly essential for cell survival (12), whereas
overexpression of a nuclear-targeted DAG kinase ablates nuclear DAG
accumulations by converting them to PtdOH and halts cell growth (16).
Control of flux through endonuclear PtdCho synthesis at key points in
the cell cycle may therefore serve to regulate this potential
endonuclear lipid second messenger, possibly by preventing its
conversion into saturated, biologically active PtdOH (40). Second, the
highly saturated nature of endonuclear PtdCho, together with the
estimations of volume occupancy that we have made, may reflect a
further specialized structural role for endogenous PtdCho within the
nuclear matrix. Finally, the same saturation offers the possibility of
a third role for saturated endogenous PtdCho, the prevention of the
propagation of oxidative damage within nuclei. Free radical generation
in close proximity to the genome is clearly undesirable. Moreover, the
presence of significant amounts of polyunsaturated lipids in the
nuclear envelope and as part of the endonuclear PtdEtn (Fig.
6b), PtdOH, and PtdIns (39) dispersed within the nuclear matrix and in close association with DNA poses significant risk of
amplification of reactive oxygen species. Saturated PtdCho in the
nuclei may provide a means of halting oxidative damage before it
overwhelms endogenous antioxidant capacity.
The presence of endonuclear CCT
appears to be universal in a range
of cultured cell types (12), and similarly, highly saturated endonuclear PtdCho synthesis may not be confined to IMR-32
neuroblastoma cells. Preliminary data from our
laboratories2 show that human
HaCaT keratinocytes and U937 cells likewise synthesize highly saturated
endonuclear PtdCho. Both the structural possibilities and the intimate
association of saturated endonuclear PtdCho synthesis with the process
of cell proliferation render this compartment of phospholipid
metabolism a challenging area of future research.
 |
ACKNOWLEDGEMENTS |
We thank Nick Barnett and Sue Cox from the
Electron Microscopy Unit, Southampton General Hospital for
preparing the electron microscopy sections.
 |
FOOTNOTES |
*
This work was supported by The Wellcome Trust (Grants 055490 and 457405).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Child Health
(803), Allergy and Inflammation Sciences Division, University of
Southampton, Level G, Centre Block, Southampton General Hospital, Tremona Rd., Southampton SO16 6YD, UK. Tel.: 44 23 8079 4178; Fax: 44 23 8079 6378; E-mail: anh@soton.ac.uk.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M009878200
2
A. N. Hunt, G. T. Clark, and A. D. Postle, unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
PtdCho, phosphatidylcholine;
PtdEtn, phosphatidylethanolamine;
PtdIns, phosphatidylinositol;
CCT, CTP:choline-phosphate
cytidylyltransferase (EC 2.7.7.15);
DAG, diacylglycerol;
MS, mass
spectrometry;
ESI-MS, electrospray ionization MS;
HBSS, Hanks'
balanced salt solution;
PtdOH, phosphatidic acid;
choline-d9, 9-deuterated methylcholine;
FCS, fetal
calf serum.
 |
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