Vol.3, No.4, 187-199 (2011) Health
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Transformations of phosphatidylinositol phosphates in
the outer and inner nuclear membrane are linked to
synthesis and restitution of cellular membranes
Amalia Slomiany, Bronislaw L. Slomiany
University of Medicine and Dentistry of New Jersey, New Jersey Dental School, Newark, New Jersey, USA; *Corresponding Author:
Received 10 February 2011; revised 18 March 2011; accept 1 April 2011.
The ultimate goal in phosphoinositides cellular
metabolism is to decipher their global func-
tional organization and coordinatio n of the com-
partmentalized signaling processes. In this re-
port we present evidence linking nuclear phos-
phoinositides cycle w ith endoplasmic reticulum
synthesis and function. The rapid transforma-
tion of [3H]inositol-labeled phosphoinositides in
the intact nuclei (IN) was captured in chase
studies for 0-5 min, followed by examination of
phosphatidylinositides in the inner nuclear me-
mbrane (INM), the outer nuclear membrane
(ONM) and endoplasmic reticulum (ER). We re-
vealed that synthesis of phosphatidylinositol
phosphates (PIPs) occurs in ONM and the de-
phosphorylation takes place in the INM. The
rapid transformation of the radiolabeled PIPs in
ONM reverberated in their appearance and suc-
cessive transformation in INM, and in the 5min
chased nuclei was tracked to ONM as the re-
emerging radiolabeled phosphatidy linositol (PI).
These chase-uncovered changes in ONM and
INM PIPs profiles allow us to conclude that the
observed conversions in the nuclear membrane
continuum are induced by the lateral movement
of the membrane and its transit from the cyto-
solic to nuclear and back to cytosolic environ-
ment. The suggested membrane synthesis-in-
duced movement provides the means to trans-
port the membrane- and the membrane lipid
ligand-associated cytosolic proteins to the in-
tranuclear spaces and renewal of INM. Export of
the nuclear components interacting with the
modified INM, by exiting from nuclear to cyto-
solic site, endows ER with a steady influx of the
membrane that is conditioned to generate vesi-
cles according to the nucleus delivered tem-
Keywords: Phosphatidylinositides; PIPs;
Transformation ; R estitution; Outer Nuclear
Membrane; Inner Nuclear Membrane; Endoplasmic
Reticulum; Cellular Membranes
The array of proteins with affinity to phosphatidy-
linositol phosphates (PIPs), including protein kinases,
phosphatases, phospholipases, ion channel proteins, scaf-
fold proteins, cytoskeletal proteins and the regulators of
membrane trafficking [1-3], attest to th ese phospholipids
pleiotropism in controlling cellular metabolism and to
the fact that the distinct molecules targeted by specific
PIPs’ must be compartmentalized in a function-depen-
dent manner. Thus, the 18 phosph oinositide interconver-
sion reactions identified thus far and mediated by as
many as 47 genes encoding 19 phosphatidylinositide
kinases and 28 phosphatidylinositide phosphatases [4],
must be restricted by cellular space and the synthetic or
catabolic action. The cell nucleus phosphoinositides seem
to function in the organelle niche that is independent and
isolated from the environment of the other cell’s mem-
branes signaling events [5,6].
The different spectrum of phosphoinositides in the
nuclear membranes than that identified in endoplasmic
reticulum (ER) [7-9] or Golgi, provides credibility to
their functional separation. But, the findings on uncon-
ventional intranuclear localization of PIPs in the nuclear
speckles, nuclear matrix, nucleoli and chromatin [2,5,6],
are still being challenged, and the current evidence sup-
ports the model that nuclear phosphoinositides remain in
the nuclear envelope [10-15]. Even the model that in-
corporates PIPs into nuclear membranes raises several
compelling questions: how is PIPs intranuclear localiza-
tion achieved, how their nuclear conversions are linked
A. Slomiany et al. / Health 3 (2011) 187-199
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to the rest of cellular events, and how is their function
expressed and metabolism regulated. The enigma is
compounded further by the fact that ER which is a con-
tinuum of outer nuclear membrane (ONM), that in turn
and beyond nuclear pore, becomes the inner nuclear
membrane (INM), is devoid of PIPs [16,17]. Thus, the
sudden transition from the nuclear presence of PIPs to
their absence in the ER is puzzling. Continuity of the
nuclear and ER membrane implies that the processes in
nuclear membrane are closely linked to the ER synthetic
responses reflected in membrane biogenesis and transport.
Yet, whilst the precise and explicit intercalation of pro-
tein into biomembrane is only possible when specific
mRNA tethered to the INM is translated concomitantly
with the synthesis of membrane lipids, the phosphoino-
sitides complexity of the nuclear membrane is not being
passed to ER membrane or to the newly synthesized bio-
membrane used to envelop transport vesicles [7-9,16,17] .
The ER membrane and its transport v esicles con sist of
phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidylinositol (PI) and ceramides (Cer) which
mature and acquire phosphatidylinositol phosphates
(PIP1s) in Golgi [16,17]. These in congruities in the co m-
position of the membrane continuum brought us to spe-
culation that the membrane biogenesis in the region of
ER transiting into ONM may be defining the outer and
inner nuclear biomembrane. In the previous study that
demonstrated the phosphatidylinositides aided cytosolic
protein transport into the nucleus and the concurrent
appearance of the transport engaged phosphatidylinosi-
tide in INM [15], we proposed that the event must be the
consequent effect of ONM synthesis-induced lateral
movement of the membrane that aided the transit of the
complex into the intranuclear space. Such a movement
of the membrane would provide continuous transport of
the membrane- and the membrane lipid-ligand associ-
ated cytosolic proteins to the intranuclear spaces, restitu-
tion of INM, and the export of the nuclear components
interacting with INM [12-14]. Moreover, it would en-
dow ER with steady influx of the membrane that would
be conditioned to generate the transport vesicles accord-
ing to the nucleus-deliv ered templates [18-27].
In order to observe the events that would proceed as a
continuous, uninterrupted transition from ONM to INM,
to ONM again, and then to ER, we have investigated
changes in nuclear membranes phosphoinositides com-
position in time. The studies of the phosphatidylinositi-
des transformation (conversions) in nuclear membranes,
prepared from intact nuclei subjected to incubation in
cytosol supporting transport, gave the credibility to our
contention that polyphosphoinositides assembled in ONM
transit into the intranuclear space, and remain in INM.
As the constituents of INM, they undergo dephosphory-
lation, and then reappear in the ONM and ER as PI.
Radiolabeled precursors of phospholipids, glycolipids,
glycoproteins and other radiochemicals were purchased
from New England Nuclear (Boston, MA). Phospholipid
standards were from Avanti (Birmingham, AL), Matreya
(Pleasant Gap, PA) or prepared in our laboratory.
Creatine phosphokinase, phenylmethylsulfonyl fluoride
(PMSF), aprotinin, pepstatin, leupeptin, ATP, CTP, GTP,
fatty acyl CoA, glycerol 3-phosphate, RNase, and RNase
free sucrose were purchased from Sigma Chemicals (St.
Louis, MO). Polyacrylamide gel electrophoresis reagents
were from Bio-Rad (Rockville Centre, NY). All other
chemicals and reagents were purchased from J.T. Baker
Chemical Co. (Phillipsburg, PA), Fisher Scientific
(Springfield, NJ), and VWR Scientific (Piscataway, NJ).
The anti-rat albumin antibodies were purchased, and the
antimucin antibodies were prepared in our laboratory
2.1. Solution Used in Preparation of Cells,
Cell Organelles, Nuclei and Nuclear
Buffered saline, with 10 mM potassium phosphate,
pH 6.8 (a), buffered saline containing 0.5 mM MgCl2
and 0.5 mM MgS04, (b), buffered saline (100 ml) con-
taining 66 mg collagenase, 80 mg hyaluron idase, and 2 g
of albumin (c), MSB, pH 6.9 buffer consisting of 0.1
Pipes, pH 6.9, 2.0 M glycerol, 1 mM Mg acetate, 0.5
mM EGTA and mixture of protease inhibitors consisting
of leupeptin, aprotinin and PMSF (d), MSB buffer con-
taining 0.2% Tr iton X 100 (e), 50 mM TRI S-HCl, pH 7.4
containing 0.25 M sucrose, 10 mM MgCl2 1 mM DTT,
10 mg/ml leupeptin and 2 mM PMSF (f).
2.2. Isolation and Separation of ER, Golgi,
Endosomes, Cellular and Outer and
Inner Nuclear Membranes.
One volume of the isolated cells was homogenized in
8 volumes of 10 mM potassium phosphate buffer, pH
6.8, 1.3 M sucrose and 1mM MgCl2 to rupture at least 80
% of cells [15]. The unbroken cells were removed by
centrifugation at 50 xg for 3 min, and the homogenate
centrifuged for 15 min at 1000 xg. The soluble cellular
material was saved for isolation of cellular organelles
and cytosol while the nuclear pellet was processed fur-
ther and the outer and inner nuclear membranes (ONM,
INM) were collected [15].
2.3. Preparation of Intact Nuclei (IN)
In the experiments employing IN, and the incubation
with radiolabel-free (cold) CC that was followed by
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separation of ONM and INM, the IN samples were sub-
jected to additional sucrose gradient purification. The
samples of IN were suspended in 40% buffered sucrose
by adding 80% sucrose in 150mM NaCl, 25 mM TRIS,
pH 7.5 buffer and ov erlaid with 10% - 30% sucro se gra-
dient in the same buffer and centrifuged at 29 000 xg for
21 h. The nuclei were recovered from 40% bottom layer
by diluting out sucrose with the buffer and centrifugation
at 1000 xg for 10min. The nuclear pellet was then sus-
pended at a protein concentration of 2 mg/ml in 1% cit-
rate and proceeded with separation of ONM and INM.
The described sucrose gradient afforded separation of
the cell membrane fragments that were trapped with nu-
clei, and thus allowed the conclusion that the radio-
labeled PIPs were strictly derived from nuclear mem-
branes. The isolated on sucrose gradient membranes
were used to determine whether the treatment with ice-
cold buffered Triton X100 (e) dissolved ONM or INM
and/or contained PIPs.
2.4. Preparation of Cells for [3H]Inositol
The cells were prepared from rat gastric mucosa and
the liver as described previously [7-9,15]. The single
cells that were separated from larger debris with aid of
specific cell size nylon mesh were centrifuged at 50 xg
for 2 min, washed twice with the enzyme-free medium
(c), twice with the Minimum Essential Medium (MEM)
and counted in he mocytometer. Thus prepared cells were
then incubated in MEM for 3 hours with cold or
[3H]inositol and used for preparation of nuclei [15] sub-
cellular organelles, cell cytosol [7-9 ,16] and cellular me-
mbranes [16,17]. In the experiments dedicated to the
determination of lipid synthesis with cell cytosol derived
from gastric epithelial cells, hepatocytes, or RNase
treated cytosol, the preparations of nuclei, ER, Golgi or
other organelles and cell membranes were additionally
rinsed with PBS (a) and urea-PBS in order to remove the
associated residual cytosolic proteins that otherwise
would remain on their membranes. Thus prepared sub-
cellular organelles and membranes were used for ex-
periments on transport vesicles synthesis [16,17], and
the preparation of ONM and the INM [15]. The synthe-
sis of phosphatidylinositides, phospholipids and protein
was determined using radiolabeled [3H]inositol, [3H]ara-
chidonate, [3H]choline, [3H]serine, [3H]palmitate and
[32P]ATP [7-9,15-17]. The chase studies on [3H]inositol
labeled intact nuclei were performed in medium con-
taining cold CC at concentration of 5 mg protein/ml of
incubation mixture enrich ed with 50 mM ATP, 250 mM
CTP, 50 mM GTP, 5 mM creatine phosphate, 8.0 IU/ml
creatine kinase, and where indicated 25 mg/ml RNase,
10 mM UDP-Glc and 10 mM palmitoyl CoA [7-9,15- 17].
The aliquots of intact inositol labeled nuclei were with-
drawn at 0, 0.5, 1.0, 3.0 and 5.0 min and diluted into 5
ml of ice cold buffer consisting of 10 mM potassium
phosphate buffer pH 6.8, 1.8 M sucrose and 1 mM mag-
nesium chloride to rinse off the incubation medium. The
IN were recovered by centrifugation for 15 min at 1000
xg and processed further to isolate outer and inner nu-
clear membranes. The same preparation of cold cytosol
was used in the chase experiments with inositol labeled
2.5. Preparation of Transport-Active Cell
Cytosol (CC)
The viable cells homogenized for 10 sec at 600 rpm in
3 volumes of buffer containing 0.25 M sucrose; 50 mM
TRIS-HCl (pH 7.4); 25 mM magnesium acetate and 10
mM each of aprotinin, leupeptin, chemostatin; and 1 mM
phenylmethylsulfonylfluoride, were centrifuged at 5 000
xg for 15 min. The supernatant, diluted with 2 volumes
of homogenization buffer, was re-centrifuged at 10 000
xg for 20 min. The resulting supernatant was then sub-
jected to centrifugation at 100 000 xg for 1h. Thus ob-
tained soluble fraction was adjusted to 15 to 18 mg pro-
tein/ml; admixed with an ATP generating system con-
sisting of 40 mM ATP, 200 mM creatine phosphate,
2000 units/ml cr eatine phosphokinase, and referred to as
transport active cell cytosol or active cytosol (CC).
2.6. Isolation and Separation of Outer
and Inner Nuclear Membranes.
The cytosol- and membrane fragments-free intact nu-
clei (IN) suspended in buffer (f) at concentration of 2mg
protein/ml, were adjusted to 1% (w/v) with sodium cit-
rate and incubated on ice with gentle stirring for 30 min
and then centrifuged at 500xg for 15 min. The obtained
supernatant contained the ONM, whereas the pellet con-
tained the INM) [12, 15]. The pellet (INM) was sus-
pended in buffer (f) at concentration of 5mg/ml and di-
gested with DNase 1 (250 mg/ml for 14h at 4˚C). The
digested material was separated on sucrose gradient con-
sisting of 0.25/1.6/2.4 M sucrose by centrifugation at 10
000 xg for 2 h. The INM were recovered at 1.6 M su-
crose boundary. The ONM was collected from citrate
supernatant by centrifugation at 100 000 xg for 20 min.
The membrane pellet was suspended in buffer (f) and
subjected to the same treatment as INM. On the average,
the preparation of INM was 30% larger than ONM.
2.7. Preparation of Cellular Membranes
The cell membranes and subcellular organelles (ER,
Golgi) were recovered from the radiolabeled cells as
described earlier [7-9,15-17]. The organelles sediment
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remaining after separation of cell cytosol, was suspended
in buffer containing 0.2 M PIPES (pH 6.9), 2 M glycerol,
1 mM EGTA and 1 mM magnesium acetate and applied
on the top of discontinuous gradient of 2.0/1.5/1.3/1.0M
sucrose and centrifuged at 100 000 xg for 16 h. The cell
membranes were recovered from 1.0 M sucrose, Smooth
Endoplasmic Reticulum (SER) from 1.3 M sucrose, RER
from 1.5 M sucrose and Golgi from the top of the 2.0 M
sucrose. Each sucrose-separated fraction was subjected
to further purification. The cell membranes were washed
with original Pipes buffer and centrifuged at 3 000 rpm
for 2 min. To separate apical epithelial membranes, the
buffer was adjusted with 0.2% Triton X-100 and the mix-
ture incubated at 4˚C for 5 min [16,17]. This treatment
resulted in breaking up the phospholipids-rich membranes
into smaller segments and that allowed us to separate
membranes containing cholesterol, glycosphingolipids
and glycoproteins. The latter membranes were recovered
by low speed centrifugation at 3 000 rpm for 2 min.
2.8. Generation and Purification of
Transport Vesicles
ER- and Golgi-derived transport vesicles were gener-
ated in the presence of radiolabeled precursors according
to procedure described previously [7-9,15-17]. The ER
or Golgi membranes incubated with cytosol, ATP-gen-
erating system, UTP, CTP GTP, fatty acyl CoA and wa-
ter soluble cold or radiolabeled lipids precursors were
incubated for 30 min at 37˚C, centrifuged over 0.3M
sucrose and treated with stripping buffer at 2˚C for 15
min followed by centrifugation at 10 000 xg for 10min
to separate transport vesicles from ER or Golgi mem-
branes. The separated from maternal membranes trans-
port vesicles were recovered from the supernatant re-
sulting from centrifugation of the supernatant mixture at
150 000 xg for 60 min. The crude fraction of the trans-
port vesicles was suspended in 55% sucrose, overlaid
with 55% - 30% gradient and centrifuged at 150 000 xg
for 16 h. The purified transport vesicles were recovered
from the gradients as reported earlier [1-9,16,17].
2.9. Fusion of Transport Vesicles with
One volume of Golgi transport vesicles (1.3-1.5 pro-
tein/ml) was suspended in one volume of active cytosol
(15 mg protein/ml) and added to one volume of cell
membranes (8 mg protein/ml of whole cell membranes
or 2 mg protein/ml of apical epithelial membranes). The
reaction was allowed to proceed from 0-30 min at 4˚C
(control) and at 37˚C in the presence of ATP regenera
ing system consisting of 40mM ATP, 200 mM creatine
phosphate, 2,000 units /ml of creatine phosphokinase, or
in the ATP depleting system containing 5mM glucose
and 500 units/ml hexokinase. After incubation, the mem-
branes were recovered by centrifugation through three
volumes of 0.5 M sucrose at 3000 rpm for 5 min. The
free vesicles were recovered from the supernatant and
used in fusion experiments with endosomes. The cell
membranes sedimented through sucrose were washed
with 25 mM Hepes-KOH buffer and treated for 5 min
with 0.2% Triton X-100 at 4˚C. The soluble fraction of
the membrane was recovered in supernatant, whereas
apical the glycosphingolipids- and glycoprotein- con-
taining membranes remained in the sediment [16,17,29].
In the experiments estimating en bloc fusion of transport
vesicles with the membrane, the associated but not fused
vesicles were released from the membrane by subjecting
the membrane fraction to treatment with 2 M urea for 30
min at 4˚C and then th e recovered membranes w ere cen-
trifuged through 0.5 M sucrose, washed and subjected to
lipid analysis.
2.10. Lipid Analysis
Preparations of IN, ONM, INM and ER from [3H]ino-
sitol labeled cells were subjected to PIPs lipid analysis
and chase-induced PIPs transformation analysis. The
lipid extracts and high performance thin layer chroma-
tography were performed exactly as described in our
previous studies [7-9,15-17,29]. The column chroma-
tography of PIPs was performed on AG1x8 formate
form columns. The lipid extracts were first subjected to
deacylation by incubation for 30 min at 53˚C with 0.75
ml of methylamine reagent containing monomethyl-
amine/ methanol/water/butanol (5/4/3/1) and after re-
moval of methylamine by evaporation to dryness the
samples were suspended in water and fatty acids ex-
tracted with mixture of n-butanol/light petroleum ether/
ethyl formate (20/4/1). The lower phase was re-extracted
with the same solvent and the deacylated lipids were
applied to column. The columns were eluted with (a)
water, (b) 5 mM disodium tetraborate/60 mM ammo-
nium formate,(c) 0.1 M formic acid/0.2 M ammonium
formate, (d) 0.1M formic acid /0.4 M ammonium for-
mate, (d) 0.1 M formic acid /1.0 M ammonium formate.
Each fraction was collected in 12 individual aliquots and
from each 0.2 ml was used to determine total radioactiv-
ity eluted with individual solvents, whereas the Bertho ld
LC radioactivity analyzer was used to determine the en-
tire elution profile.
Our previous study de monstrated that intracellular ve-
sicular transport is dependent on the finely tuned synthe-
sis of cell specific proteins and lipids that assemble pre-
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cise copies of the biomembrane, which is needed for
repair and restitution of the cell membranes and their
function [17]. Therefore, the cytosolic protein flux to the
nucleus and the signal-prompted nuclear export, are in-
timately linked with a specific membrane biogenesis
[1-5,15]. In both instances, the transp ort to and from the
nucleus are linked to nuclear membrane lipids, particu-
larly to PIPs. But, thus far, the site of nuclear PIPs gene-
sis and the specific protein affinity to th e variety of PIPs
is not determined.
As we observed previously, the PIPs which are in-
volved in cytosolic protein transport to the nuclear inner
space remain in the INM, and are either transformed
through dephosphorylation or degraded with aid of
phosphatidy l i nosi t ol-specific phosph oli pase C (PIPLC).
Hence, in this report, our studies on nuclear mem-
brane concentrate on the PIPs transformations in ONM
and INM observed during 0-5 min chase performed in
the radiolabel-free transport supporting cell cytosol (CC).
The data presented in Table 1 focus on the issue whethe r
PIPLCs induce changes in PIPs and reveal the amount of
labeled inositol released to cold cytosol. The results de-
monstrate whether during the chase incubation of la-
beled IN the phosphatidylinosito l-specific phospholipase
C (PIPLC) releases inositol phosphates (IPs) from ONM.
If PIPLC contributes to PIPs metabolism in ONM, the
recovered cytosol should contain increasingly larger
amount of the labeled inositol. As evident from the pre-
sented data, except for the residual counts displaced dur-
ing samples processing, the amount of soluble radio-
labeled inositol in the cytosol has not increased with
time of chase. Thus, the results provide convincing evi-
dence that nuclear PIPs of ONM are not metabolized
with the aid of cytosolic PIPLC.
In Table 2 we present the amount of inositol-labeled
lipids extracted from the intact nuclei (IN) follo wing 0-5
min incubation in CC. If nuclear PIPLC releases inositol
phosphates (IPs) into nuclear contents, then the total
amount of inositol labeled lipids extracted from the in-
cubated nuclei would be diminished with time. As dem-
onstrated, the amount of labeled PIPs extracted from
incubated nuclei has not declined.
Table 1. Release of [3H] inositol from IN incubated in an inac-
tive (Control) and in transport active CC for 0-5min.
(min) Control
(cpm) % cpm
released Sample+
CC (cpm) % cpm
0 1037 24.6 950 13.7
0.5 1131 19.0 1145 12.0
1.0 1178 23.3 1024 11.5
3.0 1303 24.0 1080 12.0
5.0 806 19.1 555 7.0
Table 2. PIPs extracted from IN subjected to incubation in an
inactive (Control) and in transport active CC for 0-5min.
(min) Control
(cpm) % cpm in
lipids Sample+
CC (cpm) % cpm in
0 3167 75.3 5995 86.3
0.5 4808 80.9 9545 87.9
1.0 3870 76.7 8920 88.4
3.0 4148 76.1 8965 87.9
5.0 3403 80.9 79.35 92.9
On the contrary, in the aqueous phase, we observed
some decrease in the amount of radiolabeled inositol and
an increase in an amount of radiolabel extracted in cor-
responding samples of nuclear lipids. At this stag e of the
investigation we cannot provide other explanation than
to suggest that the samples incubated with the transport
active cell cytosol retained larger fractions of water
phase, and that this inclusion appeared as an increase in
nuclear lipids.
The spectrum of PIPs present in ONM isolated from
[3H]inositol labeled IN is shown in Figure 1 and that in
INM in Figure 2. After PIPs deacylation and column
chromatography, the glycerylphosphoinositol phosphates
(GPIPs) separated into glycerylphosphoinositol (GPI),
glycerylphosphoinositol monophosphate (GPIP1), glyc-
erylphosphoinositol diphosphate (GPIP2), and glyceryl-
phosphoinositol triphosphate (GPIP3). The profiles of
the individual GPIPs suggest that each fraction is repre-
sented by more than one isomer , and the cis or trans po-
sition of the phosphate esters may cause separation of
GPIP2 and GPIP3 into two or three subfractions. How-
ever, the methods utilized in our study cannot provide
precise assignment of the phosphate substitution on the
inositol moiety, and therefore in chase study each frac-
tion is measured based on the number of phosphate
moieties identified (Figures 1 and 2).
The results of the nuclear [3H]inositol-labeled PIPs
transformations are shown in Figures 3 and 4. The data
present qualitative and quantitative changes in PIPs pro-
files in ONM and INM isolated from IN subjected to 0-5
min chase in label-free active cytosol. The initial status
(0 time) of PIPs labeling in ONM reflects low level
(5.7%) of phosphatidylinositol (PI) and the incremental
contents of phosphatidylinositol monophosphates (PIP1s),
phosphatidylinositol diphosphates (PIP2s) and phospha-
tidylinositol triphosphates (PIP3s). The PIP3s represent
largest fraction (44.6%) of the labeled PIPs.
After 30 seconds, the PIP2s increase to 53.7%,
whereas PIP3s decline to 25.2%. While throughout the
chase the PIP1s level remains almost constant, the PIP2s
decline to the level observed before chase, while PIP3s
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Figure 1. Elution profile of the deacylated PIPs, the glycerylphosphoinositol phosphates (GPIPs) present in ONM isolated
from inositol-labeled IN. The details of the stepwise elution are described in Methods.
Figure 2. Elution profile of the deacylated PIPs, the glycerylphosphoinositol phosphated (GPIPs) present in INM isolated
from inositol-labeled IN. The details of the chromatographic separation are described in Methods.
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Figure 3. Chase studies of the [3H]inositol labeled PIPs of the ONM. The radiolabeled intact nuclei were subjected to
incubation in transport active cold CC for 0-5 min. At indicated time the samples of intact nuclei were withdrawn and
subjected to isolation of inner and outer nuclear membrane followed by lipid extraction and deacylation. The PIPs con-
verted to GPIPs were separated by column chromatography described in Methods. To eliminate quantitative errors, the
external standard consisting of 20% each of radiolabeled inositol, PI, PIP1, PIP2 and PIP3 representing 5,000 cpm was
subjected to the same procedure and its recovery was used to compensate for errors in procedural recovery and sampling.
Figure 4. Chase studies of [3H]inositol labeled PIPs of the INM. The procedural details and quantitation of the PIPs are
identical with those described in Figure 3. The INM are derived from the same intact nuclei used for isolation of ONM.
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within 1 and 3 min chase continue to increase to 38.3%
at 1 min, 49.0% at 3 min, and then suddenly at 5 min
drop to the lowest level of PIPs (2.7%). That is accom-
panied by unexpected increase in PI, which at 3 min
represented 1.9% of PIPs and in 5 min chase rises to
53.5%. The results reflecting PI level in 0 to 3 min im-
pose that radiolabeled PI is used for synthesis of PIPs
and its level is not replenished through dephosphoryla-
tion of PIPs and particularly PIP3. The simultaneous and
slight decrease in th e level of PI and PIP1s an d the initial
increase in PIP2s suggest that PIP1s are further phos-
phorylated and their level is not restored through
dephosphorylation. In 1 and 3 min chase, we observed
even lower but constant level of PIP1s, while phos-
phorylative activity concentrated on PIP2s and PIP3s.
The PIP2s decrease was in concert with the continued
increase in PIP3s. In 5 min chase we observe that the
levels of PIP1s and PI P2s seem to stabilize after reaching
or diminishing to their initial value found at the zero
time, but PI level rises dramatically from 1.9% to 53.5%
and PIP3s drops precipitously from 49.0% to 2.7 %. As
evident from the values obtained in the chase between
0-3 min PI increase in ONM, in 5 min chase could not
be linked to dephosphorylation of PIP3s in ONM, since
the process would have to be detected in incremental
quantities of PIP2s, PIP1s and then PI. The obtained re-
sults suggest that the su dden drop in ONM PIP 3s and th e
appearance of PI are reflecting the process that is linked
to PIPs transformations in INM.
The INM membranes (Figure 4) purified from the
same nuclei as ONM contained 6.1%, PI, 37.7% PIP1s,
30.9% PIP2s and 25.3% PIP3s. In the 0.5 to 3 min chase,
minimal changes in PI level were detected, although
some fluctuations were evident. However, at 5 min the
level of PI rose to 15.7%. The PIP1s which represented
the largest fraction of PIPs in INM in first 30 seconds
rose from 37.7 to 52.1% and that was accompanied by
17.7% decrease in PIP2s, thus suggesting fast PIP2s
dephosphorylation to PIP1s, while PIP3s tempered in-
crease continued. In the 1 min chase, we observed PIP3s
build up to 53.7%. After reaching its highest concentra-
tion in 1 min chase, in longer chase (3 min) PIP3s drop
precipitously to 4.6% while PIP2s and PIP1s increase to
21,1 % and 57.7%, respectively. Finally, in 5min chase
the PIP3s level drop further to 2.7%, PIP2s decrease to
24.1% and PIP1s remain at 57.6%. The sudden decrease
in PIP3s, in 5 min chase is accompanied by PI generation,
which is not as dramatic as disappearance of PIP3s and
constitutes 15.7% of total PIPs. In our interpretatio n
the changes in PIPs of INM reflect dephosphorylation
reaction as the main process that causes transformation
of PIPs with final product of PI. Since the results of our
study depict also chase out of phosphorylated radio-
labeled PIPs it is reasonable to suggest that the level of
PIP3 is continuously being rebuilt by influx of phos-
phorylated PIPs from ONM. Again, as in ONM it is dif-
ficult to correlate sudden increase of PIP3s in 1min
chase while PIP1s decrease, PIP2s level undergoes tem-
pered rebuilding and PI is unchanged, except to suggest
that PIP3s are introduced from ONM. Such an infusion
of PIP3s is then reflected in the increments of PIP2s,
PIP1s and finally at 5 min in the accumulation of PI and
depletion of PIP3s. Hence, in continuation of the ex-
periments that implied that in nuclear membranes PIPs
flux was terminated by their complete dephosphorylation
and reemergence in form of PI, the studies on PIPs pro-
files in ER during 5 min chase were performed (Figure
The data presented in Figure 5 revealed that ER
membranes contain PI and some traces of PIP1s. Neither
PIP2s nor PIP3s were present at the beginning of the
chase or throughout the chase. During the chase some
decrement of radiolabeled PI was observed, which, in
our opinion, could represent the release of ER transport
vesicles or might reflect the presence of some Golgi
membranes that are highly enriched in PI and PIP1s.
If the above suggested model of the nuclear mem-
brane movement is operational, one should expect that
with chase the amount of inositol radiolabeled lipids
would diminish in INM and increase in ONM. Indeed,
we have found that the ONM fraction derived from 0.5
min chase contained 5.3 % more radiolabeled PIPs then
its matching INM fraction, 3 min chase resulted in
14.3% increase and 5 min chase produced 23.3 % in-
crease of radiolabeled PIPs in ONM fraction. Based on
the obtained evidence we propose that nuclear mem-
branes are subjected to continuous movement, which is
afforded by synthesis of PIPs in ONM and their
dephosphorylation in INM. The membrane depleted of
PIPs, but retaining their PI emerges onto cytosolic site
and thus initiates ER.
In our interpretation, the described changes in the
level of PIPs in ONM during the chase reflect two events,
the phosphorylation of PI, and the continuous movement
of the membrane containing PIP2s and PIP3s into the
nucleus. After 5 min the radiolabeled PIP3s were no
longer present in the ONM, but instead the dephos-
phorylation products from INM PIPs reentered ONM
continuum in the form of radiolabeled PI. Thus, the
metabolic processes in nuclear membranes are linked to
phosphorylation of PIs in ONM, dephosphorylation
events in the INM, and transition from PIPs of nuclear
compartment to PI of ER.
Collectively, our data suggest that nuclear membrane
represents uninterrupted continuum subjected to synthe-
sis-induced mov ement, which capacitates the sys tem with
A. Slomiany et al. / Health 3 (2011) 187-199
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Figure 5. PIPs) isolated from ER prepared from the same preparations that were used for isolation of IN. The chase
studies were performed in the same preparation of the cold transport active CC as used for ONM and INM. The prepa-
ration of PIPs, the separation of the deacylated GPIPs and their quantitation was identical with that described in Figure
1 and 3.
the ability to transport cytosolic products to nucleus,
export of nuclear components to cytosol, and growth of
ER memb rane.
Recent literature data indicate that nuclear PIPs par-
ticipate in chromatin remodeling, mRNA splicing,
mRNA 3’end processing, and the export from nucleus
[9-11,22-25]. There is also emerging evidence that some
actively transcribed genes localize to nuclear periphery
enriched with PIPs in the inner nuclear membrane and
the nuclear pore complexes, thereby positioning the
products for exit [12,14]. At the same time, the PIPs that
aid pre-mRNA processing, including splicing factors,
small nuclear ribonucleoproteins (nRNPs) and RNA
polymerase II, are not present in the ER that constitutes
nuclear membrane continuum [7-9,16,17]. Logically,
such an immediate extension of the nuclear organelle
should conserve compositional similarity. Since it does
not, then what is the deciding factor that demarcates the
nuclear and ER bound aries, while satisfying clear link in
the continuous processing of the signal in the ER. Our
uncovering of PIPs transformation in the nuclear mem-
branes and the nuclear PIPs-associated processes which
cease at ER boundary, while the membrane retains PI
core, is crucial for compartment’s functional separation
with preserved membrane continuum. It demonstrates
the assignment of nuclear and ER lipids function, while
safeguarding the paramount meaning of the continuity
and the fidelity in signaling dialog between the cellular
and nuclear membranes resulting in signal-intended re-
sponses in ER.
While the pre-mRNA processing machinery depends
on the presence of PIPs, it is obvious that such function
must be terminated with mRNA exit from the nucleus. It
has been suggested that this is achieved through the nu-
clear PIPLC activity [30]. If indeed this is the case, then
during the chase, the IN would release watersoluble ra-
diolabeled IPs and the level of radiolabeled PIPs in IN
would gradually decline. As we demonstrate in Table 1
and 2, PIPLC activity is not apparent in PIPs transfor-
mations of either ONM or INM membrane. With chase
time lapse, the labeled cytosol-soluble inositides have
not increased, whereas the amount of radiolabeled lipids
extracted from the IN increased slightly. In our opinion,
the increase in nuclear lipids, could not be factual, but
most likely reflects some discrepancies in the separation
of the org anic (lipid) an d water-soluble phase. As found,
A. Slomiany et al. / Health 3 (2011) 187-199
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the turn to unexpected opposite values is an unlikely
outcome in the chase experiments, and therefore the re-
sults provide ind isputable ev idence th at PIPs metabolism
in the nuclei is not attributed to PIPLC activity in the
cytosol or nucleus [30,31].
In analyzing the chase data, we considered two se-
quences of possible ev ents in PIPs transformations. One,
the static scenario where PIPs of ONM and INM are
independent of each other and second, the dynamic sys-
tem where nucleu s is surrounded by one membrane con-
tinuum in which PIPs transformation in ONM and INM
are inherently linked. In the static scenario, the contin-
ued synthesis of PIPs in two independent membranes
would be reflected in the loss of radiolabeled PI in ONM
and gain of the labeled PIPs. In contrast, in INM mem-
brane, depho sphorylation of PIPs would yield incr easing
quantities of PI. Indeed, in our chase study utilizing
[3H]inositol labeled nuclei subjected to 0.5-5 min incu-
bation in transport active cytosol, we observed the proc-
esses that were consistent with PI phosphorylation in th e
ONM and dephosphorylation in the INM. In ONM, the
initial phase of chase demonstrated PI phosphorylation
that was reflected in an increase of PIPs. However, with
time of chase, the level of PIPs in ONM declined, while
in the matching nuclear samples of INM their level rose
incrementally. The decline of PIPs in the ONM could
not be attributed to some PIPs-specific cytosolic phos-
phatases that could act on ONM at the same time. In a
such scenario, all chase results would generate the same
as time 0 radiolabeled PIPs profiles. The results of chase
studies on the ONM presented in Figure 3, do not reflect
neither static sequence of the events, nor phosphatase-
specific changes in time-dependent PIPs profiles. Rather,
the data express the synthesis of PIPs in the ONM facing
cytosolic environment and the active transport of the
PIPs to INM facing nuclear inner space. The sudden
increase in the amount of PIP3s in the INM and PI in the
ONM suggests that the nuclear membrane is a dynamic
continuum, which was experimentally separated into
independent fractions of the ONM and INM. The chase-
generated results capture the membrane motion by reg-
istering enrichment in PIP3s in INM and concomitant
ejection of PI-containing IMN onto cytosolic site. If nu-
clear membranes were fixed in position, the PIP3s build
up would remain in ONM and PI in INM. Together, the
chase-generated results support the dynamic mode of
PIPs’ transitions, that under in situ condition s, prior arti-
ficial separation of the nuclear membrane con tinuum, t he
PIPs synthesis-induced ONM movement introduces PIPs
onto intranuclear site while expels incremental amount
PI-containing IMN onto the cytosolic site. Such lateral
membrane movement explains reemergence of the INM
with dephosphorylated PI onto cytosolic site and transi-
tion of the INM into new segment of ER.
In previous studies we have found that PIPs of nuclear
membrane are involved in transport of cytosolic protein
to nucleus and remained in INM while their structural
profile suggested that while facing intranuclear envi-
ronment they were subjected to dephosphorylation [15].
Thus, the protein transport-associated data and the chase
results presented here, imply that transferred PIPs are
subjected to dephosphorylation while remaining in the
membrane. Moreover, the interpretation that PIPs are
confined to nuclear membranes, is also supported by
physicochemical properties of phospholipids [5,18-21].
Indeed, it is unquestionable that the amphipathic struc-
ture of PIPs render these phosp holipids en ergetically an d
thermodynamically unfavorable to move freely within
nuclear environment [6,32]. We believe that the findings
placing PIPs in the subnuclear sites originate from the
literal acceptance of the term ‘membrane stripping’. The
detergent aided membrane ‘stripping’ disintegrates phos-
pholipid-enriched membrane continuum, but does not
displace the lipids interacting with their ligands. There-
fore, the findings on the detergent-treated nuclei retain-
ing PIPs linked to subnuclear structures reinforce the
fact that phosphatidylinositides or other phospholipids
that interact with nuclear compon ents remain in the com-
plex [33,34]. Also, identification of the so-called phos-
phatidylinositides carrier proteins supports the fact that
specific complexes of lipid ligands do not easily disso ci-
ate, but normally as anchored in membrane they trans-
port cytosolic protein to the nucleus and participate in
the chromatin remodeling. Apparently, the intranuclear
interaction with PIPs remain until the lipids are trans-
formed by specific phosphatases and then enter different
set of intranuclear reactions [35]. In reality, the data that
capture the presence of pools of PIPs associated with an
unique nuclear compartments, illustrate which intranu-
clear compartments are induced by the signal to interact
with a specific and membrane-tethered PIPs ligands.
Hence, our data argue against the view that nuclear PIPs
function in membrane-free compartments, and suggest
that the nuclear transformations positioned in the prox-
imity of PIPs-containing inner leaflet of the membrane,
just as in cell cytosol, allow the specific interaction be-
tween lipid ligand and the nuclear complex. Our conten-
tion that PIPs are confined to nuclear membranes and
that outer leaflet phosphoinositides of the ONM trans-
port cytosolic protein to nucleus [15], has been elabo-
rated further. With chase experiments performed on IN
and ER in the presence of active cytosol we have de-
tected that the nuclear membrane movement associated
with PIPs assembly, dephosphorylation is inherently
connected with initiation of the membrane that becomes
part of ER. At this time, we cannot pinpoint the specific
A. Slomiany et al. / Health 3 (2011) 187-199
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interactions that take place while PIPs are transformed,
except to imply that such an interactions are terminated
once the PI is generated, and the reentrance of the INM
to the cytosolic environment is evident as the radio-
labeled PI is becoming part of ONM fraction. Therefore,
it seems more than plausible, that the membrane reen-
tering cytosolic environment is the one transiting into
ER compartment, and which is conditioned to synthesize
lipids, translate specific mRNA, and synthesize specific
transport vesicles and their cargo [36]. The support for
this conclusion emanates from several facts; firstly the
ER from the same cells that were used to isolate inosi-
tol-labeled nuclei contained PI only. Secondly, as we
have shown previously, the ER transport vesicles con-
tained PI in their membrane, which upon delivery to
Golgi was subjected to specific phosphorylatio n to PIP1s
known to direct cargo to apical, basolateral or en-
dosomal membranes [16,17]. Thirdly, the vesicular
cargo and its destination was dictated by the mRNA of
the cytosol used in the ER transport vesicles synthesis.
Finally, the ER transport vesicles were not assembled in
the RNA depleted cytosol [16,17]. As demonstrated ear-
lier, the ER membrane contains PI only [7-9,16,17]. If
the nuclear and ER phosphoinositides were degraded
through PIPLC activity, the water soluble polyphospho-
inositides would be released from the membrane and be
reflected in the loss of inositol labeled phosphatidy-
linositides in the nuclear and ER membranes. But, on the
other hand, if the processing engages dynamic act of
membrane PIPs transformation through phosphoryla-
tion/dephosphorylation reactions, the nuclear membrane
would retain their modified lipids. Such a turn of events
satisfies termination of the pre-mRNA processing at
INM, PI lipid retention in the intact nuclei, and the no-
ticeable increase of labeled PI following 5 min chase in
ONM. In situ, this is an ideal transit from nucleus mem-
brane to ER, where the compartment-specific transfor-
mation of the membrane completed nuclear signal which
in turn is translated into coordinated response in th e ER.
Compiled results of our study on the biomembrane bio-
genesis in cellular network and the nuclear membranes,
lead us to advance the hypothesis on the specificity of
the cellular membranes production and repair. All these
processes are intimately linked to PIs transformation and
movement across the nuclear space, the reappearance of
their core PI as a part of the ER conditioned to perform
the task of synthesis and transport of the vesicles, thus
restoring cellular membranes’ structure and function.
The presence of PI in the exiting inner nuclear space
INM, recovered in the membranes preparation as out-
growing ONM, provides superb transition from the
phosphoinositides metabolic transformations in the nu-
cleus to ER. As we demonstrated earlier, and again in
this study (Figure 5), ER is engaged in the synthesis of
specific cellular membranes whose delivery to multiplic-
ity of cellular sites is first dictated in ER by the protein
intercalated into the membrane and then the process is
completed in Golgi, an organelle responsible for mem-
brane maturation, protein modification and PI-specific
phosphorylation [7-9,16,17]. Considering that the above
enumerated criteria of the dynamic progression of mem-
brane synthesis and signal transfer have been satisfied, it
is apparent that the nuclear envelope constitute uninter-
rupted membrane demarcated by pore complex. The
challenge remains to determine when the pore complex
allows movement of the membrane from outer to inner
nuclear space [37], and to identify and coordinate phos-
phatidylinositides-regulated nuclear events with the
translational and synthetic events executed in the ER.
Thus, identification of the PI-sensitive components in
the nucleus and passage of the massage to the ER is cen-
tral to understanding how the cell nucleus dictates post-
transcriptional cellular responses.
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