Transformations of phosphatidylinositol phosphates in the outer and inner nuclear membrane are linked to synthesis and restitution of cellular membranes

doi:10.4236/health.2011.34035

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Vol.3, No.4, 187-199 (2011) Health

doi:10.4236/health.2011.34035

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:

slomiaam@umdnj.edu

Received 10 February 2011; revised 18 March 2011; accept 1 April 2011.

ABSTRACT

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-

plates.

Keywords: Phosphatidylinositides; PIPs;

Transformation ; R estitution; Outer Nuclear

Membrane; Inner Nuclear Membrane; Endoplasmic

Reticulum; Cellular Membranes

1. INTRODUCTION

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

188

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.

2. MATERIALS

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

[28].

2.1. Solution Used in Preparation of Cells,

Cell Organelles, Nuclei and Nuclear

Membranes

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

A. Slomiany et al. / Health 3 (2011) 187-199

189189

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

Labeling

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

ER.

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

A. Slomiany et al. / Health 3 (2011) 187-199

190

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

Membranes

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.

3. RESULTS

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-

A. Slomiany et al. / Health 3 (2011) 187-199

191191

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.

Time

(min) Control

(cpm) % cpm

released Sample+

CC (cpm) % cpm

released

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.

Time

(min) Control

(cpm) % cpm in

lipids Sample+

CC (cpm) % cpm in

lipids

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

A. Slomiany et al. / Health 3 (2011) 187-199

192

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.

A. Slomiany et al. / Health 3 (2011) 187-199

193193

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.

A. Slomiany et al. / Health 3 (2011) 187-199

194

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

5).

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

195195

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.

4. DISCUSSION

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

196

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

197197

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.

REFERENCES

[1] Di Paolo, G. and De Camilli, P. (2006) Phosphoinositi-

des in cell regulation and membrane dynamics. Nature,

443, 651-657. doi:10.1038/nature05185

[2] Vicinanza, M., D’Angelo, G., Di Campli, A. and De

Matteis, M. A. (2008) Function and disfunction of the PI

system in membrane trafficking. EMBO J., 27, 2457-

2470. doi:10.1038/emboj.2008.169

[3] Sasaki, T., Sasaki, J., Sakai, T., Takasuga, S., and Suzuki,

A. (2007) The physiology of phosphoinositides. Bio-

logical & Pharmaceutical B u ll etin, 30, 1599-1604.

doi:10.1248/bpb.30.1599

[4] Sasaki, T., Takasuga, S., Sasaki, J., Kofuji, S., Eguchi, S.,

Yamazaki, M. and Suzuki, A. (2009) Mammalian phos-

phoinositide kinases and phosphatases. Progress in Lipid

Research, 48, 307-343.

doi:10.1016/j.plipres.2009.06.001

[5] Barlow, C.A., Laishram, R.S., and Anderson, R.A. (2009)

Nuclear Phosphoinositides: a signaling enigma wrapped

in a compartmental conundrum. Trends in Cell Biology,

20, 25-35. doi:10.1016/j.tcb.2009.09.009

[6] Boronenkov I,V. Loijens, J.C., Umeda, M., and Ander-

son, R.A. (1998) Phosphoinositide signaling pathways in

nuclei are associated with nuclear specles containing

pre-mRNA processing factors. Molecular Biology of the

Cell, 9, 3547-3560.

[7] Slomiany, A., Grzelinska, E., Grabska, M., Yamaki, K.,

Tamura, S. and Slomiany, B.L. (1992) Intracellular proc-

esses associated with glycoprotein transport and proc-

essing. Archives of Biochemistry and Biophysics, 298,

167-175. doi:10.1016/0003-9861(92)90108-9

[8] Slomiany, A., Grabska M., Piotrowski, E., Morita, M.

and Slomiany, B.L. (1994) Intracellular processes asso-

ciated with vesicular transport from endoplasmic reticu-

lum to Golgi and exocytosis; Ethanol-induced changes in

membrane biogenesis. Archives of Biochemistry and

A. Slomiany et al. / Health 3 (2011) 187-199

198

Biophysics, 310, 247-255. doi:10.1006/abbi.1994.1164

[9] Slomiany, A., Sano, S., Grabska, M., Yamaki, K. and

Slomiany, B.L. (2004) Gastric mucosal cell homeostatic

physiome. Critical role of ER-initiated membrane restitu-

tion in the fidelity of cell function renewal. Journal of

Physiology and Pharmacology, 55, 837-860. 15

[10] Gonzales, M.L. and Anderson, R.A. (2006) Nuclear

phosphoinositides kinases and inositol phospholipids.

Journal of Cell Biochemistry, 97, 252-260.

doi:10.1002/jcb.20655

[11] Irvine, R.F. (2003) Nuclear lipid signaling. Nature Re-

views Molecular Cell Biology, 4, 349-361.

doi:10.1038/nrm1100

[12] Ruault, M., Dubarry, M. and Taddei, A. (2008) Re-posi-

tioning genes to nuclear envelope in mammalian cells;

impact on transcription. Trends in Genetics, 24, 574-581.

doi:10.1016/j.tig.2008.08.008

[13] Akhtar, A. and Gasser, S.M. (2007), The nuclear enve-

lope and transcriptional control. Nature Reviews Genetics,

8, 507-517. doi:10.1038/nrg2122

[14] Moore, M.J. and Proudfoot, N.J. (2009) Pre-mRNA

processing reaches back to transcription and ahead of

translation, Cell, 136, 688-700.

doi:10.1016/j.cell.2009.02.001

[15] Slomiany, A., Grabska, M. and Slomiany, B.L. (2006)

Homeostatic restitution of cell membranes. Nuclear

membrane lipid biogenesis and transport of protein from

cytosol to intranuclear spaces. International Journal of

Biological Sciences, 2, 216-226.

[16] Slomiany, A., Nowak, P., Piotrowski, E. and Slomiany,

B.L. (1998) Effect of ethanol on intracellular vesicular

transport from Golgi to apical cell membrane: Role of

phosphatidylinositol 3-kinase and phospholipase A2 in

Golgi vesicles association and fusion with the apical

membrane, Alcoholism: Clinical and Experimental Re-

search, 22, 167-175.

doi:10.1111/j.1530-0277.1998.tb03634.x

[17] Slomiany, A. and Slomiany, B.L. (2010) Cell membranes

composition is defined in ER and their restitution pro-

ceeds by en bloc fusion of ER generated transport vesi-

cles. Health, 2, 1444-1454.

doi:10.4236/health.2010.212214

[18] Bunce, M.W., Bergendahl, K. and Anderson, R.A. (2006)

Nuclear PI(4,5)P2 : A new place for an old signal. Bio-

chemica et Biophysica Acta, 1761, 560-569.

[19] Garnier-Lhomme, M., Byrne, R.D., Hobday, T.M.C.,

Gschmeissner, S., Woscholski, R.,Poccia, D.L., Dufourc,

E.J., and Larijani, B. (2009) Nuclear envelope remnants:

fluid membranes enriched in sterols and phosphatidy-

linositides. Plo S ONE 4, e4255. 16

[20] Tanaka, K., Horiguchi, K., Yoshida,T., Fujisawa, H.,

Takeuchi, K., Umeda,, M., Kato, S., Ihara, S., Nagata, S.

and Fukui, Y. (1999) Evidence that 3,4,5- triphosphate

–binding protein can function in nucleus. Journal. Bio-

logical Chemistry, 274, 3919-3922.

doi:10.1074/jbc.274.7.3919

[21] Ye, K. and Ahn, J.Y. (2008) Nuclear phosphoinositides

signaling. Frontiers in Biosciences, 13, 540-548.

doi:10.2741/2699

[22] Osborne, S.L., Thomas, C.L., Gschmeissner, S. and

Schiavo, G. (2001) Nuclear PtdIns(4,5)P2 assembles in a

mitotically regulated particle involved in pre-mRNA

splicing. Journal of Cell Science, 114, 2501-2511.

[23] Gozani, O.,Karuman, P., Jones, D.R., Lugovskoy, A.A.,

Baird, C.L, Zhu,H., Field, S.J.,Lessnick, S.L., Villasenor,

J.,Mehrotra, B., Chen, J., Rao, V.R., Brugge, J.S., Fer-

guson, C,G., Payrastre. B., Myszka, D.G., Cantley, L.C.,

Wagner, G., Divecha, N. Prestwich, G.D. and Yuan, J.

(2003) The PHD finger of the chromatin associated pro-

tein ING2 functions as a nuclear phosphoinositides re-

ceptor. Cell, 114, 99-111.

doi:10.1016/S0092-8674(03)00480-X

[24] Macbeth, M.R., Schubert, H. L., VanDemark, A.P., Lin-

gam, A.t., Hill, C.P. and Bass, B.L. (2005) Inositol-

hexakisphosphate is bound in ADAR2 core and required

for RNA editing. Science, 309, 1534-1539.

doi:10.1126/science.1113150

[25] Yu, H., Fukami, K., Watanabe, Y., Ozaki, C. and Take-

nawa, T. (1998) Phosphatidylinositol 4,5-bisphosphate

reverses the inhibition of RNA transcription caused by

histone H1. European Journal of Biochemistry, 251,

281-287. doi:10.1046/j.1432-1327.1998.2510281.x

[26] Zhao, K., Wang, W., Rando, O.J., Xue, Y., Swiderek, K.,

Kuo, A. and Crabtree, G.R. (1998) Rapid and phos-

phatidylinositol-dependent binding of the SWI/SNFlike

BAF complex to chromatin after T lymphocyte receptor

signaling. Cell, 95, 625-636.

doi:10.1016/S0092-8674(00)81633-5

[27] Mellman, D.L., Gonzales, M.L., Song, C., Barlow, C.A.,

Wang, P., Kendziorski, C., and Anderson, R.A. (2008) A

PtdIns4,5 P2-regulated nuclear poly(A) polymerase con-

trols expression of select mRNAs. Nature, 451,

1013-1017.

[28] Slomiany, A., Tamura, S., Grzelinska, E.,Piotrowski, J.,

and Slomiany, B.L. (1992) Characterization of the ‘link’

component of submandibular mucus glycoprotein, Inter-

national Journal of Biochemistry, 24, 1003-1015.

doi:10.1016/0020-711X(92)90111-D

[29] Slomiany, A. and Slomiany B.L. (2003) Lipidomic proc-

esses in homeostatic and LPS- modified cell renewal cy-

cle. Role of phosphatidylinositol 3-kinase pathway in

biomembrane synthesis and restitution of apical epithe-

lial membrane . Journal of Physiology and Pharmacology,

54, 533-551.

[30] Visnjic, D. and Banfic, H. (2007) Nuclear phospholipid

signaling : Phosphatidylinositol-specific phospholipase C

and phosphoinositide 3-kinase. Pflugers Archives, 455,

19-30. doi:10.1007/s00424-007-0288-1

[31] Sekine, T. and Raben, D.M. (2004) Nuclear production

and metabolism of diacylglycerol. European Journal of

Histochemistry, 48, 77-82.

[32] Alcazar-Roman, A.R. and Wente, S.R. (2008) Inositol

polyphosphates: a new frontier for regulating gene ex-

pression. Chromosoma, 117, 1-13.

doi:10.1007/s00412-007-0126-4

[33] Brown, D.A. (2006) Lipid rafts, detergent-resistant

membranes and raft targeting signals. Physiology, 21,

430-439. doi:10.1152/physiol.00032.2006

[34] Snoek, G.T. (2004) Phosphatidylinositol transfer protein:

emerging roles in cell proliferation, cell death and sur-

vival. IUBMB Life, 56, 467-475.

doi:10.1080/15216540400012152

[35] Yokogawa, T., Nagata, S., Nishio, Y., Tsutsumi, T.,

Ihara, S., Shirai, R., Morita, K., Umeda, M., Shirai, Y.,

A. Slomiany et al. / Health 3 (2011) 187-199

199199

Saitoh, N. and Fukui, Y. (2000) Evidence that 3’- phos-

phorylated polyphosphoinositides are generated at the

nuclear surface: use immunostaining technique with spe-

cific monoclonal antibodies specific for PI3,4P2. FEBS

Letters, 473, 222-226.

doi:10.1016/S0014-5793(00)01535-0

[36] Fagone, P. and Jackowski, S. (2009) Membrane phos-

pholipid synthesis and endoplasmic reticulum function.

Journal of Lipid Research, 50, S311-S316.

doi:10.1194/jlr.R800049-JLR200

[37] Theerthagiri, G., Eisenhardt, N., Schwarz, H. and An-

toninin, W. (2010) The nucleoporin Nup188 controls

passage of membrane proteins across the nuclear pore

complex. Journal of Cell Biology, 189, 1129-1142.

doi:10.1083/jcb.200912045