1 CNR, Institute of Neuroscience, Cellular and Molecular Pharmacology, Center of
Excellence on Neurodegenerative Diseases, Department of Medical Pharmacology,
University of Milan, Via Vanvitelli 32, 20129 Milan, Italy
2 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada M5G
1X5
3 School of Biological Sciences, University of Manchester, Manchester M13 9PT,
UK
* Author for correspondence (e-mail: p.rosa{at}csfic.mi.cnr.it)
Accepted 29 July 2002
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Summary |
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Key words: Calcium-binding proteins, Neuronal calcium sensor 1, Phosphatidylinositol 4-OH kinase, Polyphosphoinositides, Membrane traffic
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Introduction |
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An interesting insight into the physiological role of NCS-1 has come from
recent data (Hendricks et al.,
1999) demonstrating that the yeast homologue of NCS-1 can
associate with and upregulate the activity of an isoform of
phosphatidylinositol 4-OH kinase (PI4K) homologous to the mammalian PI4Kß
[a member of the so-called type III PI4Ks
(Balla, 1998
;
Fruman et al., 1998
;
Hendricks et al., 1999
)].
The members of the PI4K family catalyze the first step in the synthesis of
phosphoinositides and polyphosphoinositides that are known to play a crucial
role in exocytosis and intracellular traffic (for reviews, see
De Camilli et al., 1996;
Brodin et al., 2000
;
Huijbregts et al., 2000
;
Cremona and De Camilli, 2001
).
The first evidence that polyphosphoinositides are important in vesicular
trafficking reactions independently of their phospholipase C-mediated cleavage
came from studies of regulated exocytosis in neuroendocrine cells
(Holz et al., 1989
;
Eberhard et al., 1990
;
Hay et al., 1995
). In these
studies, Ca2+-dependent neurotransmitter release has been shown to
require an ATP-priming step. Both a PI4K and a phoshoinositide 4P
5-kinase [PI(4)P5K] are required for the priming reaction, suggesting
that phosphoinositides, mainly
phosphatidylinositol(4,5)P2, may play a role in this
process. Interestingly, a PI4K activity has been detected on chromaffin
granules (Wiedemann et al.,
1996
) and synaptic vesicles
(Wiedemann et al., 1998
).
Besides exocytosis, phosphoinositides have also been implicated in other
aspects of membrane traffic (e.g. synaptic vesicle endocytosis and
constitutive secretion), suggesting that their synthesis is highly regulated.
The molecular mechanisms underlying the synthesis of different
polyphosphoinositide pools, the subcellular localization of these pools and
the PI4Ks involved are only partially known. The recent finding in yeast
suggests that NCS-1 and PI4Kß may cooperate in modulating the exocytotic
processes. However, it is still unclear whether endogenous NCS-1 and
PI4Kß interact in vivo. The two proteins were found to form a complex
after overexpression in epithelial Madin-Darby canine kidney and COS-7 cells,
but not in cultured DRG neurons (Weisz et
al., 2000
; Bartlett et al.,
2000
; Zhao et al.,
2001
).
In order to further study the function of NCS-1 in neurosecretory cells and
its interaction with PI4Kß, we analyzed and compared the subcellular
distribution of both proteins and tested whether they are capable of forming a
complex in neurons and neuroendocrine cells. Moreover, we investigated whether
the membrane distribution of NCS-1 and PI4Kß was modulated in intact
neuroendocrine cells under conditions that are known to stimulate
polyphosphoinositide turnover and neurotransmitter secretion
(Raha et al., 1993;
Murrin and Boarder, 1992
;
Koizumi et al., 1995
).
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Materials and Methods |
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Antibodies
The rabbit polyclonal (44162) and monoclonal (3D5) antibodies against NCS-1
were prepared as described (Werle et al.,
2000). The polyclonal antibodies against ribophorin, calreticulin
and synaptotagmin were kind gifts of G. Kreibich (New York University School
of Medicine, New York), J. Meldolesi and A. Malgaroli (Department of
Neurosciences, San Raffaele Institute, Milan, Italy), respectively. Polyclonal
anti-PI4Kß antibodies were purchased from Upstate Biotechnology (Lake
Placid, NY). Mouse monoclonal antibodies against protein disulphide isomerase
(PDI), anti-tubulin, the TGN38 trans-Golgi network protein and synaptobrevin 2
were obtained from Stressgen Biotechnologies (Victoria, BC, Canada), Sigma
Aldrich (Milan, Italy), Transduction Laboratories (Lexington, KY) and Synaptic
Systems (Gottingen, Germany), respectively. The peroxidase and gold-conjugated
secondary antibodies were purchased from Jackson Immuno Research Laboratories
(West Grove, PA) or Sigma. Rabbit IgGs were purchased from Sigma.
Subcellular fractionation
Differential centrifugation
Rat brain fractionation was carried out essentially as described
(Huttner et al., 1983).
Cerebral cortices dissected from rat brains were homogenized in homogenization
buffer (4 mM Hepes-NaOH, pH 7.3, and 0.32 M sucrose). The total homogenate was
centrifuged for 10 minutes at 800 g and the post-nuclear
supernatant (S1) was collected and centrifuged as described to yield a pellet
corresponding to the synaptosomal fraction (P2) and a supernatant (S2). The S2
containing the remaining organelles from the total homogenate was centrifuged
at 165,000 g for 2 hours to yield a high-speed supernatant
corresponding to the cytosol (S3) and a pellet (P3) enriched in membrane-bound
organelles of cell bodies. P2 was subjected to hypo-osmotic shock by means of
10-fold dilution in 7.5 mM Hepes-NaOH buffer, pH 7.2. The P2-lysate was
centrifuged for 20 minutes at 25,000 g to yield a lysate
pellet (LP1) containing membrane-bound organelles/vesicles larger than
synaptic vesicles and a lysate supernatant (LS1) that was further centrifuged
at 165,000 g for 2 hours. The resulting supernatant (LS2, the
cytosolic fraction of the synaptosomal compartment) was removed and the pellet
(LP2) containing the small vesicles was resuspended in 40 mM sucrose, loaded
on top of a linear sucrose gradient (50-800 mM sucrose) and centrifuged at
65,000 g for 5 hours. After centrifugation, 20 fractions of
500 µl were collected and those equilibrated in the 200-400 mM sucrose
region were pooled and centrifuged 165,000 g for 5 hours to
yield a pellet, SG-V, highly enriched in synaptic vesicles. Equal amounts of
proteins from each fraction were separated on SDS-polyacrylamide gels and
analyzed by western blotting as described
(Rowe et al., 1999
).
Velocity gradient centrifugation
P3 was resuspended with a dounce homogenizer in 250 mM sucrose, 1 mM
Mg-acetate, 2 mM EDTA and 10 mM Hepes-KOH, pH 7.4 and then loaded on top of a
sucrose linear gradient (0.3-1.2 M). After centrifugation at 75,000
g for 20 minutes, 12 aliquots of 1 ml were collected from the
top of the gradient. Proteins from equal volumes of each fraction (300 µl)
were precipitated with acetone at -20°C and then separated on
SDS-polyacrylamide gels and analyzed by western blotting.
Discontinuous sucrose density gradient centrifugation
The S2 fraction prepared by differential centrifugation was adjusted to 1.2
M sucrose containing 1 mM EDTA loaded into an SW 27 tube and overlayed with 8
ml of 1.1 M sucrose, 10 ml of 0.85 M sucrose and 8 ml of 0.25 M sucrose. The
gradients were centrifuged at 100,000 g for 3 hours and the
band at the 0.85-1.1 M sucrose interface (fraction 1), a second band at the
1.1-1.2 M sucrose interface (fraction 2) and the pellet were collected and
analyzed by western blotting.
Immunoprecipitation
Brains from adult Sprague-Dawley rats (females) were homogenized in
ice-cold immunoprecipitation (IP) buffer (125 mM potassiumacetate, 0.1% (w/v)
Triton X-100, 20 mM Tris-HCl pH 7.2, 2 µg/ml pepstatin, 2 µg/ml
aprotinin). When required, the IP buffer was supplemented with 1 mM
CaCl2 or 5 mM EGTA. The total homogenates were incubated for 1 hour
on ice and then clarified by centrifugation (30 minutes at 14,000
g). Supernatant volumes corresponding to 0.5-1 mg of protein
were incubated for 1 hour with 50 µl of Protein A sepharose beads
(Amersham-Pharmacia). The beads were removed by centrifugation (10 minutes at
3000 g) and the `precleared' supernatants were added to 50
µl of protein A beads preincubated (2 hours at 4°C) with either the
affinity purified anti-NCS-1 IgG (2-4 µg, polyclonal 44162), the
anti-PI4Kß IgG (2-4 µg, Upstate) or rabbit IgG (2-4 µg, Sigma
Aldrich) as a control. After 16 hours at 4°C, the beads were collected by
centrifugation (5 minutes at 3000 g) and extensively washed
with IP buffer and then resuspended in Laemmli sample buffer
(Laemmli, 1970). When the
immunoprecipitation was performed on rat brain membrane or soluble protein
fractions, the tissue was homogenized in 320 mM sucrose, 4 mM Hepes-NaOH pH
7.3 supplemented with protease inhibitors. The postnuclear supernatants (S1)
were centrifuged at high speed (1 hour at 200,000 g) in order
to obtain total membrane pellets and soluble protein fractions. The S1,
membrane (resuspended in sucrose buffer to reconstitute the initial volume)
and cytosol fractions were adjusted to 1x IP buffer, incubated for 1
hour on ice and clarified by centrifugation. Immunoprecipitation was carried
out as described above by using equal volumes of S1, membrane and cytosol.
Immunoprecipitated proteins were then analyzed by western blotting.
Uridine 5'-triphosphate (UTP) stimulation
PC12 cells were grown as described
(Rowe et al., 1999). For UTP
stimulation, subconfluent cell cultures (in 35 mm petri dishes) were incubated
for 3 minutes at 37°C in 1 ml of Hepes-buffered external medium
(Krebs-Ringer buffer; KRB) with Ca2+ or without Ca2+ (+2
mM EGTA) in the absence or presence of 300 µM UTP. Cells were then cooled
on ice, scraped in ice-cold homogenization buffer (0.25 M sucrose, 1 mM
Mg-acetate, 10 mM Hepes-KOH, pH 7.4, 2 µg/ml pepstatin and 2 µg/ml
aprotinin), pelleted, and homogenized in 150 µl of homogenization buffer.
The post nuclear supernatants (120 µl) were centrifuged at 200,000
g for 1 hour. The high-speed supernatants (cytosolic
fractions) were collected and solubilized in Laemmli sample buffer (final
volume 180 µl). The pellets (membrane fractions) were resuspended in 180
µl Laemmli sample buffer. Equal volumes of cytosolic and membrane fractions
were then analyzed by western blotting. The levels of NCS-1, PI4Kß or
synaptophysin were quantified by measuring the density of the bands.
Autoradiograms showing the appropriate band intensities were acquired by means
of an ARCUS II scanner (Agfa-Gevaert, Mortsel, Germany) and the density of
each band was quantitated using the NIH Image program 1.61 (National Technical
Information Service, Springfield, VA).
Immunocytochemistry
Immunoelectron microscopy
Adult Sprague-Dawley rats (females, 150 g) were deeply anaesthetised with 2
mg xylazine and 5 mg ketamine and then perfused transcardially with 20 ml of a
solution containing 0.9% NaCl, 0.025% heparin and 2.5% polyvinyl pyrrolidone
40,000, followed by 100 ml of freshly prepared 4% paraformaldehyde and 0.2%
glutaraldehyde in 0.12 M phosphate buffer pH 7.4. The brains were dissected
and cerebral cortices and hippocampi were cut into small pieces of about 1
mm3 and fixed by immersion in the same fixative for 2 hours at
4°C. After fixation, the tissue pieces were extensively rinsed with
phosphate buffer, infiltrated overnight with 2.3 M sucrose in PBS and then
frozen in liquid nitrogen. Ultrathin frozen sections were obtained by using a
Reichert Jung Ultracut E ultramicrotome equipped with a FC4 cryochamber and
collected on Formvar-coated nickel grids. Immunolabeling experiments were
performed as described (Bassetti et al.,
1995). The specificity of staining was tested by substituting
normal rabbit or mouse IgGs for specific antibodies as well as by omitting the
primary antibody and incubating the grids with appropriate secondary
antibody.
Statistical analysis
The density of the gold particles in the perikarya is expressed as the
number of particles per square micrometer of the different organelle areas
measured using the Image 1.61 analysis program. As specified in
Table 1 a number of micrographs
were acquired for each determination using an Arcus II scanner (Agfa-Gevaert).
The data are expressed as mean values±s.e.m. The gold particle
densities at the synapses were evaluated in two different experiments using
the antibody against NCS-1 at a final dilution of 1:100, and detected using a
secondary antibody conjugated to 12 nm colloidal gold particles. Randomly
chosen synapse-rich areas were directly evaluated under the electron
microscope at a magnification of 13,500x. Three hundred synapses were
analysed in samples immunolabeled with anti-NCS-1 polyclonal antibody and 200
in control sections incubated with rabbit IgG.
|
Immunofluorescence
After fixation with 3% paraformaldehyde, cells were processed for
immunofluorescence as previously described
(Rowe et al., 1999). Images
were collected on a MRC-1024 laser scanning microscope (Bio-Rad Laboratories,
Munich, Germany) and analyzed using the Bio-Rad computer software. For
comparison of double-staining patterns, images from the FITC or TRITC channels
were acquired independently from the same area of the sample and then
superimposed.
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Results |
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NCS-1 and PI4Kß subcellular distribution in rat neurons
In order to investigate the possible interaction between NCS-1 and
PI4Kß we decided first to investigate the distribution of both NCS-1 and
PI4Kß in rat brain by subcellular fractionation assays using a procedure
originally designed for the isolation of synaptic vesicles from rat brain
cortices (Huttner et al.,
1983). During this procedure, synaptosomes (P2) were separated
from the homogenates (S1) by differential centrifugation. The supernatants
containing the remaining membrane-bound organelles (S2) were centrifuged at
high speed in order to obtain a cytosolic (S3) and a total membrane fraction
(P3). The synaptic vesicles (SG-V) were partially purified from the
synaptosomal fraction (P2) by hypo-osmotic lysis followed by differential
centrifugation and separation on a continuous sucrose density gradient. The
fractions collected during the different steps were analyzed by western
blotting by using antibodies directed against NCS-1 and PI4Kß. The
effectiveness of the purification procedure was demonstrated by the enrichment
in the synaptic vesicle fraction (SG-V) of the synaptic vesicle membrane
proteins synaptophysin and synaptotagmin
(Fig. 2A). NCS-1 was detected
in the particulate fractions (P3) containing various membrane bound
organelles, other than synaptic vesicles, indicating its widespread
distribution. Furthermore, small but consistent amounts of NCS-1 were
immunodetected in the synaptic vesicles (SG-V,
Fig. 2A) as well as in a highly
purified synaptic vesicle preparation (data not shown). Finally, NCS-1 was
also found in the fractions containing soluble proteins (S3 and LS2,
Fig. 2A), indicating that a
consistent portion of the protein is cytosolic. PI4Kß was also
immunodetected in the fractions containing membrane-bound organelles but a
larger portion of the kinase was detected in the high-speed supernatants (S3
and LS2). A band corresponding to PI4Kß was identified in the synaptic
vesicle fraction (SG-V) but only after long exposure, indicating that only a
small amount of the kinase may be associated with synaptic vesicles.
|
To study the intracellular distribution of NCS-1 and PI4Kß associated with membrane-bound organelles other than synaptic vesicles, we analyzed the high speed pellet P3 by using velocity centrifugation on a 0.3-1.2 M continuous sucrose gradient in order to separate the organelles according to their size. The fractions collected were assayed for the presence of various proteins by western blotting. As shown in Fig. 2B, NCS-1 was distributed in two peaks: the first comprising fractions 1-3 (which contain a larger portion of the protein) and the second comprising fractions 7-8. When the distribution of NCS-1 was compared with that of protein markers for the endoplasmic reticulum (ER, calreticulin and ribophorin), TGN (TGN38) and the plasma membrane (syntaxin 1), we found that NCS-1 was distributed similarly to the markers of the ER in the lighter fractions of the gradient (1-3) and to the TGN marker in the denser fractions (7-8). PI4Kß was also present in the lighter fractions as well as in the denser fractions (fractions 7-8). In contrast, the axonal membrane marker syntaxin 1 was more widely distributed in the gradient than NCS-1, PI4Kß and the markers of ER and TGN (Fig. 2B).
Under our experimental conditions, however, TGN38 was not only detected in
the denser fractions, which are expected to contain large organelles
(Tooze and Huttner, 1990), but
also at the top of the gradient. This altered distribution may be due to
fragmentation of the fragile reticular structure of the TGN into smaller
vesicles, which may have occurred during the resuspension of P3 before
centrifugation. Therefore, the organelles contained in the S2 fraction
obtained after differential centrifugation were separated by discontinuous
gradient centrifugation. After centrifugation, the two bands at the 0.8-1.1
and 1.1-1.2 M sucrose interface and the pellet were collected and analyzed by
immunoblotting with antibodies against NCS-1, PI4Kß and markers for the
ER (ribophorin), Golgi cisternae (GS-28) and TGN (TGN38). The results of
western blots demonstrated that adequate fractionation was achieved: Golgi
cisternae were mainly localized in fraction 1 (0.85-1.1 M interface), TGN
membranes were enriched in fraction 2 (1.1-1.2 M interface) and the ER
membranes were almost exclusively found in the pellet
(Fig. 2C). NCS-1 and PI4Kß
were clearly present in fractions containing TGN and ER marker proteins
(Fig. 2C).
Immunoelectron microscopy studies
To further analyze the subcellular distribution of NCS-1 in neurons, we
performed high resolution immunoelectron microscopy studies on cryosections
using colloidal gold labeling. Ultrathin frozen sections from rat brain
cortices and hippocampi were immunolabeled using the specific NCS-1 polyclonal
antibody followed by anti-rabbit IgG antibodies conjugated to gold particles.
Immunostaining for NCS-1 was detected in the majority of neurons with gold
particles being mainly observed in the perikarya. NCS-1 staining was dispersed
in the cytoplasm and partially associated with the membrane of vesicular-like
structures and stacks of tubular-like cisternae (108 gold
particles/µm2; Fig.
3A,B; Table 1). In
order to characterize this compartment, double immunolabeling was performed
using antibodies as markers for different organelles. Double immunolabeling
with a monoclonal antibody directed against the luminal ER protein PDI
demonstrated the localization of NCS-1 near to and on the membrane of ER
cisternae (Fig. 3C,D). NCS-1
was also localized in the proximity of the Golgi complex area
(Fig. 4A), and some gold
particles were detected on a reticular-like structure that, by using
double-immunolabeling with anti-TGN38 antibodies, was identified as TGN
(Fig. 4D). No significant
labeling was observed on the plasma membrane (not shown), mitochondria,
lysosomes or nuclei (Table 1;
Figs 3,
4). Under control conditions
(rabbit IgGs or no primary antibodies) very few gold particles were detected
on the ultrathin sections (Table
1; Fig. 4B).
|
|
When NCS-1 immunostaining was analyzed in the synaptic regions, not all of the synapses observed were equally immunolabeled. About 40% of the synaptic profiles examined (n=300) were positive for NCS-1, with significant labeling ranging from three to ten 12 nm gold particles/synaptic bouton against the background of less than two gold particles/bouton (Fig. 5A). In the presynaptic compartment (significant labeling ranging from two to ten gold particles/presynaptic region; Fig. 5B), NCS-1 was observed in the cytosolic matrix, near small synaptic vesicles, and also on the synaptic vesicle membranes (Fig. 6A-C).
|
|
We next analyzed the intracellular distribution of PI4Kß by
immunoelectron microscopy. Some PI4Kß immunoreactivity was detected in
dendrites and in perikarya but very few, if any, gold particles localized over
the Golgi complex (data not shown). This result suggested that either the
immunogold labeling was not sensitive enough to detect the kinase localized on
the membranes or, more likely, that the anti-PI4Kß antibodies were
working less efficiently in glutaraldehyde-fixed tissue. Therefore, we decided
to analyze the intracellular distribution of PI4Kß by confocal
immunofluorescence in cultured hippocampal neurons fixed with 3%
paraformaldehyde. Under this condition PI4Kß immunoreactivity was very
intense throughout the perikarya and dendritic trees
(Fig. 7) but barely detectable
in the axon and axon terminals. No colocalization with the presynaptic marker
synaptobrevin 2 was detected (Fig.
7D). In the perikarya, PI4Kß immunoreactivity had a clustered
perinuclear distribution that colocalized with the immunostaining of the
Golgi-marker TGN38 (Fig. 7B).
Moreover, both in perikarya and dendrites the kinase showed a patchy/punctate
signal that partially colocalized with the immuno-signal of the ER marker PDI
(Fig. 7C). These results are in
line with our biochemical data and with previous immunocytochemical studies in
rat brain neurons showing the partial distribution of PI4Kß at the Golgi
complex as well as at the membranes of the ER
(Balla et al., 2000).
|
In conclusion, the subcellular fractionation and immunocytochemical data indicate a similar widespread distribution of NCS-1 and PI4Kß in neurons. Both proteins are partly cytosolic, whereas the membrane-bound portions are localized to the ER and the TGN with only minor amounts present on synaptic vesicles.
NCS-1 interacts with PI4Kß
Although NCS-1 and PI4Kß have been shown to form a complex in vitro
and, after overexpression in epithelial Madin-Darby canine kidney and Cos-7
cells (Weisz et al., 2000;
Zhao et al., 2001
), it was
still unclear whether endogenous NCS-1 and PI4Kß interact in neuronal
cells under physiological conditions
(Bartlett et al., 2000
).
Therefore we examined whether the two mammalian proteins can be
coimmunoprecipitated from neurosecretory cell extracts. We incubated rat brain
and PC12 cells extracts with anti-NCS-1 antibodies, anti-PI4Kß antibodies
or rabbit IgGs and analyzed the immunoprecipitated proteins on western blots
by using antibodies against NCS-1 or PI4Kß. As shown in
Fig. 8, NCS-1 and PI4Kß
were specifically coimmunoprecipitated. When non-immune rabbit IgGs were used
for immunoprecipitation purposes, no specific bands were detected.
|
We next examined whether the interaction between NCS-1 and PI4Kß
occurs preferentially in the cytosol or in membranes. PI4Kß was
co-precipitated with NCS-1 from both rat brain cytosol (high speed
supernatants) and membrane fractions (Fig.
9A). To analyze whether the interaction between the two proteins
could be modulated, coimmunoprecipitation experiments were carried out in the
absence or presence of Ca2+
(Fig. 9B). Only a very small
increase in the amounts of both NCS-1 and PI4Kß was observed in
immunoprecipitates performed in the presence of 1 mM Ca2+ compared
with those performed in the presence of EGTA. Thus, NCS-1 and PI4Kß
specifically interact with each other in both cytosol and membrane fractions
and, as described in yeast (Hendricks et
al., 1999), Ca2+ does not increase the binding of NCS-1
to PI4Kß.
|
NCS-1 and PI4Kß membrane recruitment following UTP
stimulation
It has been suggested that increases in cytosolic Ca2+ may
influence the translocation of NCS-1/PI4Kß to membranes
(Meyer and York, 1999). Since
our data demonstrating the presence of a NCS1/PI4Kß complex in the
cytosol strengthen that hypothesis, we investigated the possible membrane
recruitment of NCS-1 and PI4Kß using the PC12 neuroendocrine cell line as
a model system. The distribution of NCS-1 in cytosol and membrane fractions
was analyzed upon stimulation with 300 µM UTP, a G-protein-coupled receptor
agonist known to induce a rise in [Ca2+]i, an increase in
polyphosphoinositide metabolism and concomitant release of dopamine from PC12
cells (Raha et al., 1993
;
Murrin and Boarder, 1992
;
Koizumi et al., 1995
). Upon
stimulation, membrane and cytosol fractions were separated as described in
Materials and Methods, and equal volumes of both cytosolic and membrane
fractions were analyzed by immunoblotting
(Fig. 10B). Quantitative
analysis of the blots obtained from non-stimulated cells revealed that
68.4±2.6% (n=7) of total NCS-1 was found in the membrane
fractions and 31.6±2.6% in the cytosolic fractions
(Fig. 10A), which is similar
to previous results (McFerran et al.,
1999
). On the contrary, upon stimulation with UTP about 95%
(94.9±0.6, n=7) of the total NCS-1 was detected in the
membrane fractions. Recruitment of NCS-1 to membranes was also observed after
ATP treatment (data not shown). This process appeared to be dependent on
extracellular Ca2+ since upon treatment with UTP or ATP (data not
shown), in the absence of extracellular Ca2+,
66%
(65.7±5.4, n=3) of total NCS-1 was detected in the membrane
fractions (Fig. 10A). To test
for the specificity of the membrane translocation of NCS-1 after UTP
treatment, we analyzed the cytosol and membrane distribution of tubulin in the
same blots. As shown in Fig.
10B, UTP treatment did not affect the cytosolic localization of
the cytoskeletal protein that always appeared only in the cytosolic fractions.
Together, these results indicate that stimulation of nucleotide receptors
induces the translocation of NCS-1 to membranes.
|
We next determined the effects of UTP-treatment on the membrane distribution of PI4Kß. As found for NCS-1, UTP treatment affected the membrane translocation of PI4Kß in a Ca2+-dependent way (Fig. 11A). Since at steady state the largest amount of PI4Kß was soluble, it was difficult to compare in control samples the levels of kinase in the cytosolic fractions with those in the membrane fractions because of rapid saturation reach by NCS-1 signals in autoradiograms of control cytosolic fractions. Therefore we quantified and compared the level of enzyme present in the membrane fractions of control or UTP-stimulated PC12 cells. Equal aliquots of membrane fractions were analyzed by immunoblotting using antibodies directed against PI4Kß or synaptophysin (an integral membrane protein of synaptic vesicles completely recovered in the membrane fractions; not shown). The PI4Kß signals were normalized to those of synaptophysin in the same blots and the data of multiple experiments were averaged. As shown in Fig. 11B, levels of PI4Kß in the membranes after UTP treatment were about two- to threefold higher than those detected in the untreated cells or cells upon UTP treatment in the absence of extracellular Ca2+.
|
We then analyzed whether overexpression of NCS-1 may modulate the membrane translocation of PI4Kß. PC12 cells were transfected with cDNAs coding for NCS-1 and GFP or GFP alone, respectively. Cells were used for biochemical analysis when the transfection efficiency (determined in live cells by counting the percentage of GFP-expressing cells per petri dish) was about 50%. Cytosol and membrane fractions were separated and analyzed by western blotting. In overexpressing cells, the levels of NCS-1 associated with the membrane fractions were largely increased (at least ten times more than in control cells, Fig. 12B). When PIK4ß associated with the membrane was quantified, the levels of the kinase detected in the membrane fractions of NCS-1-overexpressing cells were five- to six-times higher than those detected in GFP-transfected cells (Fig. 12A). In NCS-1-overexpressing cells, UTP-treatment was unable to further induce an increase in the levels of PI4Kß (Fig. 12A) as well as of NCS-1 (data not shown) in the membrane fractions, suggesting that the membrane-binding sites for NCS-1 and/or PI4Kß were saturated.
|
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Discussion |
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The following main findings are reported: (1) NCS-1 and PI4Kß show a similar widespread subcellular distribution; (2) endogenous NCS-1 interacts with PI4Kß to form a complex that is immunoprecipitated from rat brain and PC12 cell extracts using anti-NCS-1 or anti-PI4Kß antibodies; (3) the NCS-1/PI4Kß interaction occurs in membranes as well as in the cytosol; and (4) stimulation of regulated secretion facilitates the translocation of NCS-1 and PI4Kß from cytosolic to membrane fractions, suggesting a role for NCS-1 in the recruitment of the kinase onto target membranes.
NCS-1 and PI4Kß show a similar widespread subcellular
distribution in neurons
Although limited immunohistochemistry and in situ hybridisation data
indicate that NCS-1 is expressed in many regions of the brain
(Jeromin et al., 1999;
Martone et al., 1999
;
Paterlini et al., 2000
;
Werle et al., 2000
), less is
known about its subcellular localization. We used subcellular fractionation
and immunoelectron microscopy to investigate the subcellular localization of
NCS-1 and both methods revealed that it is partially cytosolic and partially
associated with different membrane-bound organelles. NCS-1 was distributed in
several (but not all) synaptic boutons, where it was also localized on
synaptic vesicles. The presence of NCS-1 in a subpopulation of synapses may be
related to the different physiological properties of these synapses.
Interestingly, in crustacean neuromuscular junctions, NCS-1 appears more
predominantly expressed in the phasic nerve terminals that are capable of
releasing more neurotransmitter at low frequencies than their tonic
counterparts (Jeromin et al.,
1999
). Our results show that most NCS-1 was distributed in the
perikarya, where it could be detected near to or associated with the membranes
of the ER and the TGN. A consistent amount of NCS-1 was also found in
dendrites. Although the physiological significance of this complex NCS-1
distribution is unclear, the absence of any specific compartmentalisation
suggests that it may have a general function in controlling signaling and/or
vesicle trafficking events.
Although PI4K activity has been detected in many cellular compartments
including plasma membrane (Cockcroft et
al., 1985), secretory granules
(Wiedemann et al., 1996
),
synaptic vesicles (Wiedemann et al.,
1998
), Golgi (Jergil and
Sundler, 1983
; Cockcroft et
al., 1985
) and glucose transporter 4-containing transport vesicles
(Del Vecchio and Pilch, 1991
),
little is known about the subcellular localization of PI4K proteins in neurons
and neuroendocrine cells (Balla et al.,
2000
). Different types of PI4Ks have been cloned and characterized
from yeast and mammalian cells (Balla,
1998
; Fruman et al.,
1998
; Nakagawa et al.,
1996a
; Nakagawa et al.,
1996b
; Meyers and Cantley,
1997
) and it is clear from these studies that different isoforms
of the PI4K family may synthesize different pools of polyphosphoinositides
that in turn modulate different cellular functions
(De Camilli et al., 1996
;
Anderson et al., 1999
;
Balla, 2001
;
Cremona and DeCamilli, 2001
).
The localization of distinct kinase isoforms to specific sites in cellular
compartments helps to explain the different roles that phosphoinositides play
in cells. Therefore we have characterized the distribution of PI4Kß and
compared it with that of NCS-1. Although PI4Kß is mainly enriched in the
cytosolic fraction after rat brain differential centrifugation, our results
demonstrate that PI4Kß is also present on membranes of the ER and the
late Golgi complex. This latter result is consistent with previous data
obtained in mammalian cell-free systems and yeast cells, respectively
(Godi et al., 1999
;
Walch-Solimena and Novick,
1999
; Audhya et al.,
2000
), which showed that PI4Kß plays an essential role in
Golgi complex organization and protein secretion. Our data indicate that
PI4Kß is also associated with synaptic vesicles, albeit in very small
amounts and, to our knowledge, this is the first report showing the physical
presence of an isoform of the PI4K family on synaptic vesicles. However, the
small amount of PI4Kß associated with the synaptic vesicles suggests that
this enzyme might not be the only PI4K isoform responsible for the PI4K
activity detected on synaptic vesicles
(Wiedemann et al, 1998
). In
addition, or alternatively, other kinases may be involved since several PI4K
activities have been characterized and recently a PI4K type II has been
discovered on secretory granule membranes
(Barylko et al., 2001
;
Wenk et al., 2001
).
Interaction of NCS-1 with PI4Kß and its possible function
The observed presence of both proteins on membrane-bound organelles,
together with the observed interaction of NCS-1 with PI4Kß strongly
suggests that NCS-1 could function in regulating PI4Kß activity and/or
localization. Recent data have described that myristoylated NCS-1, but not its
unmyristoylated form, is capable of stimulating the kinase activity of
recombinant PI4Kß in vivo (Balla,
2001). Similarly, a significant increase in PI4K activity was
observed when both NCS-1 and PI4Kß were exogenously expressed in COS-7
cells (Zhao et al., 2001
).
Thus the molecular interaction with NCS-1 appears to be important for
activation of the kinase. Less data are available on the possible role of
NCS-1 in the targeting of PI4Kß. Our results showing that endogenous
NCS-1 and PI4Kß can form a complex in the cytosol and that both proteins
can be translocated to membranes upon stimulation of exocytosis strongly
suggest that NCS-1 modulates the translocation of PI4Kß to membranes. Two
mechanisms by which NCS-1 may function in PI4Kß membrane localization or
activation have recently been proposed
(Meyer and York, 1999
). In the
first model, NCS-1 is prelocalized on the membrane and a Ca2+
induced-conformational change triggers its interaction with and activation of
a membrane-pre-bound pool of PI4Kß; in the second, Ca2+
binding triggers the exposure of the myristoyl group and the concomitant
translocation of the NCS-1/PI4Kß complex to the membrane. Our present
data showing that the NCS-1/PI4Kß complex is also present in the cytosol
and that both proteins are translocated to membranes upon a secretory stimulus
support the second mechanism.
At steady state, PI4Kß is mainly found in the cytosol and may thus
require protein-carrier(s) to be translocated to possible sites of action.
Recent studies have demonstrated that the small GTP-binding protein ARF-1 is
involved in the translocation of PI4Kß to Golgi membranes
(Godi et al., 1999), where the
PI4K activity is important for cisternae organization and constitutive protein
trafficking from the Golgi to the cell surface. However, at present no ARF
proteins have been shown to be concentrated at the synapse
(Cremona and De Camilli,
2001
). We suggest that NCS-1, which is mainly expressed in
neuronal and neuroendocrine cells, may modulate the localization and activity
of PI4Kß and thereby the level of a phosphoinositide pool required for
intracellular trafficking events that specifically occur in neurosecretory
cells. Neurons and neuroendocrine cells express two types of secretory
vesicles specialized in storage and release of neurotransmitters: the dense
core vesicles (or secretory granules) produced from the TGN (for reviews, see
Eaton et al., 2000
;
Tooze et al., 2001
), and the
small synaptic vesicles (or synaptic-like microvesicles) that are derived from
a recycling compartment near, or at, the plasma membrane
(Hannah et al., 1999
;
Slepnev and De Camilli, 2000
).
Upon regulated exocytosis and consumption of either secretory vesicles, the
NCS-1/PI4Kß complex may be translocated and stimulate the synthesis of
phosphoinositide pools involved in the generation of neurosecretory vesicles.
In line with this model, we found that after NCS-1 overexpression in PC12
cells, the amount of membrane-associated PI4Kß increases concomitantly
with the increase of NCS-1 in the membrane fractions. Moreover, NCS-1
overexpression enhances ATP-stimulated release from PC12 cells
(McFerran et al., 1998
) and
the levels of phosphatidylinositol 4-phosphate and phosphatidylinositol
(4,5)-bisphosphate in PC12 cells (Koizumi
et al., 2002
). Although further work is required to identify the
membrane targets of NCS-1/PI4Kß and to fully understand their role in
membrane traffic, our data demonstrate that NCS-1/PI4Kß are translocated
to membranes and suggest that the two proteins may regulate the levels of
phosphoinositides involved in regulated secretion processes; for example, by
modulating the formation of neurosecretory vesicles. This action could lead to
downstream effects on neurosecretion, and in turn could also be modulated by
Ca2+ and thus by the secretory activity of the cells.
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Acknowledgments |
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References |
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