1 INSERM U-442, Signalisation cellulaire et calcium, Bât 443,
Université Paris-Sud, 91405 Orsay Cedex, France
2 Department of Anatomy & Developmental Biology, University College London,
Gower Street Campus, London WC1E 6BT, UK
3 Laboratoire de Neurogenése et Morphogenése dans le
Développement et chez l'Adulte (NMDA, Unité Mixte de Recherche
6156), Institut de Biologie du Développement de Marseille,
Faculté des Sciences de Luminy, Université de la
Méditerranée, case 907, 13288 Marseille Cedex 09, France
Author for correspondence (e-mail:
jean-pierre.mauger{at}ibaic.u-psud.fr)
Accepted 14 March 2003
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Summary |
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Key words: Inositol 1,4,5-trisphosphate receptor, Tight junction, Polarity, Calcium, Endoplasmic reticulum, InsP3R-1-EGFP
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Introduction |
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The localization of these elementary events to specific sites indicate that
the Ca2+ stores and the proteins responsible for the
Ca2+ release are highly organized into specific domains
(Meldolesi and Pozzan, 1998).
Ca2+ is actively pumped by the SERCA proteins into endoplasmic
reticulum (ER) stores where it binds to a host of resident proteins in order
to buffer the luminal free Ca2+. In response to specific signals,
Ca2+ is quickly released from these intracellular stores through
channels. The most ubiquitous intracellular Ca2+ channel is the
inositol 1,4,5-trisphosphate receptor (InsP3R) which releases
Ca2+ in response to InsP3 produced under agonist
stimulation (Berridge,
1993
).
Three InsP3R sub-types have been identified and are expressed at
various levels among cell types (Blondel et
al., 1993; Furuichi et al.,
1989
; Maranto,
1994
; Südhof et al.,
1991
). The InsP3R subtypes assemble into tetramers to
form functional channels and are differently distributed throughout the
cytoplasm according to cell type (Joseph,
1996
; Taylor and Traynor,
1995
). In neuronal cells, InsP3Rs are associated with
the ER membrane and evenly localized throughout the cytoplasm comprising the
cell body, the perinuclear area, the dendrites and the axon
(Pozzan et al., 1994
).
Similarly, in oligodendrocytes (Simpson et
al., 1997
), HeLa cells (Thomas
et al., 2000
) and rat leukemia cells
(Wilson et al., 1998
), the
various InsP3R subtypes show a similar fine reticular distribution
throughout the cytoplasm. The formation of receptor clusters dispersed along
the ER network has been described in several cell types
(Wilson et al., 1998
) and may
correspond to cell regions that are more responsive to small increases in
InsP3 (Sheppard et al.,
1997
). In contrast, polarized epithelial cells, in particular
exocrine glandular cells or liver cells, are characterized by a distribution
of InsP3R restricted to specific areas. Subcellular fractionation
experiments with these cells have shown that the receptor does not always
co-purify with ER markers but rather with markers of the plasma membrane
(Lièvremont et al.,
1994
; Sharp et al.,
1992
). Immunocytochemical experiments have indicated that the
InsP3R is mainly concentrated at the lateral pole of polarized MDCK
cells (Bush et al., 1994
;
Zhang et al., 2003
) and near
the apical pole of other polarized cells such as hepatocytes,
(Dufour et al., 1999
;
Nathanson et al., 1994a
),
cholangiocytes (Hirata et al.,
2002
), pancreatic acinar cells and parotid cells
(Lee et al., 1997
;
Nathanson et al., 1994b
;
Yule et al., 1997
). In
pancreatic acinar cells, the three receptor sub-types displayed the same
sub-apical localization, indicating that this pattern of localization does not
depend on the receptor sub-type. These observations strongly suggest that the
subcellular localization of the InsP3R plays an important part in
determining the spatio-temporal characteristics of the Ca2+ signal
that regulates a particular Ca2+-dependent cellular function.
Although the pattern of InsP3R localization in polarized
epithelial cells is now well documented, very little is known about the
mechanisms that establish it, and the first step in studying this, was to find
an adequate polarized cell model. Such a model must fulfill several criteria:
its polarized phenotype must be stable and its polarization easy to follow and
quantify; experimental conditions that permit to disturb and restore polarity
must be available. Finally, the chosen cells must be easy to transfect,
because one of our long term aims, is to perform in vivo dynamic studies,
using cells stably expressing different versions of GFP tagged
InsP3R. Despite our previous work in liver cells study
(Decaens and Cassio, 2001;
Lièvremont et al.,
1996
) and the fact that the restricted InsP3R
localization in MDCK cells seems to differ from that in other epithelial cells
(lateral versus subapical), we have chosen to first investigate this
non-tumorigenic cell line (i.e.MDCK) because it readily fullfills all the
criteria and is by far the best known polarized cell model. In the present
work, the distribution of the InsP3R type 3 (InsP3R-3),
the most abundant InsP3R expressed in MDCK cells, was followed
during MDCK polarization and also when the polarity was disturbed and
restored. We also isolated and examined MDCK cells stably expressing a
full-length version of InsP3R type 1 tagged with EGFP
(InsP3R-1-EGFP). A clear correlation between InsP3R
distribution and polarity was found. In non-polarized cells, endogenous
InsP3R-3 and InsP3R-1-EGFP were distributed in the whole
cytoplasm and co-localized with other markers of the ER. In contrast, in
polarized cells, both receptor types are mostly distributed at the apex of the
lateral pole with tight junction markers.
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Materials and Methods |
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Cell culture, transepithelial resistance and calcium switch
Madin-Darby canine kidney (MDCK) cells or ras-transformed MDCK f3 cells,
(Hordijk et al., 1997) were
cultured on plastic culture Petri dishes (Falcon), glass coverslips (CML,
France) or inserts (Transwell filters, 30 mm diameter, 0.4 µm pore size,
Falcon) in F-12 Coon's-modified medium (Sigma-Aldrich) supplemented with 5%
FCS (Gibco BRL Invitrogen, Cergy Pontoise, France), 100 units/ml penicillin,
100 µg/ml streptomycin sulfate and 0.25 µg/ml fungizone (Gibco BRL) at
37°C in a humidified atmosphere with 7% CO2. These cultures
were found to be Mycoplasma negative using the Mycoplasma PCR ELISA
(Roche Applied Sciences, Meylan, France). Cells were confluent, and
well-polarized (except f3 cells), 10-12 days after plating at 104
cells/cm2. MDCK cells were subcloned at 2-20 cells/cm2
with a cloning efficiency of 60-90%. Chromosome content was determined as
previously described (Bender et al.,
1999
).
Transepithelial resistance (TER) was measured with a Millicel apparatus (Millipore). All TER values were obtained using at least three inserts (6-8 measurements per insert at each time) after background subtraction (i.e. filter and solution).
Calcium switch experiments were performed using calcium-free F12 Coon's
modified medium (Eurobio, Les Ulis, France), without serum. Well-polarized
MDCK cells, grown on glass coverslips or on inserts were rinsed three times
and switched in calcium-free medium ([Ca2+]e 5 µM). During a
2 hour period, TER was recorded and cells fixed for immunostaining at regular
intervals. Cells were then switched back to complete medium containing 1.12 mM
CaCl2, and at regular intervals cells were fixed and the TER
recorded.
Plasmid construction, transfection and isolation of stable
transfectants
The expression plasmid coding for the InsP3R-1-GFP chimera was
constructed using the full-length cDNA of the mouse InsP3R type 1
(Furuichi et al., 1989). This
cDNA was first subcloned from pRC/CMV vector (supplied by D Ogden, NIMR, Mill
Hill, London, UK) into the XbaI site of pBluescript SK (Stratagene).
The stop codon at the end of the InsP3R-1 coding sequence was
removed by cutting with AvrII and SalI, followed by Mung
Bean nuclease treatment to create blunt ends and then these ligated together.
The XbaI-XhoI cDNA fragment of this vector was inserted into
the NheI-XhoI site of pEGFP-N1 (Clontech) to create
pInsP3R-1-EGFP. The authenticity of the plasmid was verified by
automated nucleotide sequencing using a Perkin Elmer machine (Genome Express,
France). pInsP3R-1-EGFP codes for a chimera that is composed of the
full-length mouse InsP3R-1 attached, via a 22 amino acid linker at
its C-terminal, to EGFP.
For transfection, 5x102 cells/cm2 were seeded the day before in 6-well plates (Falcon) and transfected with 1-2 µg DNA using Superfect reagent (Qiagen). MDCK cell populations expressing InsP3R-1-EGFP or the host plasmid pEGFP-N1 were selected with 600 µg/ml G418 (Gibco BRL) 3 days after transfection, and subcloned 3 weeks later at 2 cells/cm2. Independent colonies (39 and 15 from the cell populations transfected with pInsP3R-1-EGFP and the empty pEGFP-N1, respectively) were isolated, and characterized. A few colonies well expressing the transfected gene were retained, frozen two months after transfection and studied. They were always cultured with 600 µg/ml G418, and their chromosomal content as the absence of mycoplasma were tested.
SDS-PAGE and immunoblot analysis
Cells were grown in 10 cm Petri dishes. At appropriate times, cells from
one dish were counted and cells from the remaining ones were rinsed three
times in cold PBS and lysed on ice by scraping with a rubber policeman in
either ice-cold PBS or in 10 mM Tris-HCl (pH 7.5), 5 mM EDTA containing 5
µg/ml antipain, 5 µg/ml aprotinin, 2 mM benzamidine, 1 mM DTT, 10
µg/ml leupeptin, 5 mM NEM, 10 µM pepstatin A, 5 µM 1,
10-phenanthroline, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml soybean
trypsin inhibitor, and 0.02% sodium azide. The protein concentration of the
lysates was determined using BC Assay Uptima (Interchim, France). 30-50 µg
protein/lane were subjected to SDS-PAGE
(Laemmli, 1970). Positive
controls for type 1 and type 3 inositol 1,4,5-trisphosphate receptors were rat
cerebellum membranes (Lièvremont et
al., 1994
) and HeLa cell lysate (Transduction Laboratories),
respectively.
Immunoblotting was performed following a standard method. Briefly, the proteins were electrophoretically transferred from the gel onto a Hybond-C extraTM nitrocellulose membrane (Amersham Pharmacia Biotech) in a Tris-glycine buffer with 20% methanol and 0.04% SDS. The membrane was saturated with PBS (pH 7.5) containing 10% (w/v) nonfat milk and 0.2% Tween 20 for 1 hour at 37°C, then incubated overnight at 4°C with the appropriate dilutions of the primary antibody in PBS-milk-Tween. This was followed by washing and incubation with a horseradish-peroxidase-conjugated secondary antibody (donkey anti-mouse or anti-rabbit Ig, Amersham Pharmacia Biotech) (goat anti-rabbit Ig or sheep anti-mouse Ig, Pasteur, Sanofi, France). The membranes were developed using the ECLTM system (Amersham Pharmacia Biotech) on Biomax ML films (Kodak). Prestained SDS-PAGE Standards (BioRad) were used to calibrate molecular mass.
Immunofluorescence and confocal microscopy
Cells grown on glass coverslips, were rinsed three times with cold PBS
(without Ca2+ and without Mg2+, pH 7.4), and generally
fixed on ice with 2% formaldehyde in PBS for 1 minute and methanol at 4°C
for 10 minutes (Cassio et al.,
1991), except for occludin (30 minutes in 100% ethanol at 4°C
and acetone at -20°C for 1 minute) and sec6 detection (1% Triton X-100 on
ice for 10 minutes and 100% methanol at -20°C for 10 minutes). Cells were
then incubated with single primary antibodies or a mixture of them for 45
minutes at 37°C, washed three times in PBS and incubated (20 minutes at
37°C) with the appropriate Alexa-conjugated secondary antibodies
(Molecular Probes). After three PBS washes, the coverslips were mounted in
PBS-glycerol mounting medium (Sigma) and examined with a Axioskop fluorescence
microscope (Zeiss, Germany).
Confocal microscopy was performed using either a BioRad 600 or a Zeiss 510 confocal microscope and in both cases using a x63 Plan-Apochromatic objective (NA=1.4), oil immersion. Optical sections were collected at 0.2-0.3 µm intervals and the images were processed using Photoshop 5.5 (Adobe) software.
Immunogold electron microscopy
For immunoelectron microscopy, ultrathin frozen sections of MDCK cells were
obtained and processed as described for Caco-2 cells and human colon
(Saito et al., 2001).
Ultrathin cryosections were incubated overnight at 4°C with
anti-InsP3R-3 monoclonal antibody diluted 1:15 or 1:20 in 5% goat
serum in PBS. Primary antibodies were revealed by incubation for 1 hour with
colloidal gold 6 nm-conjugated goat anti-mouse IgG diluted 1:25 in 5% goat
serum in PBS. Sections were examined under a Leo 912 electron microscope.
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Results |
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The localization of this protein was studied during the polarization of
MDCK cells. For this purpose MDCK cells were plated at 104
cells/cm2, and the establishment of polarity was monitored by
measuring the transepithelial resistance (TER), as shown in
Fig. 1B. InsP3R-3
and other ER proteins were localized during this process by indirect
immunofluorescence (Fig. 1C,D).
For InsP3R-3, a mouse monoclonal antibody was used and for ER
proteins two different antibodies were used: one polyclonal directed against a
bulk of ER resident proteins (Louvard et
al., 1982), that permits to double stain MDCK cells for ER and
InsP3R-3 (Fig. 1C)
and one monoclonal antibody specifically directed against protein disulfide
isomerase (PDI) (Fig. 1D). As
shown in Fig. 1C, the
intracellular distribution of InsP3R-3 was different, according to
the polarity state of the cells. In early cultures, when cells were sparse and
not yet polarized (day 1-3, TER less than 60 ohm cm2),
InsP3R-3 displayed a cytoplasmic distribution similar to that of ER
proteins. In contrast, in late cultures (day 10 and later), when cells were
confluent and well-polarized (stabilization of TER at 260 ohm cm2
and formation of domes), InsP3R-3 was mostly concentrated near the
plasma membrane (PM). This change in the localization of InsP3R-3
was progressive, as illustrated in Fig.
1C by the comparison of MDCK cells after 3, 6 and 10 days in
culture. This change was specific for InsP3R-3, and was not
observed for the bulk of ER proteins (Fig.
1C), or PDI (Fig.
1D).
In parallel to the localization of InsP3R-3, we evaluated the cellular content of InsP3R-3 at different times during the polarization of MDCK cells. Fig. 1A shows that the amount of InsP3R-3 (expressed either by mg of protein or by number of cells) was similar at day 3, 6 and 10 suggesting that only the cellular distribution and not the level of InsP3R-3, is modified during MDCK polarization. To examine whether this change affected all MDCK cells, several hundred colonies were examined at different times after cloning. InsP3R-3 was detected in the cytoplasm of all the cells of each colony very soon after cloning, but after a few days it had in all cases become concentrated near the PM. These results indicate that the change in localization of InsP3R-3 during polarization is a general trait of MDCK cells.
InsP3R-3 is concentrated at the tight junction level in
polarized MDCK cells
The distribution of InsP3R-3 in polarized MDCK cells was further
analyzed by confocal microscopy. The protein was first localized using two
antibodies: the monoclonal antibody used above
(Fig. 1C), directed against the
N terminal part (aa 22-230) of human InsP3R-3 and an affinity
purified polyclonal antibody (Sharp et
al., 1999), directed against a synthetic peptide corresponding to
the C-terminal portion of rat InsP3R-3 (aa 2655-2670). An identical
staining was obtained with each antibody. This is illustrated in
Fig. 2, particularly in the
merged image, that shows the distribution of InsP3R-3 in polarized
cells at day 13: as previously shown in
Fig. 1C, the receptor was
detected in clusters in the cytoplasm, and already concentrated along the PM.
This distribution was observed in all cells and was homogeneous over all the
cell population. For the fraction of InsP3R-3 present in the
vicinity of the PM, the staining was not continuous but patchy, and in some
cases one can distinguish a double row of patches
(Fig. 2), that probably
corresponds to InsP3R-3 molecules present on both sides of the
cell-cell contacts.
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To better localize the fraction of InsP3R-3 that is concentrated near the PM in well-polarized MDCK cells, double localizations were performed for InsP3R-3 and proteins specific for different PM domains. We first studied the distribution of the tight junction-associated protein ZO-1 and InsP3R-3 (Fig. 3A). We found that InsP3R-3 was present near the PM, in the same location as that of ZO-1, namely at the frontier between the apical and the basolateral pole. In contrast, the fraction of InsP3R-3 that is not concentrated near the PM was distributed in the whole cytoplasm (Fig. 3A). Concerning the staining near the PM, xy and xz sections shows a clear co-localization of InsP3R-3 and ZO-1, the InsP3R-3 staining being in some cases a little more laterally spread. However InsP3R-3 was never found all-over the lateral domain. To confirm the presence of InsP3R-3 at the tight junction level, polarized MDCK cells were double stained for InsP3R-3 and for the intrinsic tight junction protein, occludin. Fig. 3B shows that the fraction of InsP3R-3 present near the PM co-localized with occludin. Finally, double staining of InsP3R-3 with lateral or apical markers, ErbB2 and gp-114, were also performed (Fig. 3C,D) and once more showed that when MDCK cells were well-polarized, the InsP3R-3 was highly concentrated at the restricted part of the PM corresponding to the tight junction zone.
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To determine the precize localization of InsP3R-3 in polarized MDCK cells immunogoldelectronmicroscopy was used and Fig. 4 shows two representative micrographs. In all cases InsP3R-3 was concentrated in a restricted intracellular location, at the upper part of the cells and in the vicinity of tight junctions. We did not observe gold particles on the apical surface of the PM, nor along the lateral or the basal one. These results strongly suggest that InsP3R-3 in polarized cells is mostly present in a subcompartment of the ER very close to the tight junctions.
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InsP3R-3 is not concentrated near the PM when MDCK
polarity is disturbed
The relationship between MDCK polarization and the localization of
InsP3R-3 was analysed in two different cases where MDCK polarity
was disturbed. We first examined cells of the ras transformed MDCK cell line
f3, that have been shown to be unpolarized and deficient in E-cadherin
expression (Chen et al.,
2000). As shown in Fig.
5A, the localization of InsP3R-3 was similar in early
and late cultures; a cytoplasmic staining was observed with no accumulation of
InsP3R-3 near the PM, even in late confluent cultures. Moreover, as
expected, ZO-1 was not detected and E-cadherin was not expressed
(Fig. 5A). Chen et al. have
reported that the formation of tight junctions and the reexpression of
E-cadherin can be induced in f3 cells, by inhibiting the mitogen-activated
protein kinase kinase (MEK1) (Chen et al.,
2000
). Therefore f3 cells were treated with the MEK1 inhibitor
U0126 (Favata et al., 1998
)
(Fig. 5B). ZO-1 was detected at
the PM of a few percent of cells after 10 hours of treatment and of the
majority of cells after 24 hours. E-cadherin was re-expressed later but only
in a fraction of treated cells (10 and 30% after 24 and 72 hours,
respectively). No change in the localization of InsP3R-3 occurred,
except in a minority (less than 5%) of E-cadherin re-expressing cells, in
which InsP3R-3 was detected near the PM
(Fig. 5B). It should be noted
that after this treatment, the TER value, initially of 40-50 ohm
cm2, reached only 120 ohm cm2
(Fig. 5B) versus 270 ohm
cm2 for non-transformed cells
(Fig. 1B).
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Experiments were then performed on MDCK cells, in order to disrupt cellular junctions by lowering the external calcium concentration. Polarized MDCK cells were placed in calcium-deprived medium for several hours and then placed again in calcium containing-medium. During this calcium switch treatment, the TER was measured and the localization of InsP3R-3 and ZO-1 followed (Fig. 6). A rapid delocalization of InsP3R-3 was found in cells deprived of calcium, the protein no longer being concentrated near the PM, after 30 minutes of treatment. ZO-1 was also internalized but less rapidly: after two hours in calcium-deprived medium, some ZO-1 staining was still present at the PM, although these cells, compared to control ones, were less cohesive, rounded up and had a very low TER (50 ohm cm2). When cells deprived of calcium were switched into calcium-containing medium, polarity was re-established (Fig. 6). This re-establishment is a relatively slow phenomenon (TER was completely restored only after 24 hours) and is accompanied by a relocalization at the PM level of both ZO-1 and InsP3R-3. Whereas the relocalization of ZO-1 occurred rapidly, that of InsP3R-3 was more progressive; most of this protein was present near the PM, only after 8 hours in calcium containing medium, when cells exhibit a TER of 200 ohm cm2 (Fig. 6).
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Stably transfected type 1 InsP3R-EGFP behaves as the
endogenous InsP3R-3 during MDCK polarization
To investigate whether other subtypes of InsP3-R locate to the
tight junction, MDCK cells were tranfected with pInsP3R-1-EGFP, a
plasmid coding for the full-length mouse type 1 InsP3R tagged with
EGFP at the C-terminus. Some forty stable transfectants were isolated. In most
of these clones, only a minor fraction (5-10%) of cells expressed the
transfected receptor. One stable clone called 10B was retained for further
analysis: 70-80% of its cells expressed the transfected InsP3R-1
and, according to the fluorescence intensity, the protein was expressed at a
similar level in the positive cells. The chromosomal content of 10B cells was
similar to that of parental MDCK cells (mean chromosome number 74 for both
lines with a range of 60-80 and 66-77 for 10B and MDCK, respectively). MDCK
cells were also transfected with pEGFP-N1, and the stable transfectant clone
1E was analyzed to investigate the effect of EGFP on its own. EGFP was
strongly expressed by all cells of clone 1E, with a broad localization
(cytoplasm and nucleus) whatever the polarity state of the cells (results not
shown).
The type and the level of the InsP3R expressed by the two stable transfectants and their MDCK parent was analyzed by western blot using antibodies against InsP3R-1, GFP and InsP3R-3, respectively (Fig. 7A). InsP3R-3 was present in similar amounts in lysates of each clone. Using an antibody anti-type 1 receptor a robust band migrating at the same level as the cerebellar type 1 receptor, was detected only in 10B lysate (Fig. 7A). When blots were probed using an anti-GFP antibody a similar high molecular weight band was present only in 10B cells whereas, as expected, a band with an apparent molecular weight of 30 kDa was detected for the 1E lysate (Fig. 7A). This data shows that the isolated transfected MDCK clones, correctly express the EGFP exogenous proteins introduced by transfection.
The endogenous InsP3R-3 and the transfected
InsP3R-1-EGFP were localized during the polarization of 10B cells.
As shown in Fig. 7B, both
InsP3R displayed the same localization. In 3-day cultures, when
cells are non-polarized, a cytoplasmic staining was observed, as previously
shown in non-transfected MDCK cells for InsP3R-3
(Fig. 1). In late cultures (10
days), when cells are well-polarized, both types of InsP3R were
concentrated near the cell PM (Fig.
7B). This change in localization was observed in all 10B cells
expressing type 1 InsP3R. Confocal analysis was performed to refine
the localization of the transfected InsP3R-1-EGFP
(Fig. 8). In non-polarized
cells (Fig. 8A), this protein
was distributed in the cytoplasm in a similar fashion to that of ER proteins,
with a slightly more intense perinuclear localization for the
InsP3R-1-EGFP (visible when both images were merged). However, in
polarized cells most of InsP3R-1-EGFP was concentrated at the
periphery of the cells (Fig.
8A), where it co-localized with the endogenous type 3 receptor at
the tight junction level (Fig.
8B). The localization in the tight junction zone of
InsP3R-1-EGFP in 10B polarized cells was confirmed by localizing
this protein with ER markers (Fig.
8C), with lateral markers such as cadherin(s) recognized by the
pan cadherin antibody (Fig. 8D)
or with the exocyst component sec6 (Fig.
8E), known to be recruited at the tight junction in MDCK cells
(Grindstaff et al., 1998).
Moreover the concentration of the transfected type 1 InsP3R-EGFP in
the tight junction area was also seen in living polarized 10B cells
(Fig. 8F). These results
demonstrate that the presence of InsP3R at the tight junction level
in polarized MDCK cells is a general feature, at least for the type 3 and
EGFP-type 1.
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Discussion |
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Changes of InsP3R localization and MDCK polarity
Western blotting experiments indicated that the type 3 InsP3R is
easily detected in MDCK cells whereas the type 1 and the type 2 receptors are
barely detectable. We focused our study on InsP3R-3 and found that
it was redistributed during the development of MDCK polarity. In non-polarized
cells, InsP3R-3 was detected in the cytoplasm and co-localized with
different markers of the ER. In contrast, in polarized cells
InsP3R-3 was highly concentrated near the PM. Confocal microscopy
allowed us to clearly show that InsP3R-3 localized with tight
junction markers and not with lateral markers such as ErbB-2
(Fig. 3) cadherins or caveolin
(not shown). This localization was confirmed by immunoelectron microscopy. To
our knowledge, this is the first description of such a distribution of
InsP3R. The tight junction-like localization has been observed with
two different antibodies directed against different epitopes of the
InsP3R-3 and furthermore, a full-length InsP3R-1-EGFP
fusion protein stably expressed in MDCK cells displayed the same
distribution.
Other works using indirect immunofluorescence with antibodies directed
against InsP3R purified from rat cerebellum or against a peptide
sequence of mouse InsP3R-1 indicated that this receptor type
localized to the basolateral domain of the plasma membrane of polarized MDCK
cells (Bush et al., 1994;
Zhang et al., 2003
). This
suggests that InsP3R-1 and InsP3R-3 translocate to
different domains of the plasma membrane during the polarization of the MDCK
cells. However, in the present work, we have found that
InsP3R-1-EGFP co-localizes with InsP3R-3 at the tight
junction level. The discrepancy between our results and those previously
published could be due to differences between the analysed cell populations
which would differently express the protein(s) required for
InsP3R-1 translocation. Alternatively, it is possible that the
presence of EGFP at the C-terminal end of the InsP3R-1 could
inhibit the translocation of the receptor to the basolateral membrane and this
would be sufficient to permit receptor recruitment at the tight junction
level, suggesting that InsP3R-1 and InsP3R-3 have common
determinant(s) for translocation to the tight junction.
The fact that the localization of InsP3R-3 depends on the cell polarity state was confirmed by studying MDCK cells presenting polarity perturbations: calcium-deprived cells and unpolarized f3 ras transformed ones. In both cases InsP3R-3 was distributed in the cytoplasm. Calcium addition to calcium-deprived cells induces the re-establishment of polarity and the relocalization of the receptor at the tight junction level. However this relocalization was slow and occurred only when the polarity was well re-established (TER > 200 ohm cm2). The addition of MEK1 inhibitor, U0126, to f3 cells allows a partial recovery of TER, the relocalization of ZO-1 at the PM, but not that of InsP3R-3. These results suggest that the restricted distribution of InsP3R-3 at the frontier of the lateral and apical pole requires the formation of fully constituted tight junctions. Thus, InsP3R-3 recruitment would be a late event in the scaffolding of tight junctions and InsP3R-3 could be a peripheral component of tight junctions rather than an intrinsic one. This hypothesis is in agreement with the observation that InsP3R-3 is very rapidly redistributed in the cytoplasm when polarized cells are switched in calcium-deprived medium. The InsP3R-3 redistribution during the Ca2+ switch and the immunoelectron microscopy pictures showing that, in polarized cells, this receptor is localized intracellularly rather than in the plasma membrane itself, strongly suggest that during the polarization of the cells, the InsP3R concentrated in a subdomain of the ER closely associated to the tight junctions. Owing to the generation of MDCK clones expressing fluorescent InsP3R versions, as the 10B clone described in this report, it will be possible to follow in living cells the localization changes of InsP3R and thus to approach the mechanisms implicated in these changes.
Interactions of InsP3R with proteins
The tight junction-like restricted distribution of InsP3R in
polarized MDCK cells opens several questions. The first one concerns this
peculiar localization compared to the subapical distribution observed in other
epithelial cells. In fact similar results were previously obtained for the
sec6/8 complex. This complex is localized at the apical pole of pancreatic
acinar cells (Shin et al.,
2000), whereas in the case of MDCK, it is found in the cytosol of
non-polarized cells and at the apex of the lateral membrane in polarized ones
(Grindstaff et al., 1998
).
Moreover the sec6/8 complex behaves exactly as InsP3R in MDCK cells
submitted to the calcium shift (Grindstaff
et al., 1998
). This similarity could reflect a close interaction
between the two systems. The next question concerns the association and the
interactions of InsP3R with other proteins that would form an ER
subdomain in close association with tight junctions. What is the molecular
basis of this association? What are the proteins that interact with this
receptor? These questions will be approached by determining the regions of
InsP3R implicated in its recruitment in the tight junction area and
by looking for possible interactions in polarized cells between
InsP3R and several proteins, in particular tight junction
associated ones or tight junction components.
Interactions between InsP3R and several proteins have been
previously described. InsP3R has been found to be closely
associated with the actin network in different cell types and to interact with
ankyrin allowing a linkage with the cytoskeleton
(Joseph and Samanta, 1993;
Rossier et al., 1991
). A study
with ankyrin-B-deficient mice indicated that ankyrin-B is required for
intracellular targeting of Ca2+ homeostasis proteins including
InsP3R (Tuvia et al.,
1999
). In vivo and in vitro interactions between the type 1
InsP3R and 4.1N, a homolog of the erythrocyte membrane cytoskeleton
protein 4.1 have been recently reported
(Zhang et al., 2003
).
InsP3R is also involved in communication between the ER and
proteins of the plasma membrane. There is evidence that Homer proteins form a
physical tether linking metabotropic glutamate receptors with the
InsP3R; this link could contribute to the regulation of
intracellular calcium and long-term neuronal plasticity
(Tu et al., 1998
).
InsP3R also interacts with TRP Ca2+ channel and this
interaction plays an important role in the stimulation of Ca2+
entry from the extracellular medium
(Kiselyov et al., 1998
).
Moreover it has been found that InsP3R, like several
Ca2+ signaling proteins associates with the Sec6/8 complex in
neuronal cells and in pancreatic acinar cells
(Shin et al., 2000
)
Role of InsP3R at the tight junction
The presence of the Sec6/8 complex in the proximity of the tight junctions
of polarized MDCK cells has been associated with exocytosis and the delivery
of vesicles to the basolateral membranes. The co-localization of the
InsP3R and proteins of the Sec6/8 complex asks about a putative
role of Ca2+ on this delivery. In other epithelial cell types the
initiation site of the Ca2+ wave, found at the luminal pole, has
been associated to a higher sensitivity of this region to InsP3R
linked to a higher density of InsP3R
(Kasai et al., 1993;
Lee et al., 1997
;
Nathanson et al., 1994b
;
Thorn et al., 1993
). It can be
assumed that the proximity of this Ca2+ release channel will
provide the increase in the intracellular Ca2+ concentration
necessary for the fusion of vesicles with the plasma membrane. This could
participate in the establishment and maintenance of the cell polarity.
Another putative function of intracellular Ca2+-release channel
in the tight junction area is the regulation of the paracellular permeability.
In MDCK cells, intracellular Ca2+ may be necessary for intermediate
sorting and biochemical stabilization steps in tight junction and desmosome
biogenesis and therefore may regulate tight junction permeability
(Stuart et al., 1996). On the
other hand, a Ca2+-dependent phosphorylation of myosin light-chain
could regulate the contraction of the peri-junctional actomyosin ring and,
thus, the permeability of tight junctions. In monolayers of MDCK cells
expressing the catalytic domain of the myosin light-chain kinase (MLCK), the
increase in phosphorylation of myosin light chain was correlated with a
decrease of TER (Hecht et al.,
1996
). It can be assumed that a local increase of Ca2+
concentration could be a key event allowing the regulation of the MLCK
(Turner, 2000
).
Finally, tight junctions may regulate the availability of signal
transduction molecules and participate in the regulation of cell growth and
differentiation. Such a feature has been described for the transcription
factor ZONAB which interacts with ZO-1 and regulates ErbB-2 expression in
proliferating MDCK cells. The complex is recruited at the tight junctions in
polarized cells and therefore is no longer expressed in the nucleus
(Balda and Matter, 2000). It is
interesting to note that in proliferating non-polarized MDCK cells,
InsP3R is found in the cytoplasm and particularly around the
nucleus. This could provide the Ca2+ necessary for the regulation
of gene expression and proliferation of the cells. During the polarization of
the cells, InsP3R is concentrated at the tight junctions level,
away from the nucleus and thereby possibly limiting cell proliferation.
The data presented in this paper demonstrate that there is a redistribution of InsP3R during the polarization of MDCK cells independently of the type of receptor expressed. The establishment of the 10B MDCK clone, that stably expresses InsP3R-1-EGFP, provides a nice tool for studying in living cells the receptor subcellular localization, mobility and redistribution during events such as cell polarization.
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Acknowledgments |
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Footnotes |
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These authors contributed equally to this work
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References |
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