1 Department of Cell Biology, Lerner Research Institute, Cleveland, Ohio 44195,
USA
2 Urological Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195,
USA
3 Department of Ophthalmic Research, Cleveland Clinic Foundation, Cleveland,
Ohio 44195, USA
* Author for correspondence (e-mail: weimbst{at}lerner.ccf.org)
Accepted 21 August 2002
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Summary |
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Key words: Epithelial polarity, Membrane traffic, SNARE
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Introduction |
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In higher metazoan organisms a large variety of epithelial cell types perform a multitude of different functions. The apical surfaces of these cell types can face dramatically different environments. Extreme examples are epithelial cells lining the stomach, colon, bile duct, urinary tract, and the retinal pigment epithelium (RPE). Correspondingly, different epithelial cell types require different sets of apical and basolateral surface proteins to perform their unique roles in the body.
In many cases, apical and basolateral sorting signals are recognized and
interpreted identically between different epithelial cell types. In other
cases, identical proteins can be sorted to different plasma-membrane domains
in different epithelial cell types. One example is the
Na+/K+-ATPase, which is localized to the basolateral
domain in the vast majority of epithelial cells types. However, in a few
epithelia, such as the RPE and the choroid plexus epithelium, the
Na+/K+-ATPase localizes apically
(Marmorstein, 2001;
Marrs et al., 1995
). Proteins
can also be ultimately targeted to the same domain but the pathway that they
take to reach this domain differs between cell types. For example, in the
Madin Darby canine kidney (MDCK) cell line, newly synthesized influenza virus
hemagglutinin (HA) is directly targeted from the Golgi to the apical plasma
membrane (Matlin and Simons,
1984
; Misek et al.,
1984
). By contrast, in the RPE it is first transported to the
basolateral plasma membrane and subsequently endocytosed and transcytosed to
the apical surface (Bonilha et al.,
1997
). It is currently unknown how the variability in sorting
phenotypes between different epithelial cell types is accomplished.
The RPE has long been recognized to differ from many other epithelia in the
polarity of a number of proteins
(Marmorstein, 2001). A unique
characteristic of RPE cells is that their apical plasma membrane is in contact
with the extracellular matrix (the interphotoreceptor matrix) whereas almost
all other epithelia face an apical lumen devoid of matrix. Other proteins that
are sorted differently in RPE compared with most epithelia include the
extracellular matrix metalloproteinase inducer (EMMPRIN; apical)
(Marmorstein et al., 1998
),
N-CAM (apical) (Marmorstein et al.,
1998
),
vß5 integrin (apical)
(Finnemann et al., 1997
), and
possibly CFTR, which is thought to be basolateral in RPE cells but is apically
localized in other epithelia (Gallemore et
al., 1998
). Furthermore, physiological experiments have
demonstrated that Ca2+-sensitive chloride channels, which are
typically present on the apical membrane of epithelial cells, are
basolaterally polarized in RPE cells
(Gallemore et al., 1998
).
It is unknown whether the establishment of different epithelial sorting
phenotypes involves major changes in the machineries involved in polarized
targeting or simply the rerouting of proteins into different sorting pathways.
SNARE-mediated membrane fusion is the final step in all vesicle trafficking
pathways (Chen and Scheller,
2001; Jahn and Sudhof,
1999
). SNAREs belong to several related protein families
(Weimbs et al., 1997
;
Weimbs et al., 1998
), and
different family members usually exhibit a distinct subcellular localization
and function only in specific trafficking pathways. Only `matching'
combinations of v- and t-SNAREs lead to successful membrane fusion
(McNew et al., 2000
;
Scales et al., 2000
),
suggesting that the SNARE machinery plays a role not only in the mechanics of
the fusion process but also in its specificity. Members of the syntaxin family
of t-SNAREs appear to play a central role since syntaxins can interact with
virtually all other components implicated in SNARE-mediated membrane fusion.
It is reasonable to assume that the presence of a given syntaxin on a membrane
domain determines which classes of transport vesicles will be able to fuse
with that domain. We hypothesized that a feasible mechanism contributing to
the known variety of epithelial trafficking phenotypes is for different
epithelial cell types to express different sets of plasma membrane syntaxins
and/or to localize them to different domains.
The best characterized epithelial model system to date is the MDCK cell
line. We have previously shown that in these cells the t-SNAREs syntaxin 3 and
4 are specifically localized at the apical or basolateral plasma membrane
domains, respectively (Low et al.,
1996). Syntaxin 3 is functionally involved in trafficking from the
TGN to the apical plasma membrane and in apical recycling
(Low et al., 1998a
). Syntaxin
4 is involved in TGN-to-basolateral trafficking
(Lafont et al., 1999
). Two
other syntaxins, syntaxin 2 and 11, are also expressed at the plasma membrane
in MDCK cells but they are present in both domains and their function is
unknown (Low et al., 1996
;
Low et al., 2000
). In general,
the localization of these t-SNAREs appears to be well conserved in a number of
other epithelial cell lines and tissues such as Caco-2 cells, HepG2 cells,
hepatocytes, kidney epithelium and intestinal epithelium
(Delgrossi et al., 1997
;
Fujita et al., 1998
;
Galli et al., 1998
;
Lehtonen et al., 1999
;
Li et al., 2002
;
Low et al., 1998b
;
Riento et al., 1998
). Syntaxin
3, however, has been found to deviate from its normal apical plasma membrane
localization in two specialized epithelial cell types. It localizes to zymogen
granules in pancreatic acinar cells
(Gaisano et al., 1996
) and to
H+/K+-ATPase-containing tubulovesicles in non-stimulated
gastric parietal cells (Peng et al.,
1997
). Moreover, syntaxin 2 localizes to the apical
plasma-membrane domain in pancreatic acinar cells
(Gaisano et al., 1996
) in
contrast to its non-polarized distribution in MDCK cells.
We hypothesized that changes in SNARE expression and/or subcellular localization may contribute to the differential sorting phenotypes of specialized epithelial cell types. To test this, we investigated the expression and localization of the plasma membrane SNARE machinery in RPE cells in vitro and in vivo. We report here that RPE cells in vitro and in vivo differ significantly from MDCK cells and other epithelial cells in the expressed complement of SNARE proteins as well as in their subcellular localization. Altogether, our results suggest that the differential expression and subcellular localization of SNAREs is used as a general mechanism contributing to the modulation of epithelial sorting phenotypes and is at least in part responsible for the unique distribution of plasma membrane proteins in the RPE.
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Materials and Methods |
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Cell culture
RPE-J cells were from ATCC (CRL-2240) and cultured in Dulbecco's Modified
Eagle's medium containing 4 mM glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l
glucose and 1 mM sodium pyruvate. This media was further supplemented with 4%
FBS, non-essential amino acids and penicillin and streptomycin. The cells were
maintained at 32°C and 5% CO2. For polarized monolayers, the
cells were plated at 300,000 cells/cm2 on Matrigel-coated Transwell
Filters (Corning, Acton, MA) in culture medium supplemented with 2.5 nM
retinoic acid and cultured for 1 week at 32°C. The cells were then
transferred to a 40°C incubator for another week. The media was changed
every 3 days.
Tissue and cell extracts, SDS-PAGE and immunoblotting
Total protein fractions of RPE-J cells were prepared by directly lysing
cells from confluent dishes by boiling in SDS-PAGE sample buffer. DNA was
sheared by passing lysates through a 22G needle. Rat kidney and brain lysates
were prepared by dissecting out the tissues and finely mincing with a razor
blade. The tissues were then Dounce-homogenized in PBS containing 10 mM EDTA,
protease inhibitors and PMSF. SDS was added to a final concentration of 2% and
the lysate was passed through a 22G needle to shear the DNA, and the sample
was boiled for 5 minutes. Proteins were separated on 10% or 15%
SDS-polyacrylamide gels followed by transfer to PVDF membrane and incubation
with the indicated antibodies. Bands were visualized by enhanced
chemiluminescence.
RT-PCR
Total RNA was isolated from RPE-J cells, rat kidney or rat brain by using
Trizol (GIBCO) according to the manufacturer's instructions. 5 µg of total
RNA were used for reverse transcription in a 25 µl total volume using
random hexamers as primers. For the detection of syntaxin 2 isoforms, 1 µl
of the reverse-transcribed samples were used for 50 µl PCR reactions using
primers and conditions as described previously
(Quinones et al., 1999). The
following primer pairs were designed to recognize rat syntaxin 3A;
5'-GCTGAGATGTTAG-ATAACATAG-3' and
5'-TTCAGCCCAACGGACAATCCAA-3' or syntaxin 3B;
5'-CAGGGAGCCATGATTGACCGTA-3' and
5'-AAATATGCCCCCAATGGTAGAA-3', utilizing the same PCR conditions as
for syntaxin 2. 10 µl of each PCR reaction were separated on 2% agarose
gels.
Immunolocalization
Sprague-Dawley rats were anesthetized by intraperitoneal administration of
sodium pentobarbital (50 mg/kg) and perfused via the left ventricle with 4%
paraformaldehyde in PBS containing 1 mM calcium and 1 mM magnesium for 20
minutes. The eyes were enucleated, the anterior segments removed, and the
eyecups stored in the same fixative overnight at 4°C. Fixed eyecups were
then dehydrated and embedded in paraffin. Immunostaining was carried out on 5
µm sections. After deparaffinization and rehydration to PBS, the sections
were subjected to heat-mediated antigen retrieval by pressure cooking in 10 mM
citric acid buffer, pH 6.0. The sections were blocked with 3% BSA, 2% Triton
X-100 in PBS, incubated with the indicated antibodies overnight at 4°C.
The reactions were visualized with fluorescein- and Texas-Red-labeled
secondary antibodies (Jackson ImmunoResearch, West Grove, PA). SNARE-signals
were amplified by incubating with Alexa 488-labeled rabbit anti-FITC antibody
(Molecular Probes) after FITC-labeled secondary antibodies. Nuclei were
stained with DAPI. The fluorescent staining was analyzed using a confocal
laser scanning microscope (TCS-NT, Leica, Benseim, Germany). Immunostaining
for syntaxins 1B, 2, 3, 4 and endobrevin was verified using two independently
raised antibodies each of which resulted in identical staining patterns. Only
one antibody was available against syntaxin 1A.
For immunostaining of cultured RPE-J cells, cells were either fixed in methanol at -20°C or with 4% paraformaldehyde in PBS, permeabilized with 0.025% (wt/vol) saponin Sigma (St. Louis, MO) in PBS and blocked with 3% BSA followed by sequential incubations with primary antibodies and FITC- and Texas-red-conjugated secondary antibodies. Signal amplification and imaging were performed as described above.
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Results |
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Next, we investigated the possible expression of SNAREs that are typically restricted to neurons and neuroendocrine cells. Brain tissue contains very large amounts of the t-SNAREs syntaxin 1A, syntaxin 1B, SNAP-25 and of the v-SNARE synaptobrevin-2, all of which are involved in calcium-regulated synaptic vesicle exocytosis. Surprisingly, RPE-J cells expressed significant quantities of syntaxins 1A, 1B and synaptobrevin-2 (Fig. 1B). However, no SNAP-25 was detectable even after prolonged exposure of the blot (data not shown). None of these SNAREs were detectable in kidney, with the exception of low amounts of synaptobrevin-2.
In summary, the SNARE expression pattern of RPE-J cells shows two unexpected features: the absence of the normally apical-specific syntaxin 3 and the presence of `neuronal' SNAREs.
Expression of syntaxin 2 and 3 transcripts in RPE-J cells
Four syntaxin 2 isoforms derived from alternative RNA splicing have
previously been identified in rat tissues. Syntaxins 2A and 2B are membrane
anchored by hydrophobic domains, whereas syntaxin 2C and 2D lack hydrophobic
domains and are only partially membrane bound
(Quinones et al., 1999). All
four syntaxin 2 isoforms differ only in their C-termini whereas the N-terminal
bulk of their sequences are identical. Since our polyclonal syntaxin 2
antibodies react with all four isoforms (data not shown) and since the
molecular weights of these isoforms are very similar, they can not be
distinguished from each other by western blot analysis. In order to
investigate which syntaxin 2 isoforms are expressed in RPE-J cells and to
confirm the observed absence of syntaxin 3-expression, we analyzed total RNA
by RT-PCR. We used two primer combinations that have previously been shown to
distinguish between the four different rat syntaxin 2 isoforms
(Quinones et al., 1999
).
Fig. 2A shows that all four
syntaxin 2 isoforms could be detected in rat brain whereas rat kidney
expressed syntaxins 2A, B and C as previously described. By contrast, in RPE-J
cells, syntaxins 2A and a lesser amount of syntaxin 2B, but no transcripts for
syntaxins 2C or 2D, could be detected (Fig.
2). No syntaxin 3 transcripts were detectable, confirming the lack
of expression observed on the protein level.
|
SNARE localization in RPE-J cells
To determine the subcellular localization of the expressed t-SNAREs, RPE-J
cells were cultured as polarized monolayers on Matrigel-coated polycarbonate
filters, labeled with affinity-purified syntaxin antibodies and analyzed by
confocal fluorescence microscopy. As expected, syntaxin 3 was undetectable
(data not shown). Fig. 3 shows
that both syntaxins 2 and 4 localize to the plasma membrane at the regions of
cell-cell contact whereas no significant apical staining was detectable. Both
syntaxins 2 and 4 colocalize with the basolateral marker EMMPRIN as well as
with the tight junction protein ZO-1. Since RPE-J cells are typically flat
(Marmorstein et al., 1998), it
was not possible to determine by light microscopy whether syntaxin 2 and 4 are
distributed all along the lateral membrane or whether they are concentrated
close to the tight junctions. The expression levels of syntaxins 1A and 1B
were below the detection limit.
|
In conclusion, syntaxin 4 localizes to the basolateral plasma membrane domain in RPE-J cells similar to all previously investigated epithelial cell types. However, syntaxin 2 localizes to the basolateral plasma membrane domain, which is in striking contrast to its apical localization in other epithelial cell types.
SNARE localization in RPE in situ
RPE-J cells and other cultured RPE cells have been found to differ in their
protein-sorting phenotype from RPE cells in situ.
Na+/K+-ATPase, N-CAM and EMMPRIN, which are apically
polarized in RPE cells in the eye, are typically non-polar or basolateral in
culture (Marmorstein, 2001).
We therefore investigated SNARE expression and subcellular localization in rat
retina. Sections of the posterior pole of rat eyes were fluorescently
double-labeled using SNARE-specific antibodies and antibodies against various
marker proteins and analyzed by confocal microscopy. Cryosections and paraffin
sections yielded identical results. Fig.
4A shows that syntaxin 3 is abundantly expressed in photoreceptor
cells but is not detectable above background in RPE cells, in agreement with
our results on RPE-J cells.
|
As predicted from our studies on RPE-J cells, syntaxins 2, 4, 1A and 1B are all clearly detectable in RPE cells in situ. Syntaxins 2 and 4 exhibit a surprising subcellular localization. Syntaxin 2 is restricted to a narrow band that localizes closely with the tight junctional protein occludin. At higher magnification it was apparent that these two proteins do not exactly overlap but that syntaxin 2 localizes to a region of the lateral plasma membrane basal to the tight junctions (Fig. 4D). Since the tight junctions represent the border between the apical and basolateral plasma membrane domains in epithelial cells, syntaxin 2 is therefore a basolateral SNARE in RPE cells in contrast to its apical localization in other epithelial cells. This is in agreement with our results on RPE-J cells.
Syntaxin 4 also localizes to the same narrow band underneath the tight junctions. However, in contrast to syntaxin 2, it is also clearly present at the basal plasma membrane of RPE cells (Fig. 4E). Both syntaxin 1A (Fig. 4B) and syntaxin 1B (Fig. 4C) are localized throughout the apical plasma membrane in RPE cells, which constitutes 75% of the total RPE plasma membrane owing to microvilli that interdigitate with the photoreceptor outer segments. The outer segments themselves are negative for both syntaxins.
Endobrevin is a v-SNARE that has been found to be most highly expressed in
epithelial cells. It has been implicated in endosome fusion in non-epithelial
cells (Antonin et al., 2000)
but has also been shown to cycle through the apical plasma membrane in MDCK
cells (Steegmaier et al.,
2000
). Fig. 4F
shows that endobrevin was surprisingly also highly concentrated underneath the
tight junctions in RPE cells similar to syntaxins 2 and 4. In addition,
endobrevin localized to intracellular vesicles throughout the cytoplasm.
Immunostaining results for syntaxins 1B, 2, 3, 4 and endobrevin were confirmed using independently raised antibodies and yielded identical results (see Materials and Methods, data not shown).
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Discussion |
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In the present work we tested our hypothesis that the expression of different sets of plasma membrane syntaxins and/or their localization to different domains is a mechanism contributing to the known variety of epithelial trafficking phenotypes. Our investigation of this possibility using RPE cells as an example of an epithelium with a polarity phenotype that diverges substantially from the prototypic MDCK cell is in agreement with this hypothesis. Fig. 5 shows a schematic summary of the results.
|
RPE cells both in vitro and in vivo lack expression of the normally
apical-specific syntaxin 3. This may suggest that a trafficking pathway that
normally involves syntaxin 3 is absent in this cell type. This finding is in
excellent agreement with a known difference in the targeting of the influenza
virus hemagglutinin (HA) in RPE cells. We have previously shown that syntaxin
3 functions in transport from the trans-Golgi network to the apical plasma
membrane in MDCK cells (Low et al.,
1998a) (route A in Fig.
5). This route is taken by newly synthesized HA in MDCK cells
(Matlin and Simons, 1984
;
Misek et al., 1984
), and
indeed Lafont et al. could demonstrate that apical HA-trafficking in MDCK
cells is syntaxin 3 dependent (Lafont et
al., 1999
). By contrast, newly synthesized HA is initially
transported to the basolateral plasma membrane domain in RPE-J cells and in
RPE cells in situ (Bonilha et al.,
1997
) (route B1 or B2) and is subsequently
transcytosed to the apical domain (route T). HA therefore bypasses the direct
TGN-to-apical route in this cell type. Importantly, our previous results
indicated that syntaxin 3 is not involved in basolateral-to-apical
transcytosis (route T) in MDCK cells, suggesting that this step requires
another (unidentified) syntaxin. The absence of syntaxin 3 in RPE cells
therefore agrees well with the itinerary of HA in this cell type, which avoids
syntaxin-dependent trafficking routes.
A direct TGN-to-apical route does exist in RPE cells, however, and is taken
by cargo proteins such as p75-NTR, VEGF-165, TGF-ß,
(Marmorstein et al., 2000;
Marmorstein et al., 1998
) and
retinol binding protein and transthyreitin
(Jaworowski et al., 1995
).
Since this occurs in the absence of syntaxin 3 we propose that an alternative
route exists in RPE cells (route A2) that utilizes a different
syntaxin and may or may not exist in MDCK cells. Likely candidates are
syntaxin 1A or 1B, which we found to be expressed at the apical plasma
membrane in RPE cells. These two syntaxins are usually expressed in neurons
and neuroendocrine cells and function in calcium-regulated exocytosis such as
synaptic vesicle fusion with the presynaptic plasma membrane. An intriguing
speculation is that apical vesicle fusion in RPE cells may be calcium
regulated.
A surprising finding is that syntaxin 2 is localized to the basolateral
plasma membrane domain in RPE cells. Of the four known syntaxin 2 splice
isoforms, we found that only syntaxins 2A and 2B were expressed in RPE-J
cells; these isoforms differ only in their C-terminal transmembrane domains
(Quinones et al., 1999). In
pancreatic acinar cells, syntaxin 2 localizes to the apical plasma membrane
but it was not investigated which isoforms are expressed in these cells
(Gaisano et al., 1996
).
Syntaxin 2 is therefore the first known syntaxin whose polarity can be
reversed depending on the epithelial cell type. In MDCK cells, syntaxin 2A
expressed by stable transfection localizes to both the apical and basolateral
plasma membrane domains (Low et al.,
1996
). It is interesting to note that syntaxin 2A expressed by
adenovirus-mediated transfection was found to be concentrated at the apical
plasma membrane of MDCK cells whereas syntaxin 2B was more evenly distributed
on both domains (Quinones et al.,
1999
). From these data it is unlikely that the reversed polarity
of syntaxin 2 in RPE cells can be explained simply by the expression of
alternative isoforms. Other mechanisms must be responsible for this
differential targeting, for example, differential recognition of targeting
signals within the amino-acid sequence of syntaxin 2. This mechanism remains
to be identified. It also remains unknown which cargo proteins utilize a
syntaxin-2-dependent fusion step. To date, syntaxin 2 has been implicated in
only two fusion events, zymogen granule exocytosis in acinar cells
(Hansen et al., 1999
) and the
fusion of the acrosome with the plasma membrane of spermatozoa
(Katafuchi et al., 2000
).
Since the major contents of both zymogen granules and the acrosome are
hydrolytic enzymes it is tempting to speculate that syntaxin 2 is used for the
secretion of similar cargo in RPE and other epithelial cells.
Unexpectedly, we found that both syntaxins 2 and 4 localize to a narrow
band just underneath the tight junctions in RPE cells in vivo. Confocal
microscopy of double-stained sections showed that both syntaxins do not
overlap with the tight junction protein occludin; instead they are immediately
adjacent on the lateral membrane, indicating that both are involved in
basolateral trafficking pathways. On the basis of the localization of other
proteins implicated in vesicle fusion on the tight junctions of epithelial
cells, it has been proposed that the tight junctions or structures in
close proximity may be preferred sites of vesicle exocytosis. These
proteins include rab8 (Huber et al.,
1993), rab3b (Weber et al.,
1994
), rab13 (Zahraoui et al.,
1994
), the sec6/8 complex or exocyst
(Grindstaff et al., 1998
) and
VAP-A (Lapierre et al., 1999
).
Direct evidence of vesicle fusion events at the tight junctions, however, has
been lacking, and the fact that syntaxin 4 localizes all along the lateral
membrane in MDCK and other epithelial cells seemed to be at odds with this
hypothesis. Recently, fusion events of post-Golgi transport vesicles carrying
GFP-tagged apical and basolateral marker proteins were monitored in polarized
MDCK cells using time-lapse microscopy (G. Kreitzer, J. Schmoranzel, S.-H.
Low, Y. Chen et al., unpublished). The results demonstrated that fusion of
basolateral vesicles occurs all along the lateral plasma membrane in agreement
with the lateral localization of syntaxin 4. It is still possible that more
specialized trafficking pathways may be directed towards the tight junctions
in MDCK cells, but this remains to be investigated. Our striking finding that
syntaxins 2 and 4 localize close to the tight junctions in RPE cells strongly
suggests that a tight junctional `fusion patch' indeed exists in this
epithelial cell type and that exocytic pathways that depend on these syntaxins
will be directed toward this site (route B1 in
Fig. 5). Syntaxin 4, but not
syntaxin 2, was also present at the basal membrane in RPE cells, suggesting
that basolateral fusion events occur either at the `tight junctional fusion
patch' or at the basal membrane (route B2). It is possible that
syntaxin 4 is not only involved in TGN-to-basolateral trafficking in
epithelial cells but also in recycling pathways as could be deduced from its
known involvement in GLUT-4 translocation from endosomes to the plasma
membrane in adipocytes (Macaulay et al.,
1997
; Olson et al.,
1997
; Tellam et al.,
1997
). In this case, routes B1 and B2 may
differ in that one of them originates from endosomes rather than the TGN.
In conclusion, we have shown that RPE cells differ in their complement of plasma membrane syntaxins and in the polarity of one syntaxin from other epithelial cell types. Furthermore, they exhibit the prominent localization of two syntaxins at a putative `sub-tight junctional fusion patch'. These differences suggest that epithelial cell types can indeed differ in the molecular machineries controlling membrane trafficking. We consider it therefore unlikely that the known variability of epithelial trafficking phenotypes is solely due to re-routing of cargo proteins for example, by differential recognition of sorting signals into otherwise identical trafficking pathways. A more likely scenario may be that epithelial cells can regulate the presence/absence, preponderance and/or direction of entire trafficking pathways. This would include the expression and localization of plasma membrane syntaxins, which serve as `end points' of all exocytic pathways. It is unlikely, however, that regulation of syntaxin expression/localization alone can accomplish the diversity of epithelial trafficking phenotypes. Membrane trafficking pathways consist of a succession of mechanisms such as vesicle budding, transport along cytoskeletal elements etc. many of which may need to be modulated depending on the epithelial cell type.
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Acknowledgments |
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