Department of Physiological Chemistry, Faculty of Medical Sciences, University of Groningen, Groningen, The Netherlands
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Abstract |
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In polarized HepG2 cells, the fluorescent sphingolipid analogues of glucosylceramide (C6-NBD-GlcCer) and sphingomyelin (C6-NBD-SM) display a preferential localization at the apical and basolateral domain, respectively, which is expressed during apical to basolateral transcytosis of the lipids (van IJzendoorn, S.C.D., M.M.P. Zegers, J.W. Kok, and D. Hoekstra. 1997. J. Cell Biol. 137:347-457). In the present study we have identified a non-Golgi-related, sub-apical compartment (SAC), in which sorting of the lipids occurs. Thus, in the apical to basolateral transcytotic pathway both C6-NBD-GlcCer and C6-NBD-SM accumulate in SAC at 18°C. At this temperature, transcytosing IgA also accumulates, and colocalizes with the lipids. Upon rewarming the cells to 37°C, the lipids are transported from the SAC to their preferred membrane domain. Kinetic evidence is presented that shows in a direct manner that after leaving SAC, sphingomyelin disappears from the apical region of the cell, whereas GlcCer is transferred to the apical, bile canalicular membrane. The sorting event is very specific, as the GlcCer epimer C6-NBD-galactosylceramide, like C6-NBD-SM, is sorted in the SAC and directed to the basolateral surface. It is demonstrated that transport of the lipids to and from SAC is accomplished by a vesicular mechanism, and is in part microtubule dependent. Furthermore, the SAC in HepG2 bear analogy to the apical recycling compartments, previously described in MDCK cells. However, in contrast to the latter, the structural integrity of SAC does not depend on an intact microtubule system. Taken together, we have identified a non-Golgi-related compartment, acting as a "traffic center" in apical to basolateral trafficking and vice versa, and directing the polarized distribution of sphingolipids in hepatic cells.
Key words: sphingolipid; immunoglobulin A; sorting; transcytosis; HepG2 cell ![]() |
Introduction |
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THE plasma membrane (PM)1 of polarized cells is divided into a basolateral and an apical PM domain. Each membrane domain has its specific protein and lipid composition, an essential feature for the polarized function of these cells. To generate and maintain such unique membrane compositions, proteins and lipids have to be sorted and targeted to the appropriate PM domains. In polarized cells apical delivery may occur via two routes, including a direct transport from the TGN, and an indirect pathway, in which before apical delivery, newly synthesized components are first transported from the TGN to the basolateral surface. Subsequently, apical constituents are endocytosed, sorted into transcytotic vesicles, and transported to the apical pole of the cell. To add to the complexity of trafficking in polarized cells, proteins, and lipids from each membrane domain are continuously reinternalized, recycled, or redistributed, reflecting a dynamic equilibrium indispensable for maintaining a dynamic membrane composition capable of adapting to different needs. As a consequence, apical (and basolateral) components must be sorted into or excluded from vesicles with preferential targets at various intracellular sites, each site thus contributing to the polarized distribution of PM components.
For both proteins and distinct (glyco)sphingolipids, the
main intracellular sorting site for polarized transport is believed to be the TGN. Specifically, in this compartment,
several apical proteins appear to associate with glycosphingolipid (GSL)-enriched domains (Brown and Rose,
1992), so-called GSL rafts (Simons and Ikonen, 1997
).
Budding of these rafts will give rise to vesicles, with an
apical destination (Simons and Wandinger-Ness, 1990
). Whereas MDCK cells predominantly use the direct pathway for the delivery of apical components (Matlin and Simons, 1984
; Misek et al., 1984
; Pfeiffer et al., 1985
; Simons
and Fuller, 1985
), other polarized cells may use a combination of the direct and the indirect, transcytotic pathway
(van't Hof and van Meer, 1990
; Weimbs et al., 1997
). Hepatic cells are believed to rely mainly on the indirect, transcytotic pathway for the apical delivery of newly synthesized proteins (Bartles et al., 1987
; Cariappa and Kilberg,
1992
; Schell et al., 1992
), although evidence is accumulating which indicates that direct Golgi to apical transport of
proteins and lipids also occurs (Ali and Evans, 1990
; Zaal et al., 1993
; Zegers and Hoekstra, 1997
). The overall
mechanism involved in the apical flow of proteins and lipids via the indirect, transcytotic route is still largely unclear. For example, whether GSL-rafting plays a role in
this route remains to be determined.
To study transcytosis, the polymeric immunoglobulin receptor (pIgR) and its ligand IgA is often used as a marker
of this pathway (Mostov and Deitcher, 1986; Apodaca et
al., 1991
; Mostov et al., 1995
). It has thus been demonstrated that transcytosis proceeds along routes, which constitute part of other pathways used by non-transcytosing
molecules. pIgR-IgA complexes, endocytosed from the
basolateral surface, are delivered to basolateral endosomes and reach via transcytosis sub-apical endosomal
compartments, termed apical recycling compartments
(ARC) in MDCK cells, before their delivery to the apical
PM (Apodaca et al., 1994
; Barroso and Sztul, 1994). Other
apical proteins such as dipeptidyl peptidase IV and the
glycosylphosphatidylinositol-anchored protein 5' nucleotidase have been suggested to use the same transcytotic
pathway as pIgR-IgA (Barr et al., 1995
). As revealed in
MDCK cells, the sub-apical compartments (SAC) are not
only accessible to proteins with a preference for apical localization. Also a considerable portion of the basolaterally
endocytosed transferrin receptor was found to be transported to the apical recycling compartment. However, in
contrast to the apically targeted pIgR-IgA, the transferrin receptor returns to the basolateral surface (Apodaca et al.,
1994
). Hence, these data suggest a role for the sub-apical
compartment in the generation and/or maintenance of the
polarized distribution of PM components.
Previous studies in MDCK (van Genderen and van
Meer, 1995) and the human hepatoma-derived cell line,
HepG2 (van IJzendoorn et al., 1997
; Zegers and Hoekstra,
1997
) revealed that sphingolipids such as glucosylceramide
(GlcCer) and sphingomyelin (SM) also use the transcytotic pathway to traverse the cell in either direction. However, very little is known of the mechanisms involved in
transcytotic transport of lipids. In a recent study, we demonstrated that in well-polarized HepG2 cells fluorescent
acyl chain-labeled analogues of glucosylceramide (C6-NBD-GlcCer) and sphingomyelin (C6-NBD-SM) are segregated in the apical to basolateral transcytotic pathway (van IJzendoorn et al., 1997
). The observed segregation
event did not involve the Golgi apparatus, hinting at the
existence of another, yet unidentified subcellular compartment where sphingolipids are sorted. In the present work
we provide evidence that SAC, similar to ARC as defined
in MDCK cells, are also operative in HepG2 cells. Specifically, it is demonstrated that these SAC represent the major intracellular site in the transcytotic pathway where GlcCer and SM are sorted and directed to distinct PM domains.
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Materials and Methods |
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Materials
Sphingosylphosphorylcholine, 1--glucosylsphingosine, cerebroside type
II, asialofetuin type I, and geneticin (G-418) were from Sigma Chemical
Co. (St. Louis, MO). Albumin (from bovine serum, fraction V) was
bought from Fluka Chemie AG (Buchs, Switzerland). 6-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino) hexanoic acid (C6-NBD) was obtained from
Molecular Probes (Eugene, OR). DME was purchased from GIBCO-BRL (Paisley, Scotland). FCS was bought from BioWhittaker (Verviers,
Belgium), and Na2S2O4 was from Merck (Darmstadt, Germany). Nocodazole was obtained from Boehringer Mannheim (Mannheim, Germany). PSC 833 and MK 571 were gifts from Dr. E. Vellenga (University of
Groningen, The Netherlands). Texas red-labeled IgA was kindly provided by Dr. K. Dunn (Indiana University Medical Center, Indianapolis, IN). pIgR cDNA and an mAb against pIgR was kindly provided by Dr. K. Mostov (University of California, San Francisco, CA). All other chemicals
were of analytical grade.
Cell Culture
HepG2 cells were grown at 37°C under a 5% CO2-containing, humidified atmosphere in DME, supplemented with 10% heat-inactivated (at 56°C) FCS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Medium was replaced every other day. For experiments, cells were used 3 d after plating. At this time the cells had reached an optimal ratio of polarity versus density, i.e., No. of bile canalicular spaces (BCs) versus No. of cells.
Synthesis of C6-NBD-labeled Sphingolipids
C6-NBD-GlcCer and C6-NBD-SM were synthesized from C6-NBD and
1--glucosylsphingosine and sphingosylphosphorylcholine, respectively, as described elsewhere (Kishimoto, 1975
; Babia et al., 1994
). 1-
-D-galactosylceramide was prepared from cerebroside (type II) (Goda et al.,
1987
). The C6-NBD-lipids were stored at
20°C and routinely checked for
purity.
Cell Labeling and Lipid Transport Assays
For microscopy, cells were plated onto glass coverslips. For biochemical
analysis cells were cultured in 25-cm2 culture flasks (Costar, Cambridge,
UK). After 3 d, cells were washed three times with a PBS solution. C6-NBD-GlcCer or C6-NBD-SM were dried under nitrogen, redissolved in
absolute ethanol, and then injected into HBSS under vigorous vortexing.
The final concentration of ethanol was kept below 0.5% (vol/vol). Cells
were incubated with 4 µM of either lipid analogue at 37°C for 30 min (see
Fig. 1, step 1). To monitor transport of apical membrane-associated lipid
analogues, the basolateral pool of fluorescent lipid analogues was depleted by incubating the cells in HBSS, supplemented with 5% (wt/vol)
BSA at 4°C twice for 30 min (back exchange, Fig. 1, step 2). Then, cells
were washed three times with ice-cold PBS, rewarmed to the desired temperature, and then incubated in HBSS, with 5% (wt/vol) BSA (Fig. 1, step
3). The 18°C temperature block was dictated by previous observations in
MDCK cells showing that an incubation at this temperature causes a selective inhibition of transcytotic transport, as revealed by the accumulation of the transcytotic marker pIgA-R (Apodaca et al., 1994; Barosso and
Sztul, 1994
). In some experiments, NBD fluorescence associated with BC
was eliminated by incubating the cells with 30 mM sodiumdithionite (diluted from a stock solution of 1 M Na2S2O4 in 1 M Tris, pH 10) at 4°C for 7 min (Fig. 1, step 4). As described previously, sodiumdithionite is able to
pass the tight junctional complexes in HepG2 cells (van IJzendoorn et al.,
1997
), whereas BSA does not affect the labeling of BC (see Results; compare with van IJzendoorn et al., 1997
). Sodiumdithionite-treated cells
were then washed extensively (>10 times) with ice-cold HBSS, after
which transport was re-activated by rewarming and further incubating the
cells in HBSS at 37°C (Fig. 1, step 5). BSA (5% wt/vol) was included in the
medium to prevent the lipid analogues from re-entering the cells at the basolateral surface. After all final incubations, cells were washed with ice-cold HBSS and kept on ice until use (within 30 min).
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To study the involvement of microtubules on transport of BC-associated lipids, nocodazole (33 µM, diluted from a 10 mM stock solution) was added during step 2 of the incubation scheme depicted in Fig. 1 and kept present in all subsequent steps. In parallel experiments, cells were treated with 33 µM nocodazole at 4°C for 1 h after the 18°C chase of BC-associated lipid analogues (Fig. 1, step 3). Indirect immunofluorescence, using a mAb directed against fl-tubulin (Sigma Chemical Co.), was performed to verify the microtubule-disrupting potency of nocodazole in HepG2 cells (not shown).
To study the effect of PSC 833 and MK 571, which are specific inhibitors of the multi-drug resistance proteins MDR1/P-glycoprotein and MRP1, respectively, on the different transport steps, cells were incubated with MK 571 (25 µM) or PSC 833 (5 µg/ml) before step 1, with either inhibitor between steps 2 and 3, or between steps 4 and 5. The concentration of PSC 833 was sufficient to completely block the excretion of rhodamine 123 into BC of HepG2 cells by MDR1/P-glycoprotein activity (not shown).
Quantification of Fluorescent BC Labeling
As a measure for transport of the lipid analogues to and from the BC
membranes, the percentage of NBD-positive BC membranes was determined as described elsewhere (van IJzendoorn et al., 1997). In brief, BC
were first identified by phase contrast illumination, and then classified as
NBD-positive or NBD-negative under epifluorescence illumination. Furthermore, distinct pools of fluorescence are discerned, present in vesicular
structures adjacent to BC, which in this work are defined as SAC. Together, BC and SAC thus constitute the apical, bile canalicular pole
(BCP) in HepG2 cells. Therefore, within the BCP region the localization
of the fluorescent lipid analogues will be defined as being derived from
BC, SAC, or both. For this kind of quantification, at least 50 BCP were
analyzed per coverslip. All data are expressed as the mean ± SD of four independent experiments, carried out in duplicate.
Lipid Extraction and Quantification
Lipids were extracted according to the method of Bligh and Dyer (1959)
and analyzed by thin layer chromatography using CHCl3/methanol/ NH4OH/H2O (35:15:2:0.5, vol/vol/vol/vol) as running solvent. For quantification, fluorescent lipids were scraped from the TLC plates, followed by
vigorous shaking in 1% (vol/vol) Triton X-100 in H2O for 60 min to remove the lipids from the silica. Silica particles were then spun down and
NBD fluorescence was measured spectrophotometrically in an SLM fluorometer at excitation and emission wavelengths of 465 nm and 530 nm, respectively.
Transfection of HepG2 Cells with the pIgR
HepG2 cells were plated at low density (±20% confluency) in medium,
containing 20% FCS. Cells were allowed to attach and spread for 24 h.
The cells were then transfected with the cDNA encoding wild-type rabbit
pIgR, inserted into a pCB6 expression vector, which contained a neomycin resistance marker. The synthetic pyridinium-based amphiphile
SAINT-2 (a gift from Saint BV, Groningen, The Netherlands) was used as
DNA carrier. Preparation of small unilamellar SAINT-2/dioleylphosphatidyl ethanolamine (DOPE) vesicles and transfection was performed
as described by van der Woude et al. (1997). Cells were selected in culture
media, supplemented with 600 µg/ml geneticin (G-418). Successful transfection of HepG2 cells was evidenced by indirect immunofluorescence microscopy using a mAb against pIgR, and by the ability of the cells to internalize Texas red-labeled IgA (TxR-IgA) in the presence of a 100-fold
excess asialofetuin (see Results). The polarity of the transfected cells was
verified by determination of the ratio BC/cells (Zegers and Hoekstra,
1997
).
Internalization of Texas Red-labeled IgA
Cells transfected with the pIgR as described above were washed and asialoglycoprotein receptors were saturated with excess asialofetuin at 37°C for 30 min to prevent uptake of IgA via these receptors (see Results). Cells were incubated with TxR-IgA (50 µg/ml) at 4°C for 30 min. Cells were then washed to remove non-bound TxR-IgA and further incubated at 18°, 37°C, or a combination of both for various time intervals, depending on the experiment.
Microscopical Analysis and Image Processing
Cells were examined microscopically using a conventional Olympus Provis AX70 fluorescence microscope. Photomicrographs were taken using Illford HP5-plus films and scanned. For confocal laser scanning microscopy a TCS Leica (Heidelberg, Germany) apparatus equipped with a argon/krypton laser coupled to a Leitz DM IRB-inverted microscope was used. All images were converted to tagged-information-file format before printing on a Fujix P3000 printer.
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Results |
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Apical Membrane-derived C6-NBD-GlcCer and C6-NBD-SM Are Delivered to SAC
In a previous study we demonstrated that C6-NBD-GlcCer
and C6-NBD-SM display a preferential apical and basolateral localization, respectively, in HepG2 cells. This preferential localization was established during apical to basolateral transcytosis, a novel pathway in hepatic cells. A
tentative role for non-Golgi-related SAC in the apical recycling of C6-NBD-GlcCer was proposed (van IJzendoorn
et al., 1997). However, neither the identity of these compartments nor their functional involvement in the apical to basolateral transcytotic pathway of either lipid was established. To address these issues, the following experiments
were performed.
Cells were labeled with 4 µM C6-NBD-SM or C6-NBD-GlcCer at 37°C for 30 min. In this way, >70% of the BC,
identified by phase-contrast microscopy, became labeled,
the lipid analogues thus becoming inserted into the canalicular membrane. In addition, fluorescently labeled vesicular structures are observed in the cytoplasm, presumably representing compartments of the endocytic internalization pathway and transport vesicles still en route to their
target membranes. (Fig. 2, a and b; compare with Fig. 2 in
van IJzendoorn et al., 1997). To subsequently monitor the
fate of the apical membrane-associated lipid analogues,
the basolateral pool was depleted by incubating the cells in
HBSS, supplemented with 5% BSA at 4°C twice for 30 min (back exchange). The cells were then washed, rewarmed to either 18° (see Materials and Methods) or 37°C,
and further incubated in HBSS, supplemented with 5%
BSA, for 60 min. At 18°C, basolateral delivery of the
lipid analogues, relative to that at 37°C, was inhibited by
~70%, as calculated from the amount of C6-NBD-SM and
C6-NBD-GlcCer that could be retrieved in the BSA-containing medium. Fluorescence microscopical analysis revealed that upon chasing BC-associated lipid analogues
at 18°C, a major part of both C6-NBD-GlcCer and C6-NBD-SM remained localized at the BCP of the cells, i.e.,
in BC and/or sub-apically located vesicular structures (Fig.
2, d and f: thin arrows, BC; arrowheads, SAC). To reveal a
net directional movement of the lipid analogues in the
apical bile canalicular area, BCP labeling was distinguished into labeling of only BC, only SAC or labeling of
both BC and SAC. (See Materials and Methods, and Figs.
2, a [BC] and f [BC + SAC]; 4 b [SAC].) Such semi-quantitative analysis of labeling of the apical region revealed
that before the chase, >90% of the label detected in the
BCP region, was found to be solely associated with BC
(Fig. 3 A; see also the labeling pattern in Fig. 2, a and b).
Thus, the prominent labeling of sub-apical structures, as
revealed by accumulation of the lipid analogues, was not
observed before the chase at 18°C was started (also compare with van IJzendoorn et al., 1997
). After the 18°C
chase, the majority (±85%) of the C6-NBD-GlcCer or C6-NBD-SM present in the BCP was associated with both
SAC and BC (Fig. 3 B, BC + SAC and SAC), while only
~15% was due to the exclusive labeling of BC only (Fig. 3
B, BC). Also note that, for either lipid analogue, the percentage of labeled BCP did not change during the chase.
In conjunction with the fact that the presence of BSA in
the incubation medium prevented potential re-internalization of the lipid analogues from the basolateral surface
and subsequent transport to the SAC, the data thus indicate that the lipid analogues were transported via a direct
(i.e., basolateral membrane-independent) route from BC
to SAC.
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Not all cells participate in the formation of BC (Zegers
and Hoekstra, 1997). Remarkably, however, also in cells
lacking a distinct apical PM domain a significant part of
the intracellular C6-NBD-GlcCer and C6-NBD-SM fractions accumulated in juxtanuclear compartments after a
60-min incubation at 18°C (Fig. 2 f, wide arrows), but not
at 37°C (data not shown).
Finally, it is important to note that in the experiments at both 18° and 37°C, the only fluorescent lipid analogue identified in the incubation media and cell fractions was the one that was exogenously administered, indicating that no metabolism of either lipid had occurred during the time span of the experiments (data not shown). Taken together, the data clearly demonstrate that low temperature (18°C) can selectively impede apical to basolateral transcytosis of both C6-NBD-GlcCer and C6-NBD-SM. The observed accumulation of the analogues in the same subcellular compartments (SAC) located adjacent to the BC membrane, indicates that at least early in the trafficking pathway during apical to basolateral transcytosis, both C6-NBD-GlcCer and C6-NBD-SM pass through the same compartments, while before their arrival in SAC, significant sorting, proceeding their preferential rerouting to apical and basolateral region, respectively, has not yet occurred. The latter is supported by the striking similarity in semi-quantitative distribution of the two analogues (Fig. 3 B).
C6-NBD-SM and C6-NBD-GlcCer Flow from SAC to Distinct PM Domains
Since GlcCer and SM display a distinct preferential localization within the cell, we next examined the possibility as
to whether the compartments identified as SAC by accumulation of either lipid analogue, were functionally involved in redirecting their trafficking. Hence, to address
the issue of SAC acting as a sorting compartment, the cells
were labeled with C6-NBD-GlcCer or C6-NBD-SM, similarly as described above. After chasing the lipid analogues
into SAC at 18°C, NBD fluorescence still present in the
BC was irreversibly abolished by incubating the cells with
30 mM sodiumdithionite at 4°C for 10 min (Fig. 4; see Materials and Methods). After treatment, ~80-90% of the
identified BCP was still fluorescently labeled (Fig. 5 A),
but <10% of the total fluorescence (C6-NBD-GlcCer or
C6-NBD-SM) located in the bile canalicular area after
dithionite quenching, was due to labeling of BC (Fig. 5,
B and C, 0 min; for fluorescence images compare Fig. 4,
b/d vs. Fig. 2, b/d). Indeed, dithionite effectively abolishes
NBD fluorescence appearing at the luminal surface of the
bile canalicular space, while it does not gain access to intracellular sites at the conditions of the treatment (van
IJzendoorn et al., 1997). Consequently, labeling with the
lipid analogues was predominantly in SAC alone (Fig. 4),
representing >80% of the NBD-positive BCP fraction
(Fig. 5, B and C, SAC).
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The virtually exclusive labeling of SAC in the BCP region, as accomplished by this treatment thus allowed us, after extensive washing of the cells to remove the dithionite, to investigate the subsequent fate of C6-NBD-GlcCer and C6-NBD-SM associated with the SAC. To this end, transport was re-activated by rewarming and further incubating the cells in HBSS at 37°C for various time intervals. BSA was included in the medium to prevent re-internalization of the lipid analogues from the basolateral PM and subsequent basolateral to apical transcytosis. Over a 30-min time span, the percentage of BCP labeled with C6-NBD-GlcCer remained fairly constant at ~90%, whereas the percentage of C6-NBD-SM-labeled BCP decreased to ±55% after 30 min (Fig. 5 A). Thus, after re-activation of transport, C6-NBD-SM, in contrast to C6-NBD-GlcCer, disappeared from the apical membrane region. Indeed, while after a 30-min time interval the localization of the remaining fraction of C6-NBD-GlcCer in the BCP compartments was about equally distributed between BC and SAC (Fig. 5 B), <25% of the remaining fraction of the C6-NBD-SM fluorescence at the BCP was associated with BC. Rather, nearly 80% was found to be associated with SAC over the entire 30-min time interval (Fig. 5 C). From these data we conclude that after re-activation of transport of the lipid analogues from the SAC, C6-NBD-SM trafficking prefers a sub-apical to basolateral direction, while C6-NBD-GlcCer recycles from the SAC to BC thus remaining in the apical region of the cell. Hence, the results suggest that in the sub-apical vesicular structures C6-NBD-GlcCer and C6-NBD-SM are sorted and flow preferentially to the apical and basolateral membrane domains, respectively.
Sorting of Glycosphingolipid in SAC Is Highly Specific
To further determine the specificity of the sorting event in
the SAC, the trafficking of C6-NBD-GalCer was examined. Whereas C6-NBD-GlcCer and C6-NBD-SM have entirely different head groups (glucose versus phosphocholine, respectively), C6-NBD-GlcCer and C6-NBD-GalCer
are epimers (i.e., they differ in the spatial orientation of
only one hydroxyl moiety in the carbohydrate head
group). To label the bile canalicular membrane, the cells
were labeled with C6-NBD-GalCer at 37°C for 30 min and
back exchanged (according to Fig. 1, step 1 and 2). C6-NBD-GalCer was found to be transcytosed to the BC with
similar kinetics as those of the short chain analogues of
GlcCer and SM (not shown), implying bulk membrane
transport. BC-associated C6-NBD-GalCer (±85% of the
NBD-positive BCP was due to labeling of only BC) was
subsequently chased at 18°C for 60 min in BSA-containing
HBSS (Fig. 1, step 3). Similar to C6-NBD-GlcCer and -SM,
C6-NBD-GalCer was found to accumulate in SAC (not
shown). Cells were then subjected to steps 4 and 5 of the
incubation scheme (Fig. 1) to examine the exiting of the
lipid analogue from the SAC. Over a 30-min time interval, the percentage of C6-NBD-GalCer-labeled BCP decreased from 85% to 65% (Fig. 6 A). Compared with the
trafficking of C6-NBD-GlcCer and C6-NBD-SM (compare
with Fig. 5), C6-NBD-GalCer thus appeared to disappear
from the BCP, similarly as observed in the case of C6-NBD-SM. Consistently, as reported previously for the latter
analogue, GalCer also preferentially locates to the basolateral membrane, as revealed by fluorescence microscopy (not shown; compare with Fig. 3 in van IJzendoorn et al.,
1997). Examination of the distribution of the remaining
fraction of C6-NBD-GalCer in the apical area revealed,
that the vast majority (60-70%) of this fraction resided exclusively in SAC (Fig. 6 B), indicative of a lack of a substantial movement of C6-NBD-GalCer toward BC, similarly as observed for the SM analogue, and emphasizing
that like C6-NBD-SM, the glycolipid, after exiting SAC, disappeared from the apical region of the cell. Hence, the
data indicate that C6-NBD-GalCer, like C6-NBD-SM, is
preferentially targeted from the SAC to the basolateral
PM domain and, thus, appears to be sorted in this compartment from C6-NBD-GlcCer.
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Sorting at the Luminal Leaflet of SAC; Transcytotic Trafficking of C6-NBD-SM and C6-NBD-GlcCer Occurs by Vesicular Means
To further define the exclusiveness and specificity of SAC
in lipid sorting in the reverse transcytotic pathway in
HepG2 cells, it is important to establish the mechanism of
trafficking of both sphingolipids in HepG2 cells. This mechanism could involve monomeric flow or vesicle-mediated
transport or both. Monomeric flow could entail flip-flop
mechanisms across basolateral and apical membranes, mediated by the translocating activities (in outward direction) of MRP1 and MDR1, respectively (van Helvoort et al.,
1996; Roelofsen et al., 1997
). The evidence indicates that
the lipid pool, derived from the apical membrane and
arriving in SAC is derived from the exoplasmic leaflet of
the apical membrane. Thus, treatment of the cells before the
incubation with the lipid analogues (Fig. 1, step 1) with the
MDR1 inhibitor PSC 833, under conditions that completely blocked the expulsion of the MDR substrate
Rhodamine 123, did not affect delivery of either lipid analogue to the lumenal leaflet of BC as judged by the ability
of sodiumdithionite to reduce the entire pool of BC-associated NBD fluorescence (Fig. 7 A; compare with van
IJzendoorn et al., 1997
). This implies that the analogues
reached the apical membrane by vesicular transport, the
NBD-tagged lipids residing in the inner leaflet of the vesicular membrane. Note that NBD fluorescence, had it
been located in the cytoplasmic leaflet of the BC membranes, in contrast to that with a lumenal orientation,
would not be accessible to quenching by sodiumdithionite
under the experimental conditions (see Materials and
Methods; compare with Fig. 4). Therefore, the presence of any NBD fluorescence in the cytoplasmic BC leaflet resulting from impaired translocator activity would have
prompted us to classify such BC as NBD-positive. Identical results were obtained when the cells had been preincubated with MK 571 (compare with Fig. 7 A), a specific inhibitor of the function of basolaterally located MRP1,
which has been shown to partly overlap with the exclusively at the BC membrane localized MDR1 (Roelofsen
et al., 1997
). Thus neither MDR1 nor MRP1 were involved
in the delivery of C6-NBD-GlcCer and C6-NBD-SM to the
lumenal leaflet of BC.
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It is then reasonable to assume that subsequent transport from apical membrane to SAC is vesicle mediated, delivering the exoplasmic lipids to the inner leaflet of the SAC. Indeed, given the capacity of the analogues to readily engage in monomeric transfer and yet, their specific retainment in SAC at 18°C, the data are entirely consistent with a localization that is restricted to the inner leaflet of SAC membranes. Subsequent transport after sorting in this compartment most likely occurred by a vesicle-mediated mechanism. This was inferred from the following experiments. Monomeric trafficking from SAC would have resulted in a preferential delivery of C6-NBD-GlcCer to the cytoplasmic leaflet of the apical membrane and of C6-NBD-SM to the corresponding leaflet of the basolateral membrane. The subsequent appearance at the exoplasmic leaflet would then require translocation via MDR1 and MRP1, respectively. To investigate whether any fraction of the lipid analogues that had left the apical membrane could reach the inner leaflet of the basolateral PM, and thus become susceptible to translocation by MRP1, cells were first incubated according to steps 1 and 2 (see Fig. 1). MK 571 was added during step 2. The cells were then rewarmed to 37°C and further incubated in HBSS, supplemented with BSA and MK 571 for 1 h. As shown in Fig. 7 B, the fraction of lipid analogues that could be back exchanged from the basolateral surface was similar to that of non-treated cells. Subsequently, we examined transport of SAC-accumulated lipids. Thus, the cells were treated subsequent to step 4 (i.e., before chasing the lipid analogues from the SAC), with either PSC 833 or MK 571. However, in neither case did the treatment affect the preferential trafficking of C6-NBD-GlcCer and C6-NBD-SM to the apical or basolateral PM domain, respectively (compare with Fig. 5). Taken together, the results indicate that C6-NBD-SM and C6-NBD-GlcCer were not used as a substrate for the multi-drug resistance proteins at either PM domain and therefore must have been residing in the lumenal leaflet of vesicular compartments during all transport steps. Hence, the observed sorting of C6-NBD-SM and C6-NBD-GlcCer must have been restricted to the lumenal leaflet of the SAC.
Transcytosing pIgR-IgA Colocalizes with Apically Derived C6-NBD-Sphingolipids in the SAC
In MDCK cells, SAC (also referred to as ARC) have been
described that are part of the transcytotic pathway, as revealed by the trafficking of the pIgR-IgA receptor-ligand
complex, an established transcytotic marker (Apodaca
et al., 1994; Barosso and Sztul, 1994
). To further define the
identity of the SAC through which both C6-NBD-SM and
C6-NBD-GlcCer pass, the intracellular trafficking of fluorescently labeled IgA in HepG2 was examined, and related to that of the lipid analogues. HepG2 cells do not endogenously express the pIgR. Consistently, we were unable
to detect pIgR by indirect immunofluorescence using an
anti-pIgR mAb (not shown). However, some internalization of TxR-IgA was observed. Yet, this uptake could be
completely abolished by adding excess asialofetuin (not
shown), indicating that in HepG2 cells IgA can presumably also become internalized by the asialoglycoprotein
receptor, consistent with previous observations (Tomana
et al., 1988
). We therefore stably transfected HepG2 cells
with the pIgR (see Materials and Methods), which at
steady-state was distributed throughout the cytoplasm
with a preference for the apical region of the cell, and
monitored the internalization of IgA in the presence of excess asialofetuin. When cells were incubated with 50 µg/ml
TxR-IgA at 4°C for 30 min, washed, and then incubated at
37°C for short time intervals only (20 min or less), a substantial fraction of the intracellular fluorescence could already be detected in SAC (Fig. 8 a). Prolonged incubations at 37°C (Fig. 8 b), but not 18°C (Fig. 8 c), resulted in a
pronounced labeling of BC, indicating that pIgR-IgA traveled through SAC before being delivered to BC. Moreover, the delivery of sub-apically located pIgR-IgA to BC was impaired at 18°C. To determine whether the TxR-IgA-labeled SAC were the same as those in which apically
derived lipid analogues were found to accumulate (i.e.,
SAC [see Fig. 2, d and f]), cells were incubated with TxR-IgA at 4°C for 30 min, washed, and then incubated with
C6-NBD-SM at 37°C, allowing both compounds to enter the cells. After a back exchange of the fluorescent lipid
pool associated with the basolateral PM (see Fig. 1, step 2),
C6-NBD-SM was found to label intracellular vesicular
structures and BC (compare with Fig. 2, c and d; van
IJzendoorn et al., 1997
). Note that pretreatment with excess asialofetuin did not affect uptake and intracellular localization of the lipid analogue (not shown). In the same
cells, TxR-IgA was detected in both BC and vesicular structures located near the BC (Fig. 8 a). The cells, thus
containing intracellular pools of IgA and sphingolipid,
were subsequently incubated at 18°C for 1 h to allow apically derived lipid analogues to accumulate in SAC as described above. Interestingly, as evidenced by confocal laser scanning microscopy, TxR-IgA distribution displayed a
substantial overlap with C6-NBD-SM in the SAC (Fig. 8,
d-g, arrows). Similar results were obtained with C6-NBD-GlcCer and C6-NBD-GalCer (not shown). Note that the
data in Fig. 8 demonstrate a colocalization of lipid and
IgA, particularly in the prominently present SAC. Some
differences in localization of lipid analogue and IgA can
be readily explained by the notion that the lipid is transported from BC to SAC, whereas IgA enters SAC, arriving from the basolateral membrane. Also, the fate of the
subsequent flow differs, SM traveling to the basolateral
membrane, whereas IgA travels to the apical membrane
(see also Discussion). In conclusion, the results indicate
that the sorting compartment for simple monohexoylsphingolipids in the reverse transcytotic pathway in
HepG2 cells, SAC, displays the same identity as pIgR-IgA-
accumulating compartments identified in MDCK as ARC.
|
Transport of Apically Derived C6-NBD-GlcCer and -SM to SAC, but Not Apical Endocytosis of C6-NBD-GlcCer and -SM Is Affected by Nocodazole
The aforementioned results indicate that the mechanism
of sphingolipid trafficking between apical membrane and
SAC, is accomplished by vesicular transport. Many vesicular transport events appear to require an intact microtubule network. To investigate the involvement of microtubules in transport between the apical PM and the SAC, cells were incubated with C6-NBD-SM or -GlcCer at 37°C
for 30 min, after which the pool of fluorescent lipid analogues in the basolateral PM was depleted with BSA (Fig.
1, step 1 and 2). During this back exchange procedure 33 µM
nocodazole was included, a concentration sufficient to disrupt microtubules as revealed by immunofluorescence using a mAb directed against -tubulin (not shown). After
extensive washing, the cells were incubated at 18°C and
BC-associated lipid analogues were chased in back exchange medium (HBSS + 5% BSA), supplemented with
nocodazole for 1 h. Internalization of either lipid analogue
from the apical PM was not inhibited by nocodazole, as
judged by the similarities in the decrease of the percentage
of NBD-labeled BC, when compared with non-treated
cells (Table I). However, whereas in non-treated cells, intensely labeled SAC was observed after a 1-h chase at 18°C (Fig. 2, d and f), in nocodazole-treated cells the labeling of SAC was conspicuously absent. Rather, numerous
small vesicular structures, labeled with either C6-NBD-SM
or -GlcCer, were seen, distributed throughout the cytoplasm (Fig. 9, a-d). The effect of nocodazole was reversible, and C6-NBD-GlcCer- and -SM-labeled SAC appeared after a prolonged incubation in nocodazole-free
medium (Fig. 9, c and f, arrows). Concomitantly, immunofluorescence experiments to visualize
-tubulin (as described above) revealed that intact microtubules reappeared during this prolonged incubation (not shown).
|
|
Previously, it was demonstrated that the organization of
the sub-apical compartments (ARC) as described in
MDCK cells requires an intact microtubule network.
Thus, whereas the SAC in control cells were concentrated
in a centralized spot, they were found to be dispersed
throughout the apical cytoplasm in the nocodazole-treated cells (Apodaca et al., 1994). To investigate whether the organization of the SAC, as shown in Figs. 2 (d and f) and 4, was also dependent on intact microtubules, SAC were first
pre-loaded with either lipid analogue by chasing BC-associated lipid at 18°C for 1 h as described above. After
quenching of label in BC with dithionite (Fig. 1, step 4),
the cells were incubated in nocodazole-containing HBSS
at 4°C or 18°C for 1 h. Although the microtubule organization was effectively disrupted at either condition (see
above), nocodazole did not change the appearance of labeled SAC, implying that their appearance was indistinguishable from that in non-treated cells (Fig. 10, a and b).
However, under identical experimental conditions, nocodazole was found to be effective in disrupting C6-NBD-Cer-preloaded Golgi apparatus (Fig. 10, c and d; Turner
and Tartakoff, 1989
). The results thus indicate that rather
than (early) apical endocytosis or the organization of the
SAC, (a) distinct transport step(s) after endocytic internalization, but before the delivery to SAC, is dependent on
intact microtubules.
|
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Discussion |
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---|
In polarized HepG2 cells, the sphingolipid analogues
C6-NBD-GlcCer and C6-NBD-SM display a preferential
plasma membrane localization, the GlcCer derivative accumulating primarily at the apical membrane, while the
SM derivative prefers the basolateral membrane (van IJzendoorn et al., 1997). The mechanism underlying this
preferential distribution involves their internalization
from the apical, BC surface. For the trafficking of GlcCer,
a tentative role for a non-Golgi-related compartment was
proposed. In the present study, we have established the
identity of these compartments and their functional involvement in determining the distinct fate of several sphingolipids in the apical to basolateral pathway. Furthermore, the data demonstrate that the processing of C6-NBD-GlcCer and C6-NBD-SM in HepG2 cells is accomplished
by vesicular transport and that the preferential localization in either plasma membrane domain is not mediated
by interference of multi-drug resistance proteins, which
have been reported to display the ability to translocate lipid analogues from inner to outer leaflet across plasma
membranes (van Helvoort et al., 1997). Rather, after having reached the apical membrane by transcytosis, after initial insertion in the basolateral membrane, the lipid analogues were transported from the BC toward SAC, where
they accumulate at low (18°C) temperature, reflecting a
temperature-sensitive step in the exit of the lipids from
this compartment. In Fig. 11, a model depicting the central involvement of SAC in apical to basolateral transcytosis of
sphingolipids in HepG2 cells is presented. In this figure,
transport of the sphingolipids from BC to SAC is represented by steps 1 and 2. Transport to the SAC is microtubule dependent (Fig. 11, step 2) and represents a direct
pathway, i.e., it does not involve trafficking via the basolateral PM, as the presence of BSA in the incubation medium precludes any re-uptake and subsequent trafficking
of the lipids back to the apical pole. The results indicate that
SAC constitute an intrinsic part of the apical to basolateral
transcytotic pathway through which (glyco)sphingolipids
traverse the cells, bearing reminiscence of a sub-apical
compartment, previously identified as ARC in polarized
protein transport in MDCK cells (Apodaca et al., 1994
;
Barosso and Sztul, 1994
). However, an important distinction between both compartments is that the structural organization of ARC depends on an intact microtubule system, in marked contrast to SAC, the integrity of which
does not depend on microtubules. In this Golgi-unrelated
compartment, lipid sorting occurs, implying that GlcCer is
recycled to the apical membrane, whereas NBD derivatives from SM and GalCer are processed by vesicular
transport to the basolateral membrane (Fig. 11, step 3).
Hence, SAC appear to play a prominent role in maintaining lipid polarity in polarized hepatic cells.
|
Characteristics of the SAC
The identity of SAC as a compartment specifically associated with the transcytotic pathway in HepG2 cells, is supported by the observation that the transcytotic marker
IgA/pIgR traverses this compartment in a temperature-dependent manner. In polarized MDCK cells, its validity
as a transcytotic marker has been demonstrated, and low
temperature inhibits transcytosis of receptor-bound IgA by an impaired exit of this receptor-ligand complex from
structures located near the apical PM, i.e., ARCs. Indeed,
inhibitory effects of low temperature on transcytotic transport of proteins in polarized cells, without affecting endocytosis, have been shown before (Maratos-Flier, 1987;
Breitfeld et al., 1989; Hunziker and Mellman, 1989
; Apodaca et al., 1994
; Barosso and Sztul, 1994
). Moreover, IgA-
and transcytosing pIgR-containing vesicular structures located subjacent the apical PM have been described in rat
hepatic cells (Hoppe et al., 1985
; Barr and Hubbard, 1993
;
Geuze et al., 1984
; Hemery et al., 1996
) and in polarized
WIF-B cells (Ihrke et al., 1998
). HepG2 cells are of human
origin and do not express the pIgR. Expression was accomplished however by transfection and transfected
HepG2 cells were found to internalize TxR-IgA, which
was rapidly transported to the apical pole (Fig. 8 a), consistent with the anticipated fate of a transcytotic marker.
Interestingly, whereas intense labeling of BC was observed after longer incubations at 37°C (Fig. 8 b), TxR-IgA was not fully transcytosed to the apical surface at 18°C
but accumulated in SAC instead. Since TxR-IgA was observed in SAC before it appeared in the BC, the data suggest that SAC-associated TxR-IgA originated directly
from the basolateral area rather than from the apical PM
from which IgA (at least in rat hepatocytes) can be internalized (Jones et al., 1984). This conclusion is entirely compatible with the fate of pIgR in hepatic WIF-B cells
(Ihrke et al., 1998
). Importantly, apically derived C6-NBD-lipids were found to colocalize with transcytosing TxR-IgA in the SAC (Fig. 8, d vs. e, and f vs. g). Remarkably, in
contrast to apically derived C6-NBD-lipids, which were
found to colocalize with transcytosing TxR-IgA in SAC
(Fig. 8, d-g), basolaterally derived lipid was far less prominently trapped in SAC at 18°C. As demonstrated before, basolateral to apical trafficking of the sphingolipids proceeds by bulk flow, whereas segregation is seen in the apical to basolateral pathway (van IJzendoorn et al., 1997
).
The appreciation of the involvement of SAC in apically directed bulk flow trafficking of the lipid analogues therefore remains to be determined. The data emphasize however the involvement of SAC as a sorting compartment in
the segregation of the lipids, as occurs in the reverse pathway. Thus, the striking similarity with respect to the intracellular (sub-apical) localization and the temperature-dependent characteristics concerning the trafficking of
IgA via these compartments supports the view, that the
lipid-labeled SAC in HepG2 cells and the ARC in MDCK
cells are analogous. Ihrke et al. (1998)
proposed that SAC
in hepatic WIF-B cells are mainly a one-way sorting station in the basolateral to apical pathway, as they were unable to detect recycling resident apical proteins in similar
SAC by biochemical means (Barr and Hubbard, 1993
; Barr
et al., 1995
). However, our experiments, in which we are
able to directly monitor the endocytic flow of apical membrane in living cells, indicate that the transcytotic trafficking pathways between apical and basolateral membranes and vice versa do appear to merge in SAC. The relationship between lipid and protein traffic via these compartments is clearly an important issue that needs to be addressed in future studies.
Golgi markers are excluded from the apical recycling
compartments in MDCK cells, and an (apical) endosomal
nature of the compartment was proposed (Apodaca et al.,
1994). Two pieces of evidence indicate that also in HepG2
cells SAC appear to be unrelated to the Golgi apparatus.
First, whereas monensin inhibits sphingolipid transport from
the Golgi-apparatus to BC in HepG2 cells (van IJzendoorn et al., 1997
) as well as to the PM in non-polarized
cells (Lipsky and Pagano, 1985
; Kok et al., 1992
), apical to
basolateral transcytosis of C6-NBD-GlcCer and C6-NBD-SM in HepG2 cells is unaffected by this treatment. Second,
treatment of the cells with nocodazole, a microtubule-disrupting compound, clearly affected the morphology of the
Golgi apparatus, but had no visible effect on the morphology of SAC preloaded with fluorescent lipid analogue
(Fig. 10). Intriguingly however, this very feature distinguishes SAC from ARC in MDCK cells in that the integrity of the latter entirely depends on an intact microtubular organization (Apodaca et al., 1994
).
Interestingly, nocodazole inhibited transport of lipid analogues from BC to SAC (Fig. 9), but not apical endocytosis (Table I; Fig. 11, steps 1 and 2). Transport of proteins
and lipids from the PM to early "sorting" endosomes is
typically microtubule-independent (Hunziker et al., 1990;
Matter et al., 1990
; Kok et al., 1992
). By contrast, transport
of proteins and lipids from such early sorting endosomes
to a functionally and morphologically distinct set of endosomal compartments located in the perinuclear region (Gruenberg and Maxfield, 1995
) requires an intact microtubule network (Gruenberg et al., 1989
; Hunziker et al.,
1990
; Kok et al., 1992
). Moreover, the ARC appears to
share a striking homology with the perinuclear recycling
endosome, described in non-polarized cells (Zacchi et al.,
1998
). These features also seem to hold for the present system, when comparing the localization of lipid analogues
and pIgR in polarized and non-polarized HepG2 cells (Fig. 2, see below). Hence, in conjunction with its localization in the juxtanuclear/Golgi area facing the apical PM
(Gruenberg and Maxfield, 1995
), SAC thus bear reminiscence to a recycling endosomal compartment rather than
to an early sorting endosome, correlating with the pericentriolar localization of recycling endosomes in other polarized cells (Hughson and Hopkins, 1990
). Obviously, a detailed characterization of the localization of specific
markers (e.g., mannose-6-phosphate receptor; different rab proteins, etc.) will be required to obtain further insight into the exact nature of this compartment. However, given
that apical membrane derived lipid analogues and basolateral membrane derived IgA are both delivered to SAC
(Fig. 8), it would thus appear that the compartment acts as
a "traffic center" in (reverse) transcytotic trafficking in polarized cells. Fully compatible with this conclusion is a previous hypothesis that pathways originating from the apical
and basolateral pathways merge at a perinuclear endosomal compartment that is distinct from early (sorting) endosomes in polarized cells (Parton et al., 1989
; Hughes and
Hopkins, 1990).
Intriguingly, accumulation of the lipid analogues and
IgA was also observed in non-polarized HepG2 cells, i.e.,
cells that had not formed an apical, biliary PM domain.
This accumulation was typically in the juxtanuclear area of
the cells (Fig. 2, wide arrows), and was abolished in nocodazole-pretreated cells (Fig. 9), similarly as observed for
sphingolipid transport from early to perinuclear endosomes in non-polarized BHK cells (Kok et al., 1992).
These observations underscore that SAC is not a unique compartment, but rather that its specific functional features, including sorting in (reverse) transcytosis, are becoming apparent only in polarized cells. Indeed, after submission of this manuscript, Zacchi et al. (1998)
reported
that rab17, a small GTPase that has been shown to be induced upon cell polarization (Lütcke et al., 1993), associates
with both the ARC in polarized cells and the perinuclear
recycling endosome when expressed in non-polarized cells.
Sorting of Sphingolipids in SAC
After treatment with sodiumdithionite, NBD fluorescence
in BC, but not in SAC, was nearly completely eliminated
(Figs. 4 and 5, B and C, 0 min; compare with van IJzendoorn et al., 1997). This approach thus allows to monitor
subsequently the fate of the lipid analogues that have accumulated into SAC (at 18°C). Indeed, after a 30-min
chase at 37°C, the extent of BCP labeling with C6-NBD-SM
or C6-NBD-GalCer had decreased by >30% and 20%, respectively, compared with that with C6-NBD-GlcCer (Figs. 5
A and 6 A). This indicated that C6-NBD-SM and C6-NBD-GalCer, relative to C6-NBD-GlcCer, were transported out
of the bile canalicular region (Fig. 5 A and 6 A). Indeed,
whereas the fractions of both SM and GalCer that remained in the BCP area were primarily restricted to SAC,
that of GlcCer showed a different fate. Not only did the latter lipid show a strong preference for a localization in
the apical region of the cell, as reflected by the fact that
~90% of the BCP remained labeled with this lipid during
a chase of at least 30 min, there was also an apparent trend
of its time-dependent translocation from SAC to the BC.
Evidently, these kinetic changes in pool size and BCP localization (SAC versus BC) are entirely consistent with
the preferential localization of GlcCer in the apical and
that of SM in the basolateral region of the cell (van IJzendoorn et al., 1997
). Here, we show that GalCer also shows a distinct preference for a basolateral localization, although less pronounced than SM. The results indicate that
SAC determine the subsequent fate of the lipid analogues,
involving their flow from the SAC to preferential PM domains, and implying that sorting occurs in these compartments.
Presumably, segregation of the sphingolipids takes place
within the lumenal leaflet of the SAC, since inhibitors of
MDR1/P-glycoprotein and MRP1, previously shown to be
able to translocate C6-NBD-lipids over the PM (van Helvoort et al., 1996), did not affect the outcome of the experiments (Fig. 7). Hence, monomeric trafficking to the inner
leaflet of either plasma membrane, followed by outward
translocation by either MDR1 or MRP1, can be excluded.
Thus, a highly specific sorting event, given the remarkable
distinction that becomes apparent in the sorting of GlcCer
and its epimer, GalCer, and vesicular packaging underlie
the overall processing of the lipid analogues by SAC. In
this context it is interesting to mention that the calmodulin
antagonist W7 appears to have distinct effects on the trafficking of C6-NBD-GlcCer and C6-NBD-SM from the
SAC, thus supporting the proposed model in which
GlcCer and SM are segregated into distinct pools within
the SAC membranes, which can be separately recognized
by mechanisms that regulate trafficking (van IJzendoorn, S.C.D., and D. Hoekstra, unpublished results).
In the biosynthetic pathway, apical sorting in polarized
cells has mainly focused on a mechanism involving targeting of glycosphingolipid-enriched clusters or rafts, originating from the TGN (Simons and Ikonen, 1997; Weimbs
et al., 1997
). In addition, glycosphingolipid-enriched rafts
were also postulated to be involved in endocytotic and
transcytotic transport routes, mediated by caveolae. In the
present study, we present for the first time evidence that
clearly indicates a role for a non-Golgi-related intracellular compartment in the sorting and, consequently, polarized distribution of sphingolipids. However, the ubiquity
of this phenomenon remains to be determined, the site
and extent of sorting being dependent on cell type and nature of transport route (biosynthetic pathway, basolateral
to apical membrane flow, i.e., transcytosis, or the reverse
pathway). For example, sphingolipid sorting in the transcytotic pathway could not be demonstrated in MDCK cells
(van Genderen and van Meer, 1995
). Possibly, sorting of
sphingolipids in this pathway is restricted to hepatic cells,
which are thought to rely heavily on transcytosis for the
correct targeting and delivery of apical proteins (Bartles et
al., 1987
; Schell et al., 1992
). It is yet of interest however,
that in MDCK cells, C6-NBD-GlcCer and C6-NBD-SM, endocytosed from either PM domain reach the apical recycling compartments, where they colocalize with the IgA/
pIgR complex, before delivery to the opposite surface
(van IJzendoorn, S.C.D., K.E. Mostov, and D. Hoekstra,
manuscript in preparation). In MDCK cells, sorting of
newly synthesized sphingolipids is believed to occur predominantly in the biosynthetic pathway and the actual
sorting compartment was proposed to be the TGN (Simons and van Meer, 1988
). Although newly synthesized
C6-NBD-GlcCer and C6-NBD-SM can be directly transported to either PM domain in HepG2 cells (Zaal et al.,
1994; Zegers and Hoekstra, 1997
), sorting of sphingolipids
has not been demonstrated in hepatic cells yet. Also, the
direct Golgi to apical transport pathway is poorly characterized and the intriguing question remains as to whether
SAC are part of this route in HepG2 cells. Further research is imperative for a better understanding of the functional involvement of the SAC/ARCs in the different polarized cell types with respect to lipid trafficking in distinct
transport routes as well as the integration of lipid and protein trafficking. Such work is currently in progress in our
laboratory. The identification in HepG2 cells of a non-Golgi-related intracellular compartment, SAC, that is involved in the trafficking and sorting of sphingolipids and,
moreover, its similarity to the ARC in MDCK cells (Apodaca et al., 1994
), will be of great importance to further reveal and understand mechanisms underlying the generation and maintenance of membrane polarity.
![]() |
Footnotes |
---|
Received for publication 19 February 1998 and in revised form 29 June 1998.
Address all correspondence to Dick Hoekstra, Department of Physiological Chemistry, University of Groningen, A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands. Tel.: +31-50-3632741. Fax: +31-50-3632728. E-mail: d.hoekstra{at}med.rug.nlWe wish to thank Dr. K. Mostov for his kind gift of pIgR cDNA and anti-pIgR antibody; Dr. K. Dunn for kindly providing us with TxR-labeled IgA; Dr. E. Vellenga for the PSC833 and MK 571; Dr. H. Roelofsen for advice and assistance with confocal laser scanning microscopy; P.v.d. Syde and D. Huizinga for photographical work, and W. Visser for expert technical assistance with the transfections. We thank Drs. K. Mostov, J.W. Kok, and M. Zegers for helpful and stimulating discussions.
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Abbreviations used in this paper |
---|
ARC, apical recycling compartment; BC, bile canaliculus; BCP, bile canalicular pole; GalCer, galactosylceramide; GlcCer, glucosylceramide; GSL, glycosphingolipid; pIgR, polymeric immunoglobulin receptor; PM, plasma membrane; SAC, sub-apical compartments; SM, sphingomyelin; TxR, Texas red-labeled.
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