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Segregation of glucosylceramide and sphingomyelin occurs in the apical to basolateral transcytotic route in HepG2 cells.

van IJzendoorn SC, Zegers MM, Kok JW, Hoekstra D - J. Cell Biol. (1997)

Bottom Line: The redirection of C6-NBD-GlcCer did not involve trafficking via the Golgi apparatus.The data unambiguously demonstrate that segregation of GlcCer and SM occurs in the reverse transcytotic route, i.e., during apical to basolateral transport, which results in the preferential localization of GlcCer and SM in the apical and basolateral region of the cells, respectively.A role for non-Golgi-related, sub-apical vesicular compartments in the sorting of GlcCer and SM is proposed.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, University of Groningen, The Netherlands.

ABSTRACT
HepG2 cells are highly differentiated hepatoma cells that have retained an apical, bile canalicular (BC) plasma membrane polarity. We investigated the dynamics of two BC-associated sphingolipids, glucosylceramide (GlcCer) and sphingomyelin (SM). For this, the cells were labeled with fluorescent acyl chain-labeled 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]hexanoic acid (C6-NBD) derivatives of either GlcCer (C6-NBD-GlcCer) or SM (C6-NBD-SM). The pool of the fluorescent lipid analogues present in the basolateral plasma membrane domain was subsequently depleted and the apically located C6-NBD-lipid was chased at 37 degrees C. By using fluorescence microscopical analysis and a new assay that allows an accurate estimation of the fluorescent lipid pool in the apical membrane, qualitative and quantitative insight was obtained concerning kinetics, extent and (intra)cellular sites of the redistribution of apically located C6-NBD-GlcCer and C6-NBD-SM. It is demonstrated that both lipids display a preferential localization, C6-NBD-GlcCer in the apical and C6-NBD-SM in the basolateral area. Such a preference is expressed during transcytosis of both sphingolipids from the apical to the basolateral plasma membrane domain, a novel lipid trafficking route in HepG2 cells. Whereas the vast majority of the apically derived C6-NBD-SM was rapidly transcytosed to the basolateral surface, most of the apically internalized C6-NBD-GlcCer was efficiently redirected to the BC. The redirection of C6-NBD-GlcCer did not involve trafficking via the Golgi apparatus. Evidence is provided which suggests the involvement of vesicular compartments, located subjacent to the apical plasma membrane. Interestingly, the observed difference in preferential localization of C6-NBD-GlcCer and C6-NBD-SM was perturbed by treatment of the cells with dibutyryl cAMP, a stable cAMP analogue. While the preferential apical localization of C6-NBD-GlcCer was amplified, dibutyryl cAMP-treatment caused apically retrieved C6-NBD-SM to be processed via a similar pathway as that of C6-NBD-GlcCer. The data unambiguously demonstrate that segregation of GlcCer and SM occurs in the reverse transcytotic route, i.e., during apical to basolateral transport, which results in the preferential localization of GlcCer and SM in the apical and basolateral region of the cells, respectively. A role for non-Golgi-related, sub-apical vesicular compartments in the sorting of GlcCer and SM is proposed.

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Effect of db-cAMP on the trafficking of BC-derived C6-NBD-SM. Cells were labeled with 4 μM C6-NBD-SM at 37°C for 30  min, washed, and subjected to a back exchange procedure. During the back exchange 100 μM db-cAMP was added. To monitor intracellular trafficking of the BC-derived lipid analogue, cells were rewarmed to 37°C and further incubated in HBSS, supplemented with  100 μM db-cAMP for 60 min. C6-NBD-SM labeled mostly BC and BC-surrounding vesicles (b; arrowhead) (a, phase contrast to b). Bar, 15 μm.
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Figure 9: Effect of db-cAMP on the trafficking of BC-derived C6-NBD-SM. Cells were labeled with 4 μM C6-NBD-SM at 37°C for 30 min, washed, and subjected to a back exchange procedure. During the back exchange 100 μM db-cAMP was added. To monitor intracellular trafficking of the BC-derived lipid analogue, cells were rewarmed to 37°C and further incubated in HBSS, supplemented with 100 μM db-cAMP for 60 min. C6-NBD-SM labeled mostly BC and BC-surrounding vesicles (b; arrowhead) (a, phase contrast to b). Bar, 15 μm.

Mentions: Studies on apical endocytosis in various polarized cells have demonstrated that (a) apically internalized membrane proteins can recycle from a tubulovesicular compartment, located subjacent to the apical PM (Hunziker et al., 1990; Apodaca et al., 1994; Barroso and Sztul, 1994) and (b) transport between sub-apical compartments and the apical PM can be affected by modulators of distinct signal transduction pathways (Barroso and Sztul, 1994; Cardone et al., 1994; Hansen and Casanova, 1994). cAMP and stimulators of protein kinase A (PKA) which are both members of the adenylate cyclase-cAMP-PKA signal transducing pathway, have been shown to positively modulate apically directed transport (Brignoni et al., 1995; Pimplikar and Simons, 1994). To investigate a possible role for cAMP in the segregation of C6-NBD-GlcCer and C6-NBD-SM in the reverse transcytotic pathway, cells were labeled with either lipid after which the basolateral PM pool of C6-NBD-lipid was depleted by a back exchange as described above. During the back exchange procedure, which is carried out at 4°C, cells were preincubated with 100 μM db-cAMP. After the back exchange, the cells were rewarmed and further incubated in HBSS in the presence of 100 μM db-cAMP. As shown in Fig. 7, the percentage of NBD-positive BC in cells labeled with C6-NBD-GlcCer remained constant for 90 min, as was the case in control cells (cf. Fig. 3). However, whereas the percentage of NBD-positive BC in cells labeled with C6-NBD-SM rapidly decreased in control cells (cf. Fig. 3), this percentage of labeling remained virtually the same, for up to 90 min, in db-cAMP–treated cells (Fig. 7). Although (fractions of) both lipids could still be transcytosed to the basolateral surface, as reflected by their back exchangeability after chasing BC-associated C6-NBD-lipid in the presence of BSA, basolateral trafficking of both lipids was severely slowed down with regard to rate and extent (Fig. 8) in comparison to the basolateral flow in nontreated cells (cf. Fig. 6). Microscopic examination revealed that in cells, not treated with dbcAMP, fluorescently labeled vesicles surrounding the BC were most prominently seen in cells labeled with C6-NBD-GlcCer (Fig. 5). Interestingly, although essentially no BC-surrounding C6-NBDSM–labeled vesicles could be observed in nontreated cells (Fig. 5 b), such sub-apical NBD-fluorescent vesicles were readily distinguishable in db-cAMP–treated cells (Fig. 9). Hence, db-cAMP favored and/or promoted apically directed trafficking of both sphingolipids.


Segregation of glucosylceramide and sphingomyelin occurs in the apical to basolateral transcytotic route in HepG2 cells.

van IJzendoorn SC, Zegers MM, Kok JW, Hoekstra D - J. Cell Biol. (1997)

Effect of db-cAMP on the trafficking of BC-derived C6-NBD-SM. Cells were labeled with 4 μM C6-NBD-SM at 37°C for 30  min, washed, and subjected to a back exchange procedure. During the back exchange 100 μM db-cAMP was added. To monitor intracellular trafficking of the BC-derived lipid analogue, cells were rewarmed to 37°C and further incubated in HBSS, supplemented with  100 μM db-cAMP for 60 min. C6-NBD-SM labeled mostly BC and BC-surrounding vesicles (b; arrowhead) (a, phase contrast to b). Bar, 15 μm.
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Related In: Results  -  Collection

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Figure 9: Effect of db-cAMP on the trafficking of BC-derived C6-NBD-SM. Cells were labeled with 4 μM C6-NBD-SM at 37°C for 30 min, washed, and subjected to a back exchange procedure. During the back exchange 100 μM db-cAMP was added. To monitor intracellular trafficking of the BC-derived lipid analogue, cells were rewarmed to 37°C and further incubated in HBSS, supplemented with 100 μM db-cAMP for 60 min. C6-NBD-SM labeled mostly BC and BC-surrounding vesicles (b; arrowhead) (a, phase contrast to b). Bar, 15 μm.
Mentions: Studies on apical endocytosis in various polarized cells have demonstrated that (a) apically internalized membrane proteins can recycle from a tubulovesicular compartment, located subjacent to the apical PM (Hunziker et al., 1990; Apodaca et al., 1994; Barroso and Sztul, 1994) and (b) transport between sub-apical compartments and the apical PM can be affected by modulators of distinct signal transduction pathways (Barroso and Sztul, 1994; Cardone et al., 1994; Hansen and Casanova, 1994). cAMP and stimulators of protein kinase A (PKA) which are both members of the adenylate cyclase-cAMP-PKA signal transducing pathway, have been shown to positively modulate apically directed transport (Brignoni et al., 1995; Pimplikar and Simons, 1994). To investigate a possible role for cAMP in the segregation of C6-NBD-GlcCer and C6-NBD-SM in the reverse transcytotic pathway, cells were labeled with either lipid after which the basolateral PM pool of C6-NBD-lipid was depleted by a back exchange as described above. During the back exchange procedure, which is carried out at 4°C, cells were preincubated with 100 μM db-cAMP. After the back exchange, the cells were rewarmed and further incubated in HBSS in the presence of 100 μM db-cAMP. As shown in Fig. 7, the percentage of NBD-positive BC in cells labeled with C6-NBD-GlcCer remained constant for 90 min, as was the case in control cells (cf. Fig. 3). However, whereas the percentage of NBD-positive BC in cells labeled with C6-NBD-SM rapidly decreased in control cells (cf. Fig. 3), this percentage of labeling remained virtually the same, for up to 90 min, in db-cAMP–treated cells (Fig. 7). Although (fractions of) both lipids could still be transcytosed to the basolateral surface, as reflected by their back exchangeability after chasing BC-associated C6-NBD-lipid in the presence of BSA, basolateral trafficking of both lipids was severely slowed down with regard to rate and extent (Fig. 8) in comparison to the basolateral flow in nontreated cells (cf. Fig. 6). Microscopic examination revealed that in cells, not treated with dbcAMP, fluorescently labeled vesicles surrounding the BC were most prominently seen in cells labeled with C6-NBD-GlcCer (Fig. 5). Interestingly, although essentially no BC-surrounding C6-NBDSM–labeled vesicles could be observed in nontreated cells (Fig. 5 b), such sub-apical NBD-fluorescent vesicles were readily distinguishable in db-cAMP–treated cells (Fig. 9). Hence, db-cAMP favored and/or promoted apically directed trafficking of both sphingolipids.

Bottom Line: The redirection of C6-NBD-GlcCer did not involve trafficking via the Golgi apparatus.The data unambiguously demonstrate that segregation of GlcCer and SM occurs in the reverse transcytotic route, i.e., during apical to basolateral transport, which results in the preferential localization of GlcCer and SM in the apical and basolateral region of the cells, respectively.A role for non-Golgi-related, sub-apical vesicular compartments in the sorting of GlcCer and SM is proposed.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, University of Groningen, The Netherlands.

ABSTRACT
HepG2 cells are highly differentiated hepatoma cells that have retained an apical, bile canalicular (BC) plasma membrane polarity. We investigated the dynamics of two BC-associated sphingolipids, glucosylceramide (GlcCer) and sphingomyelin (SM). For this, the cells were labeled with fluorescent acyl chain-labeled 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]hexanoic acid (C6-NBD) derivatives of either GlcCer (C6-NBD-GlcCer) or SM (C6-NBD-SM). The pool of the fluorescent lipid analogues present in the basolateral plasma membrane domain was subsequently depleted and the apically located C6-NBD-lipid was chased at 37 degrees C. By using fluorescence microscopical analysis and a new assay that allows an accurate estimation of the fluorescent lipid pool in the apical membrane, qualitative and quantitative insight was obtained concerning kinetics, extent and (intra)cellular sites of the redistribution of apically located C6-NBD-GlcCer and C6-NBD-SM. It is demonstrated that both lipids display a preferential localization, C6-NBD-GlcCer in the apical and C6-NBD-SM in the basolateral area. Such a preference is expressed during transcytosis of both sphingolipids from the apical to the basolateral plasma membrane domain, a novel lipid trafficking route in HepG2 cells. Whereas the vast majority of the apically derived C6-NBD-SM was rapidly transcytosed to the basolateral surface, most of the apically internalized C6-NBD-GlcCer was efficiently redirected to the BC. The redirection of C6-NBD-GlcCer did not involve trafficking via the Golgi apparatus. Evidence is provided which suggests the involvement of vesicular compartments, located subjacent to the apical plasma membrane. Interestingly, the observed difference in preferential localization of C6-NBD-GlcCer and C6-NBD-SM was perturbed by treatment of the cells with dibutyryl cAMP, a stable cAMP analogue. While the preferential apical localization of C6-NBD-GlcCer was amplified, dibutyryl cAMP-treatment caused apically retrieved C6-NBD-SM to be processed via a similar pathway as that of C6-NBD-GlcCer. The data unambiguously demonstrate that segregation of GlcCer and SM occurs in the reverse transcytotic route, i.e., during apical to basolateral transport, which results in the preferential localization of GlcCer and SM in the apical and basolateral region of the cells, respectively. A role for non-Golgi-related, sub-apical vesicular compartments in the sorting of GlcCer and SM is proposed.

Show MeSH
Related in: MedlinePlus