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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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Labeling of HepG2 cells with exogenously administered C6-NBD-GlcCer and C6-NBD-SM. Cells were labeled with 4 μM C6NBD-GlcCer or C6-NBD-SM at 37°C for 30 min. Both lipid analogues were present in the BC (arrowheads), the basolateral PM and intracellular vesicles (b, C6-NBD-GlcCer; d, C6-NBD-SM) (a and c, phase contrast to b and d). Note that in order to visualize intracellular  fluorescence in b and d, basolateral membrane-located lipid analogues were partly removed by a brief back exchange procedure (5–10  min, 4°C). Alternatively, cells incubated as in a and c were subsequently subjected to a back exchange procedure at 4°C for 30 min to  completely remove the basolateral PM pool of lipid analogues. After back exchange, the cells were incubated in HBSS, supplemented  with 30 mM sodiumdithionite at 4°C for 7 min. After this incubation, the cells were washed 15 times with ice-cold HBSS to remove the  sodiumdithionite. Note that after sodiumdithionite treatment C6-NBD-GlcCer (f, phase contrast to e) nor C6-NBD-SM (h, phase contrast to g) were detectable in the BC (arrowheads). Bar, 15 μm.
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Figure 1: Labeling of HepG2 cells with exogenously administered C6-NBD-GlcCer and C6-NBD-SM. Cells were labeled with 4 μM C6NBD-GlcCer or C6-NBD-SM at 37°C for 30 min. Both lipid analogues were present in the BC (arrowheads), the basolateral PM and intracellular vesicles (b, C6-NBD-GlcCer; d, C6-NBD-SM) (a and c, phase contrast to b and d). Note that in order to visualize intracellular fluorescence in b and d, basolateral membrane-located lipid analogues were partly removed by a brief back exchange procedure (5–10 min, 4°C). Alternatively, cells incubated as in a and c were subsequently subjected to a back exchange procedure at 4°C for 30 min to completely remove the basolateral PM pool of lipid analogues. After back exchange, the cells were incubated in HBSS, supplemented with 30 mM sodiumdithionite at 4°C for 7 min. After this incubation, the cells were washed 15 times with ice-cold HBSS to remove the sodiumdithionite. Note that after sodiumdithionite treatment C6-NBD-GlcCer (f, phase contrast to e) nor C6-NBD-SM (h, phase contrast to g) were detectable in the BC (arrowheads). Bar, 15 μm.

Mentions: Fluorescent BC labeling was quantified by the use of two different approaches: (1) Determination of the percentage NBD-positive BC and (2) calculation of BC-associated NBD-fluorescence. To determine the percentage of BC labeled with C6-NBD-lipid, BC were first identified by phase contrast illumination, and then classified as being NBD-positive or NBD-negative under epifluorescence illumination using a filter set for blue excitation (BP 470-490/LP515). BC were only classified as being NBD-positive when labeled microvilli could be observed (cf. Figs. 1 and 2 B). Quantitative determination of BC-associated C6-NBD-lipid was performed by using the capacity of sodiumdithionite to rapidly reduce NBDfluorescence in the lumen and/or lumenal leaflet of the BC (see Results). For this, cells were grown in plastic 25 cm2 culture flasks and labeled with the fluorescent lipid analogues as described above. To determine the amount of BC-associated lipid, the cells were first subjected to a BSA/ HBSS back exchange procedure, as described above, to remove the basolateral PM pool of C6-NBD-lipid. Note that BSA has no access to the BC domain (see Results). By contrast, we observed that the irreversible NBDquencher sodiumdithionite (McIntyre and Sleight, 1991) does acquire access to this membrane domain. Hence, after a BSA-back exchange, the cells were subsequently incubated with 30 mM sodiumdithionite in HBSS (diluted from a stock solution of 1 M dithionite in 1 M Tris-buffer, pH 10) at 4°C for 7 min. As a control, cells were incubated in HBSS in the absence of sodiumdithionite at 4°C for 7 min. After extensively washing (15 times with HBSS), cells were scraped, lipid was extracted, and fluorescence was measured as described above. The amount of C6-NBD-lipid (expressed as pmol/mg protein) that was associated with BC was calculated from the following equation: NBD-fluorescenceBC = NBD-fluorescencecontrol − NBDfluorescencedithionite-treated.


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)

Labeling of HepG2 cells with exogenously administered C6-NBD-GlcCer and C6-NBD-SM. Cells were labeled with 4 μM C6NBD-GlcCer or C6-NBD-SM at 37°C for 30 min. Both lipid analogues were present in the BC (arrowheads), the basolateral PM and intracellular vesicles (b, C6-NBD-GlcCer; d, C6-NBD-SM) (a and c, phase contrast to b and d). Note that in order to visualize intracellular  fluorescence in b and d, basolateral membrane-located lipid analogues were partly removed by a brief back exchange procedure (5–10  min, 4°C). Alternatively, cells incubated as in a and c were subsequently subjected to a back exchange procedure at 4°C for 30 min to  completely remove the basolateral PM pool of lipid analogues. After back exchange, the cells were incubated in HBSS, supplemented  with 30 mM sodiumdithionite at 4°C for 7 min. After this incubation, the cells were washed 15 times with ice-cold HBSS to remove the  sodiumdithionite. Note that after sodiumdithionite treatment C6-NBD-GlcCer (f, phase contrast to e) nor C6-NBD-SM (h, phase contrast to g) were detectable in the BC (arrowheads). Bar, 15 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139765&req=5

Figure 1: Labeling of HepG2 cells with exogenously administered C6-NBD-GlcCer and C6-NBD-SM. Cells were labeled with 4 μM C6NBD-GlcCer or C6-NBD-SM at 37°C for 30 min. Both lipid analogues were present in the BC (arrowheads), the basolateral PM and intracellular vesicles (b, C6-NBD-GlcCer; d, C6-NBD-SM) (a and c, phase contrast to b and d). Note that in order to visualize intracellular fluorescence in b and d, basolateral membrane-located lipid analogues were partly removed by a brief back exchange procedure (5–10 min, 4°C). Alternatively, cells incubated as in a and c were subsequently subjected to a back exchange procedure at 4°C for 30 min to completely remove the basolateral PM pool of lipid analogues. After back exchange, the cells were incubated in HBSS, supplemented with 30 mM sodiumdithionite at 4°C for 7 min. After this incubation, the cells were washed 15 times with ice-cold HBSS to remove the sodiumdithionite. Note that after sodiumdithionite treatment C6-NBD-GlcCer (f, phase contrast to e) nor C6-NBD-SM (h, phase contrast to g) were detectable in the BC (arrowheads). Bar, 15 μm.
Mentions: Fluorescent BC labeling was quantified by the use of two different approaches: (1) Determination of the percentage NBD-positive BC and (2) calculation of BC-associated NBD-fluorescence. To determine the percentage of BC labeled with C6-NBD-lipid, BC were first identified by phase contrast illumination, and then classified as being NBD-positive or NBD-negative under epifluorescence illumination using a filter set for blue excitation (BP 470-490/LP515). BC were only classified as being NBD-positive when labeled microvilli could be observed (cf. Figs. 1 and 2 B). Quantitative determination of BC-associated C6-NBD-lipid was performed by using the capacity of sodiumdithionite to rapidly reduce NBDfluorescence in the lumen and/or lumenal leaflet of the BC (see Results). For this, cells were grown in plastic 25 cm2 culture flasks and labeled with the fluorescent lipid analogues as described above. To determine the amount of BC-associated lipid, the cells were first subjected to a BSA/ HBSS back exchange procedure, as described above, to remove the basolateral PM pool of C6-NBD-lipid. Note that BSA has no access to the BC domain (see Results). By contrast, we observed that the irreversible NBDquencher sodiumdithionite (McIntyre and Sleight, 1991) does acquire access to this membrane domain. Hence, after a BSA-back exchange, the cells were subsequently incubated with 30 mM sodiumdithionite in HBSS (diluted from a stock solution of 1 M dithionite in 1 M Tris-buffer, pH 10) at 4°C for 7 min. As a control, cells were incubated in HBSS in the absence of sodiumdithionite at 4°C for 7 min. After extensively washing (15 times with HBSS), cells were scraped, lipid was extracted, and fluorescence was measured as described above. The amount of C6-NBD-lipid (expressed as pmol/mg protein) that was associated with BC was calculated from the following equation: NBD-fluorescenceBC = NBD-fluorescencecontrol − NBDfluorescencedithionite-treated.

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