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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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BC-associated C6-NBD-GlcCer and C6-NBD-SM after  a chase in HBSS. HepG2 cells were grown in plastic 25 cm2 culture flasks for 3 d. Cells were loaded with 4 μM C6-NBD-GlcCer  or C6-NBD-SM at 37°C for 30 min. Then, C6-NBD-lipid residing  at the basolateral surface was depleted by BSA and cells were  subsequently rewarmed to 37°C and further incubated in HBSS  for 0, 30, 60, or 90 min. After another back exchange, the cells  were incubated in HBSS (control) or HBSS supplemented with  30 mM sodiumdithionite (treated) at 4°C for 7 min. The amount  of BC-associated C6-NBD-GlcCer (filled symbols) or C6-NBD-SM  (open symbols) was then determined as described in Materials  and Methods. Data are given as mean ± SD of 4–6 experiments.  *P < 0.05.
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Figure 4: BC-associated C6-NBD-GlcCer and C6-NBD-SM after a chase in HBSS. HepG2 cells were grown in plastic 25 cm2 culture flasks for 3 d. Cells were loaded with 4 μM C6-NBD-GlcCer or C6-NBD-SM at 37°C for 30 min. Then, C6-NBD-lipid residing at the basolateral surface was depleted by BSA and cells were subsequently rewarmed to 37°C and further incubated in HBSS for 0, 30, 60, or 90 min. After another back exchange, the cells were incubated in HBSS (control) or HBSS supplemented with 30 mM sodiumdithionite (treated) at 4°C for 7 min. The amount of BC-associated C6-NBD-GlcCer (filled symbols) or C6-NBD-SM (open symbols) was then determined as described in Materials and Methods. Data are given as mean ± SD of 4–6 experiments. *P < 0.05.

Mentions: After labeling the cells (30 min, 37°C) with either C6NBD-GlcCer or C6-NBD-SM and a subsequent depletion of the C6-NBD-lipid from the basolateral PM, the cells were rewarmed to 37°C and incubated in HBSS for 30, 60, or 90 min. Over a 90-min incubation period, the percentage of BC labeled with C6-NBD-GlcCer, as determined by fluorescence microscopy, remained nearly constant (Fig. 3, hatched bars). In cells labeled with C6-NBD-SM (Fig. 3, cross-hatched bars), the percentage of fluorescently labeled BC progressively decreased from 71.5 ± 8.8% at time zero to 44.0 ± 2.4% after 60 min, after which the number of fluorescently labeled BC remained constant. However, although the percentage of C6-NBD-SM–labeled BC, as detected by microscopical examination, did not further decline, the BC-associated fluorescence intensity did. As shown in Fig. 4, after a 60-min incubation period, the amount of BC-associated C6-NBD-SM was hardly detectable anymore, as revealed by using the sodiumdithionite quenching assay. By contrast, and consistent with the microscopic observations, the amount of BC-associated C6NBD-GlcCer slightly decreased from 40 to ∼30% of the total pool of cell-associated fluorescence (Fig. 4, filled symbols). Indeed, fluorescence microscopical analysis revealed that C6-NBD-GlcCer remained mainly associated with BC and was also found in clusters of vesicles surrounding the BC (Fig. 5 a). Only a relatively faint labeling of the basolateral PM could be observed. After 60 min, C6NBD-SM was found to label mostly the basolateral PM while labeling of BC was hardly distinguishable (Fig. 5 b, compare to Fig. 1 d). Apparently, apically located C6NBD-GlcCer and C6-NBD-SM were segregated for either the apical or basolateral domains in HepG2 cells, respectively. Moreover, this segregation step appears to occur subsequent to basolateral to apical transport of the lipid analogues, i.e., after insertion of the lipid analogues in the apical membrane.


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)

BC-associated C6-NBD-GlcCer and C6-NBD-SM after  a chase in HBSS. HepG2 cells were grown in plastic 25 cm2 culture flasks for 3 d. Cells were loaded with 4 μM C6-NBD-GlcCer  or C6-NBD-SM at 37°C for 30 min. Then, C6-NBD-lipid residing  at the basolateral surface was depleted by BSA and cells were  subsequently rewarmed to 37°C and further incubated in HBSS  for 0, 30, 60, or 90 min. After another back exchange, the cells  were incubated in HBSS (control) or HBSS supplemented with  30 mM sodiumdithionite (treated) at 4°C for 7 min. The amount  of BC-associated C6-NBD-GlcCer (filled symbols) or C6-NBD-SM  (open symbols) was then determined as described in Materials  and Methods. Data are given as mean ± SD of 4–6 experiments.  *P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139765&req=5

Figure 4: BC-associated C6-NBD-GlcCer and C6-NBD-SM after a chase in HBSS. HepG2 cells were grown in plastic 25 cm2 culture flasks for 3 d. Cells were loaded with 4 μM C6-NBD-GlcCer or C6-NBD-SM at 37°C for 30 min. Then, C6-NBD-lipid residing at the basolateral surface was depleted by BSA and cells were subsequently rewarmed to 37°C and further incubated in HBSS for 0, 30, 60, or 90 min. After another back exchange, the cells were incubated in HBSS (control) or HBSS supplemented with 30 mM sodiumdithionite (treated) at 4°C for 7 min. The amount of BC-associated C6-NBD-GlcCer (filled symbols) or C6-NBD-SM (open symbols) was then determined as described in Materials and Methods. Data are given as mean ± SD of 4–6 experiments. *P < 0.05.
Mentions: After labeling the cells (30 min, 37°C) with either C6NBD-GlcCer or C6-NBD-SM and a subsequent depletion of the C6-NBD-lipid from the basolateral PM, the cells were rewarmed to 37°C and incubated in HBSS for 30, 60, or 90 min. Over a 90-min incubation period, the percentage of BC labeled with C6-NBD-GlcCer, as determined by fluorescence microscopy, remained nearly constant (Fig. 3, hatched bars). In cells labeled with C6-NBD-SM (Fig. 3, cross-hatched bars), the percentage of fluorescently labeled BC progressively decreased from 71.5 ± 8.8% at time zero to 44.0 ± 2.4% after 60 min, after which the number of fluorescently labeled BC remained constant. However, although the percentage of C6-NBD-SM–labeled BC, as detected by microscopical examination, did not further decline, the BC-associated fluorescence intensity did. As shown in Fig. 4, after a 60-min incubation period, the amount of BC-associated C6-NBD-SM was hardly detectable anymore, as revealed by using the sodiumdithionite quenching assay. By contrast, and consistent with the microscopic observations, the amount of BC-associated C6NBD-GlcCer slightly decreased from 40 to ∼30% of the total pool of cell-associated fluorescence (Fig. 4, filled symbols). Indeed, fluorescence microscopical analysis revealed that C6-NBD-GlcCer remained mainly associated with BC and was also found in clusters of vesicles surrounding the BC (Fig. 5 a). Only a relatively faint labeling of the basolateral PM could be observed. After 60 min, C6NBD-SM was found to label mostly the basolateral PM while labeling of BC was hardly distinguishable (Fig. 5 b, compare to Fig. 1 d). Apparently, apically located C6NBD-GlcCer and C6-NBD-SM were segregated for either the apical or basolateral domains in HepG2 cells, respectively. Moreover, this segregation step appears to occur subsequent to basolateral to apical transport of the lipid analogues, i.e., after insertion of the lipid analogues in the apical membrane.

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