Segregation of Glucosylceramide and Sphingomyelin Occurs in the Apical to Basolateral Transcytotic Route in HepG2 Cells

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 chainlabeled 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°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 C6NBD-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.

Oncostatin M-stimulated Apical Plasma Membrane Biogenesis Requires p27Kip1-Regulated Cell Cycle Dynamics

“…Apical BC PM domains form between two adjacent cells, as readily visualized by staining the underlying actin network with fluorescently labeled phalloidin (van IJzendoorn et al, 1997). As shown in Figure 2A, HepG2 cells acquire PM polarity in time, as evidenced by the ratio BC/100 cells, and reach optimal polarity 3 d after plating (Figure 2A; c.f.…”

“…Briefly, BC were first identified by phase contrast illumination and then categorized as NBDpositive or NBD-negative under epifluorescence illumination. Note that a BC is categorized as fluorescently labeled, i.e., NBD-positive, when microvilli-like structures, characteristic of the BC, can be detected that are seemingly fluorescent in the wake of the fluorescence derived from the lipid analog, present in the apical membrane (van IJzendoorn et al, 1997). Such microvilli-like structures are typically and readily observed upon gradual photobleaching of the BC-associated NBD fluorescence.…”

“…To quantitate transport of the lipid analog to and from the BC, the percentage of NBD-positive BC was determined as described previously (van IJzendoorn et al, 1997;van IJzendoorn and Hoekstra, 1998). Briefly, BC were first identified by phase contrast illumination and then categorized as NBDpositive or NBD-negative under epifluorescence illumination.…”

Anchoring of Protein Kinase A-Regulatory Subunit IIα to Subapically Positioned Centrosomes Mediates Apical Bile Canalicular Lumen Development in Response to Oncostatin M but Not cAMP

“…To this end, the cells were incubated for 30 min at 4°C with 5% BSA in HBSS, followed by washing with ice-cold HBSS. This procedure was repeated once (van IJzendoorn et al, 1997). …”

“…After the preincubation, the cells were cooled to 4°C by washing with ice-cold HBSS. Subsequently, the basolateral plasma membrane (PM) was labeled with 4 M C 6 -NBD-SM for 30 min at 4°C in HBSS supplemented or not with OSM, which allows incorporation of the lipid probe in the exoplasmic leaflet of the basolateral plasma membrane while preventing its internalization (van IJzendoorn et al, 1997). When cells had been pretreated with OSM, the cytokine was included in all further incubations.…”