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. 2003 Jul;14(7):2689-705.
doi: 10.1091/mbc.e02-12-0816. Epub 2003 Apr 4.

Transcytotic efflux from early endosomes is dependent on cholesterol and glycosphingolipids in polarized hepatic cells

Lydia K Nyasae  1 , Ann L Hubbard, Pamela L Tuma
Affiliations

Transcytotic efflux from early endosomes is dependent on cholesterol and glycosphingolipids in polarized hepatic cells

Lydia K Nyasae et al. Mol Biol Cell. 2003 Jul.

Abstract

We examined the role that lipid rafts play in regulating apical protein trafficking in polarized hepatic cells. Rafts are postulated to form in the trans-Golgi network where they recruit newly synthesized apical residents and mediate their direct transport to the apical plasma membrane. In hepatocytes, single transmembrane and glycolipid-anchored apical proteins take the "indirect" route. They are transported from the trans-Golgi to the basolateral plasma membrane where they are endocytosed and transcytosed to the apical surface. Do rafts sort hepatic apical proteins along this circuitous pathway? We took two approaches to answer this question. First, we determined the detergent solubility of selected apical proteins and where in the biosynthetic pathway insolubility was acquired. Second, we used pharmacological agents to deplete raft components and assessed their effects on basolateral-to-apical transcytosis. We found that cholesterol and glycosphingolipids are required for delivery from basolateral early endosomes to the subapical compartment. In contrast, fluid phase uptake and clathrin-mediated internalization of recycling receptors were only mildly impaired. Apical protein solubility did not correlate with raft depletion or impaired transcytosis, suggesting other factors contribute to apical protein insolubility. Examination of apical proteins in Fao cells also revealed that raft-dependent sorting does not require the polarized cell context.

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Figures

Figure 3.
Figure 3.
Cholesterol is rapidly depleted in WIF-B cells treated with mβCD, but the GPI-anchored apical residents remain detergent insoluble. (A) WIF-B cells were treated for the indicated times (in minutes) in LPDM containing 5 mM mβCD. Total lipids were extracted, separated by TLC and visualized by charring. Duplicate samples for each time point are shown. (B) Coverslips processed in parallel to those in A were extracted in 1% Triton X-100 for 30 min on ice and the soluble and insoluble fractions were separated by centrifugation. The soluble (S) and pelleted (P) fractions were analyzed by Western blotting with the indicated antibodies. (C) The relative levels of immunoreactive species in the soluble and insoluble fractions as shown in B were determined by densitometric comparison of immunoreactive bands, and the values for the insoluble populations are plotted. Values are expressed as the mean ± SD. Measurements were done on at least three experiments each performed in duplicate. std, cholesterol standard.
Figure 5.
Figure 5.
Glycosphingolipids are depleted in WIF-B cells treated with FB1, but the solubility properties of the apical residents do not change. (A) WIF-B cells were treated for the indicated times in medium containing 25 μM FB1. Total lipids were extracted, separated by TLC and visualized by charring. Duplicate samples for each time point are shown. (B) WIF-B cells were treated for 48 h in the absence or presence of FB1, extracted in 1% Triton X-100 at 4°C for 30 min and the soluble and insoluble fractions were separated by centrifugation. The soluble (S) and pelleted (P) fractions were analyzed by Western blotting with the indicated antibodies. (C) In the left-hand panels, WIF-B cells were treated with LPDM containing 5 mM mβCD and 25 μM FB1. The mβCD was added in the final hour of the 48-h FB1 treatment. In the middle panels, cells were treated with 10 μM cytochalasin D (CD) or latranculin B (lat B) for 60 min and in the right hand panels, cells were treated in LPDM containing both cytochalasin D and mβCD. Detergent extractions and sample processing were performed as described in B. Representative TLC and Western blotting results from three independent experiments are shown. std, standard; chol., cholesterol.
Figure 1.
Figure 1.
A subset of apical PM residents in polarized WIF-B cells is insoluble in Triton X-100. (A) WIF-B cells were extracted in 1% Triton X-100 at 4°C for 30 min and the soluble and insoluble fractions were separated by centrifugation. The soluble (S) and pelleted (P) fractions were analyzed by Western blotting with the indicated antibodies. The fraction of the total population that was detergent insoluble for each molecule is indicated on the right (% Insoluble). Values are expressed as the mean ± SD. Measurements were done on at least three experiments each performed in duplicate. (B) 5′NT steady-state distributions into the S and P fractions after Triton X-100 extraction are shown (–IP). The pelleted fraction was resuspended in solubilization buffer and the soluble fraction corrected for solubilization buffer components (see MATERIALS AND METHODS). The fractions were then incubated for 3 min at 100°C (S1, P1) or at room temperature for 30 min (S2, P2) or 60 min (S3, P3). The fractions were immunoprecipitated with 5′NT monoclonal antibodies and immunoblotted with 5′NT polyclonal antibodies (+IP). None of the 5′NT in the pelleted fractions was immunoprecipitated (compare P with P1, P2 or P3). (C) WIF-B cells were extracted in 1% Triton X-100 for 30 min at 4 or 37°C and the soluble and insoluble fractions separated by centrifugation. The S and P fractions were analyzed by Western blotting with 5′NT antibodies (–IP). The resultant soluble fractions were immunoprecipitated with 5′NT monoclonal antibodies and immunoblotted with 5′NT polyclonal antibodies (+IP). Duplicate samples are shown. 5′NT was quantitatively immunoprecipitated from both the 4 and 37°C soluble fractions. Only 16% of the starting material was loaded in the –IP samples, whereas the total immunoprecipitation was analyzed in the +IP lanes.
Figure 2.
Figure 2.
Apical residents acquire detergent insolubility with different kinetics and at different places in the biosynthetic pipeline. (A) WIF-B cells were pulse-labeled with 35S-amino acids for 10 min and chased at 37°C for the indicated times. Cells were then extracted in 1% Triton X-100 for 30 min at 4°C or 15 min at 37°C and the soluble and insoluble fractions separated by centrifugation. Immunoprecipitations were performed on the fractions with the indicated antibodies and processed for autoradiography. Values are expressed as the mean ± SD. Measurements were done on at least three experiments each performed in duplicate. Autoradiographs from representative experiments are shown. For each time point in a and c, the soluble (left lane) and insoluble (right lane) fractions are shown. In b, the immunoprecipitates from extracts performed at 4°C (soluble) and 37°C (total) are shown. Arrows are pointing to the precursor (p) and mature (m) forms of the newly synthesized proteins. (B) WIF-B cells were pulse-labeled with 35S-amino acids for 10 min at 37°C. After washing, cells were incubated for 2 h at 20°C to accumulate newly synthesized proteins in the Golgi. The 20°C medium was replaced with medium prewarmed to 37°C and the labeled proteins chased for the indicated times at 37°C. Samples were processed as described in A. Values in d and f are expressed as the mean ± SD and were obtained from measurements done on at least three experiments each performed in duplicate. In e, values are averages of measurements obtained from two experiments, both performed in duplicate. Representative autoradiographs as described in A are shown.
Figure 4.
Figure 4.
Transcytosis of newly synthesized apical residents is dramatically impaired by cholesterol depletion. WIF-B cells were pretreated for 5 min in LPDM in the absence (a) or presence of 5 mM mβCD (b–i). The indicated apical residents or recycling receptors present at the basolateral PM were continuously labeled with specific antibodies for 60 min at 37°C. The cells were fixed, permeabilized, and the trafficked antibody–antigen complexes visualized with secondary antibodies. In j, the medium containing mβCD was removed, the cells were rinsed in LPDM and then reincubated in LPDM containing 365 μg/ml cholesterol-loaded mβCD for 60 min at 37°C. APN was continuously antibody labeled for 60 min in the presence of the mβCD-cholesterol (chol). The cells were fixed, permeabilized, and the trafficked antibody–antigen complexes visualized with secondary antibodies. Asterisks are marking bile canaliculi (BC). Arrows in e and f are pointing to intracellular puncta enlarged in the insets approximately twofold. Bar, 10 μm.
Figure 6.
Figure 6.
Transcytosis of newly synthesized apical residents is impaired by glycosphingolipid depletion. WIF-B cells were pretreated for 48 h in the absence (a) or presence of 25 μM FB1 (b–h). The indicated apical residents or recycling receptors present at the basolateral PM were continuously labeled with specific antibodies for 60 min at 37°C. The cells were fixed, permeabilized and the trafficked antibody–antigen complexes visualized with secondary antibodies. Asterisks are marking bile canaliculi (BC). Arrows in e–h are pointing to intracellular puncta enlarged in the insets approximately twofold. Bar, 10 μm.
Figure 7.
Figure 7.
Apical residents are internalized in mβCD-treated cells and found in early endosomes. (A) To measure internalization, WIF-B cells were continuously labeled with biotinylated antibodies diluted in LPDM in the absence or presence of 5 mM mβCD for the indicated times at 37°C. The remaining PM-associated antibodies were eluted with isoglycine for 5 min at room temperature and the cells lysed. Aliquots of the eluate and lysate (the internalized population) were assayed for amounts of biotinylated antibodies using streptavidin-coated 96-well plates and colorimetric detection of HRP-conjugated secondary antibodies. The total amount (in nanograms) of antibody internalized is plotted relative to the maximum observed at 60 min in control cells, which was set to 100%. Values are expressed as the mean ± SD. Measurements were done on at least three experiments each performed in duplicate. (B) WIF-B cells were pretreated for 5 min in LPDM in the absence (a, b, e, f, i, j, m, and n) or presence of 5 mM mβCD (c, d, g, h, k, l, o, and p). The indicated apical residents or recycling receptors present at the basolateral PM were continuously labeled with specific antibodies for 60 min at 37°C. The cells were fixed, permeabilized, and the trafficked antibody–antigen complexes visualized with secondary antibodies. Arrows are pointing to intracellular clusters enlarged in the insets approximately twofold. In c, d, g, and o, images were intentionally overexposed to highlight the intracellular population of the transcytosing proteins. Thus, the apparent apical labeling is exaggerated. The insets in m and n are highlighting a region where Tf-R and ASGP-R do not significantly overlap. Arrowheads are pointing to colocalized structures. Bar, 10 μm.
Figure 8.
Figure 8.
Cholesterol depletion impairs apical protein trafficking in nonpolarized hepatic Fao cells. (A) Fao cells were treated with LPDM in the absence (control) or presence of 5 mM mβCD for 60 min at 37°C. Cells were extracted in 1% Triton X-100 for 30 min and the soluble and insoluble fractions separated by centrifugation. The soluble (S) and pelleted (P) fractions were analyzed by Western blotting with the indicated antibodies. The fraction of the total population that was detergent insoluble for each molecule is indicated on the right (% Ins.). (B) Fao cells were treated in LPDM in the absence or presence of 5 mM mβCD as indicated. The steadystate (ss) distributions of 5′NT are shown. In C, the indicated apical residents or recycling receptors present at the PM were continuously labeled with specific antibodies for 60 min at 37°C. The cells were fixed, permeabilized, and the trafficked antibody–antigen complexes visualized with secondary antibodies. In general, the treated Fao cells seemed to have altered cell-to-cell adhesion, resulting in the formation of gaps between adjacent cells, which are not to be confused with the BC present in polarized WIF-B cells. Bar, 10 μm.
Figure 9.
Figure 9.
The mβCD defect is reversible and cholesterol depletion does not impair recycling in nonpolarized Fao cells. The cells in a and b were assayed for recovery as described in the legend to Figure 4. In c, APN present at the PM was continuously antibody-labeled for 60 min at 37°C. The remaining PM-associated antibodies were stripped with isoglycine for 5 min at room temperature (d). Only the protected, internalized APN–antibody complexes were detected. Cells were incubated an additional hour at 37°C in the absence (e) or presence (f) of 5 mM mβCD. The cells were fixed, permeabilized, and the antibody–antigen complexes visualized with secondary antibodies. Bar, 10 μm.
Figure 10.
Figure 10.
Intracellular itineraries of apical proteins in control and lipid-depleted WIF-B cells. In control cells, newly synthesized apical proteins are delivered from the TGN to the basolateral PM. They are selectively retrieved by endocytosis and transcytosed to the apical PM. Only two intermediates in the basolateral-to-apical transcytotic pathway have been identified: the basolateral early endosome and SAC. In treated cells, Golgi-to-basolateral PM delivery is unchanged and transcytosing proteins are internalized from the basolateral PM and delivered to the early endosome. Transport from the early endosome to the SAC is inhibited, and we propose that the apical residents recycle back to the basolateral PM. Except for the transcytotic efflux from the compartment, all other pathways are largely unaffected by cholesterol or glycosphingolipid depletion. EE, early endosome; Lys, lysosome; RE, recycling endosome.
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