Actin filaments and microtubules are involved in different membrane traffic pathways that transport sphingolipids to the apical surface of polarized HepG2 cells

Abstract

In polarized HepG2 hepatoma cells, sphingolipids are transported to the apical, bile canalicular membrane by two different transport routes, as revealed with fluorescently tagged sphingolipid analogs. One route involves direct, transcytosis-independent transport of Golgi-derived glucosylceramide and sphingomyelin, whereas the other involves basolateral to apical transcytosis of both sphingolipids. We show that these distinct routes display a different sensitivity toward nocodazole and cytochalasin D, implying a specific transport dependence on either microtubules or actin filaments, respectively. Thus, nocodazole strongly inhibited the direct route, whereas sphingolipid transport by transcytosis was hardly affected. Moreover, nocodazole blocked "hyperpolarization," i.e., the enlargement of the apical membrane surface, which is induced by treating cells with dibutyryl-cAMP. By contrast, the transcytotic route but not the direct route was inhibited by cytochalasin D. The actin-dependent step during transcytotic lipid transport probably occurs at an early endocytic event at the basolateral plasma membrane, because total lipid uptake and fluid phase endocytosis of horseradish peroxidase from this membrane were inhibited by cytochalasin D as well. In summary, the results show that the two sphingolipid transport pathways to the apical membrane must have a different requirement for cytoskeletal elements.

Figure 1

Effect of cytoskeletal drugs on the distribution of actin filaments and microtubules in HepG2 cells. The effect of cytD on actin filaments was determined by incubating the cells with 10 μg/ml cytD in HBSS for 30 min at 37°C. After the incubation, the cells were washed and fixed in ethanol for 10 s at −20°C. Actin filaments were stained with 100 ng/ml TRITC-labeled phalloidin in PBS for 30 min at room temperature. Note the extensive staining of actin filaments around the bile canaliculus (arrowhead) and the additional staining at the basolateral membrane in control cells (A), whereas in cytD-treated cells (B) mainly small aggregates and a strongly reduced labeling around the bile canaliculus are observed. By staining cells with anti-β-tubulin, the effect of nocodazole on microtubules was evaluated. Cells were preincubated with 33 μM nocodazole in HBSS for 2 h at 37°C. The cells were then fixed with 3% paraformaldehyde for 30 min at room temperature and permeabilized with 0.1% Triton X-100. Indirect immunofluorescence staining with an antibody against β-tubulin revealed that in control cells, the microtubules formed a delicate network (C) that was severely disrupted after nocodazole treatment (D). (E–H) Phase-contrast images of A–D, respectively. Bar, 10 μm.

Figure 2

Effect of nocodazole and cytD on apical sphingolipid transport. HepG2 cells were preincubated with 10 μg/ml cytD for 30 min or 33 μM nocodazole or 1 μM colchicine for 2 h in HBSS at 37°C. The compounds were kept present during further incubations. (A) Effect of the cytoskeletal drugs on direct transport of newly synthesized sphingolipids. Cells were labeled with 3 μM C₆-NBD-Cer at 4°C, as described in MATERIALS AND METHODS. After an incubation at 37°C for 60 min in back-exchange medium, the cells were washed, and apical transport was determined as described. Data represent the mean ± SEM of three or four independent experiments of cells treated with 0.1% DMSO (control), cytD, nocodazole, or colchicine. (B) Effect of cytD or nocodazole on transcytosis of exogenously inserted (i.e., in the basolateral membrane) C₆-NBD-SM and C₆-NBD-GlcCer. After preincubation with cytD or nocodazole, cells were labeled with 3 μM C₆-NBD-GlcCer (white bars) or C₆-NBD-SM (black bars) at 4°C. After warming at 37°C for 15 min in HBSS, the cells were subsequently washed, cooled, and back-exchanged. Apical transport was determined as described in MATERIALS AND METHODS. Data represent the mean ± SEM of three to five independent experiments.

Figure 3

Effect of cytD and nocodazole on the internalization of basolateral membrane-inserted C₆-NBD-SM and C₆-NBD-GlcCer. HepG2 cells were preincubated with or without 10 μg/ml cytD or 33 μM nocodazole in HBSS at 37°C for 30 min or 2 h, respectively. Cells were then cooled and, in the presence of the drugs, labeled with 3 μM C₆-NBD-SM or C₆-NBD-GlcCer at 4°C. Subsequent incubations were carried out at 37°C for the indicated time intervals. After the incubation, the cells were washed with cold PBS, back-exchanged, and scraped from the culture dish. Lipids were extracted and NBD-lipids were quantified as described in MATERIALS AND METHODS. Each value represents the mean of two independent experiments with variations between 3 and 14%. (A) Internalization of C₆-NBD-GlcCer in control (○), cytD (□), and nocodazole-treated (▵) cells. (B) Internalization of C₆-NBD-SM in control (•), cytD (▪), and nocodazole-treated (▴) cells.

Figure 4

Effect of nocodazole, dB-cAMP, or both on apical sphingolipid transport. Cells were preincubated at 37°C with or without 33 μM nocodazole for 2 h and/or with 1 mM dB-cAMP for 30 min. Then, in the absence or presence of either nocodazole or dB-cAMP or both (noc/dBcAMP), direct, apical transport of newly synthesized C₆-NBD-sphingolipids (A) or transcytotic transport of C₆-NBD-SM (B) was determined as described in MATERIALS AND METHODS and in the legend of Figure 2. Data represent the mean ± SEM of three independent experiments.