Volume 119, Issue 3, Pages 782-793 (September 2000) Copper-induced apical trafficking of ATP7B in polarized hepatoma cells provides a mechanism for biliary copper excretion  Han Roelofsen, Henk Wolters, Marja J.A. Van Luyn, Naoyuki Miura, Folkert Kuipers, Roel J. Vonk  Gastroenterology  Volume 119, Issue 3, Pages 782-793 (September 2000) DOI: 10.1053/gast.2000.17834 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Increased copper levels induce relocation of ATP7B from the TGN to the apical membrane. HepG2 cells grown on coverslips were cultured in the absence of copper (−Cu2+) or for 4 hours in the presence of 20 μmol/L CuS04 (+ Cu2+). Cells were fixed and double stained for ATP7B and the apical transporter MRP2 or the TGN marker TGN38. Images of polarized cells were taken with a confocal laser microscope. (A) In the absence of copper, no overlap between ATP7B (green) and MRP2 (red), which stains apical vacuoles, was observed. In the presence of 20 μmol/L Cu2+, ATP7B partially overlapped with the apical MRP2 (yellow in merged image). (B) In the absence of copper, ATP7B (green) staining almost completely overlaps with TGN38 (red), evident from the yellow of the merged images. In the presence of 20 μmol/L Cu2+, the overlap between the 2 labels is lost. Arrowhead indicates an ATP7B-positive apical vacuole. Bar = 10 μm. Gastroenterology 2000 119, 782-793DOI: (10.1053/gast.2000.17834) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 The presence of ATP7B at the apical membrane and in a vesicular pool remains after prolonged incubation with increased copper concentrations. Cells were incubated for 20 hours with 50 μmol/L Cu2+ and double-labeled for (A) ATP7B and (B) MRP2 to identify apical vacuoles. Images were taken with a confocal laser microscope. The apical vacuole, containing microvilli that fill up the lumen, clearly stains for ATP7B. ATP7B is still located to relatively large intracellular vesicles that have a tendency to cluster at the apical pole of the cell (arrows). Bar = 10 μm. Gastroenterology 2000 119, 782-793DOI: (10.1053/gast.2000.17834) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Ultrastructural localization of ATP7B to apical vacuoles. The apical localization of ATP7B was studied at an ultrastructural level using electron microscopy. Cells were cultured for 4 hours with 20 μmol/L Cu2+ and processed for immunogold labeling and electron microscopy (see Materials and Methods). The image shows an apical vacuole containing microvilli that clearly label for ATP7B, represented by the black dots. This indicates that ATP7B is present in the apical membrane at increased copper concentrations. Gastroenterology 2000 119, 782-793DOI: (10.1053/gast.2000.17834) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Characteristics of the copper-induced redistribution of ATP7B. To determine the concentration and time dependency of the redistribution process, (A) cells were cultured for 4 hours with copper concentrations ranging from 0 to 40 μmol/L or (B) with 20 μmol/L of Cu2+ for the indicated time periods, or (C) cells were incubated for 4 hours with 20 μmol/L Cu2+ and subsequently cultured for the indicated periods in copper-free media containing 100 μmol/L of the copper-chelator bathocuproinedisulfonic acid. The cells were fixed and double-labeled for ATP7B and MRP2 to identify apical vacuoles. The number of apical vacuoles that stained for ATP7B was scored and expressed as the percentage of the total number of apical vacuoles counted. Each point represents the mean ± SD of 3–6 independent incubations and 50 vacuoles per incubation. Gastroenterology 2000 119, 782-793DOI: (10.1053/gast.2000.17834) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 The ATP7B redistribution process is reversible upon removal of copper. To study the redistribution process in more detail, cells were incubated with 20 μmol/L Cu2+ for 0, 0.5, 2, and 4 hours. Furthermore, cells were first incubated for 4 hours with 20 μmol/L Cu2+ and subsequently cultured for the indicated time periods (−1 h, −2 h, −3 h, −16 h) without copper in the presence of 100 μmol/L of the copper-chelator bathocuproinedisulfonic acid. The cells were fixed and double-labeled for ATP7B (green) and MRP2 (red), to identify apical vacuoles. Images of both labels were taken with a confocal laser microscope and subsequently merged. Overlap between the 2 labels is indicated by the orange (relatively low abundance of ATP7B) or yellow (high abundance of ATP7B). Bar = 10 μmol/L. Gastroenterology 2000 119, 782-793DOI: (10.1053/gast.2000.17834) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 BFA inhibits copper-induced trafficking of ATP7B. (A) To determine the effect of BFA on the copper-induced redistribution of ATP7B, cells were incubated with for 4 hours with 20 μmol/L BFA in the absence (4h −Cu2+ +BFA) or presence (4h +Cu2+ +BFA) of 20 μmol/L Cu2+. To determine the effect of BFA on trafficking of ATP7B outside the TGN, ATP7B was first chased out of the TGN by incubating the cells for 1 hour with 20 μmol/L Cu2+, before 20 μmol/L BFA was added for the remaining 3 hours of incubation (1h +Cu2+ → 3h Cu2+ +BFA). We also examined whether BFA affects the new steady-state distribution of ATP7B, reached after 4 hours of Cu2+. Cells were cultured for 4 hours with 20 μmol/L Cu2+, then BFA was added and cells were incubated for another 3 hours (4h +Cu2+ → 3h +Cu2+ +BFA). Subsequently, the cells were fixed and double-labeled for ATP7B (green) and MRP2 (red) to identify apical vacuoles. Images of both labels were taken with a confocal laser microscope and merged. (B) For each condition, the effect of BFA on apical trafficking was quantified by scoring the number of apical vacuoles that stained for ATP7B. Results are expressed as the percentage of the total number of apical vacuoles counted. Each point represents 3–6 independent incubations and 50 vacuoles per incubation. *P < 0.001 compared with 4h +Cu2+; ‡P < 0.001 compared with 7h +Cu2+ (unpaired 2-sided Student t test). Arrows indicate subapical compartments that accumulate ATP7B. Bar = 10 μm. Gastroenterology 2000 119, 782-793DOI: (10.1053/gast.2000.17834) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Nocodazole inhibits copper-induced trafficking of ATP7B. (A) To determine the effect of nocodazole on the copper-induced redistribution of ATP7B, cells were incubated for 4 hours with 20 μmol/L nocodazole in the absence (4h −Cu2+ +noco) or presence (4h +Cu2+ +noco) of 20 μmol/L Cu2+. To determine the effect of nocodazole on trafficking of ATP7B outside the TGN, ATP7B was first chased out of the TGN by incubating the cells for 1 hour with 20 μmol/L Cu2+, before 20 μmol/L nocodazole was added for the remaining 3 hours of incubation (1h +Cu2+ → 3h +Cu2+ +noco). We also examined whether nocodazole affects the new steady-state distribution of ATP7B, after 4 hours of incubation with Cu2+. Cells were cultured for 4 hours with 20 μmol/L Cu2+, then nocodazole was added and cells were incubated for another 3 hours (4h +Cu2+ → 3h +Cu2+ +noco). The cells were then fixed and double-labeled for ATP7B (green) and MRP2 (red) to identify apical vacuoles. Images of both labels were taken with a confocal laser microscope and were merged. (B) For each condition, the effect of BFA on apical trafficking was quantified by scoring the number of apical vacuoles that stained for ATP7B, using an epifluorescence microscope. Results are expressed as the percentage of the total number of apical vacuoles. Each point represents 3–6 independent incubations and 50 vacuoles per incubation. *P < 0.001 compared with 4h +Cu2+; ‡P < 0.001 compared with 7h +Cu2+ (unpaired 2-sided Student t test). Arrows indicate subapical compartments that accumulate ATP7B. Bar = 10 μm. Gastroenterology 2000 119, 782-793DOI: (10.1053/gast.2000.17834) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Schematic representation of copper-induced apical trafficking of ATP7B in HepG2. Based on the data in our study, a model was constructed for copper-induced redistribution of ATP7B. At low copper levels, ATP7B is predominantly localized to the TGN. When copper concentration increases, ATP7B leaves the TGN and is sorted via a vesicular compartment to the apical membrane. ATP7B-containing vesicles become larger and fewer, suggesting they fuse. A new steady-state distribution is reached within 3 hours. Studies with BFA and nocodazole indicate that ATP7B continuously recycles between the apical membrane and a vesicular compartment when copper is increased. When copper levels are lowered again, ATP7B returns to the TGN via the intermediate vesicular compartment. Copper-induced redistribution of ATP7B may provide a mechanism to preserve copper when it is scarce, by facilitating the incorporation of copper into cupro-enzymes such as ceruloplasmin in the TGN, or to prevent copper toxicity when levels become too high, by mediating the apical (or biliary) excretion of copper. N, nucleus; AV, apical vacuole. Gastroenterology 2000 119, 782-793DOI: (10.1053/gast.2000.17834) Copyright © 2000 American Gastroenterological Association Terms and Conditions