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First published online June 8, 2005
doi: 10.1242/10.1242/jcs.02403

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Three-dimensional polarization sensitizes hepatocytes to Fas/CD95 apoptotic signalling

¹ Institut de Génétique Moléculaire, CNRS UMR5535, IFR122, 1919 route de Mende, 34293 Montpellier Cedex 5, France
² UMR 5539 Centre National de la Recherche Scientifique, Dynamique Moléculaire des Interactions Membranaires, Université Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France
³ Service d'Anatomie Pathologique, Groupe Hospitalo-Universitaire Caremeau, Place du Pr Robert Debré, 30029 Nîmes Cedex 9, France
⁴ CRBM, CNRS FRE2593, IFR122, 1919 Route de Mende, 34293 Montpellier Cedex 5, France
⁵ UMR 5539 CNRS/UM2, IFR122, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France

View larger version (53K): [in a new window]   Fig. 1. MhAT3F hepatocytes form organoids in Matrigel. Phase-contrast microscopy of monolayer grown cells (a) and organoids formed after 8 days of culture in Matrigel (b). After 4 days of culture, most of the organoids were filled with cells (c), whereas, at day 8, most were hollow (d) as visualized by Hoechst 33258 nuclear staining (blue). Multiple lumens are visualized on semi-thin sections of day 8 organoids (e,f). Apoptotic bodies (ab) and debris can be seen within some lumens (L) (e), whereas others contained no dense material (f). n, nucleus. Before lumen formation, cells in the interior of the organoids contain activated caspase 3 (green) (g). Bars, 20 µm (a-d,g), 3.5 µm (e,f).

View larger version (81K): [in a new window]   Fig. 2. Hepatocytes in the organoids are strongly polarized. MhAT3F cells were grown either in a monolayer or included in Matrigel or a collagen sandwich. The cells had an epithelial phenotype, visualized by the cortical actin distribution (phalloidin-TRITC, red), both in monolayer (2D) and in the organoids (3D). (A) Radixin (fluorescein-isothiocyanate labelled, green), a marker of the apical membrane domains in hepatocytes, showed a strong apical labelling of a subset of monolayer cells, easily visible in 3D-reconstructed (3D reconst.) image and a z-axis sections (below). It decorated the lumen-exposed membranes of all cells in 3D organoids. Nuclei are stained with Hoechst 33258 (blue). 3D-reconstructed images are shown, except for overlay panels, which show single confocal stacks. (B) ZO-1 (fluorescein-isothiocyanate labelled, green), a tight-junction marker, had a heterogeneous distribution in monolayer cells but was strongly localized around the lumen in the organoids. (C) Transmission electron microscopy of monolayer (a,b) and organoids (c,d) allows visualization of microvilli (mv) and tight junctions (tj). Multiple small (a) or larger (b) lumens (IL) lined with microvilli can be seen in the organoids. n, nucleus. (D) Phase-contrast microscopy of cells in the collagen-sandwich culture. Two types of structures can be seen: spheroid organoids (arrows) and extended sheets of cells. (E) Immunofluorescence analysis of collagen-sandwich cultures. Radixin labelling (green) is punctate in extended cells (top) and strongly localized to the interior of the organoids (bottom). Bars, 20 µm (A,B,E), 1 µm (C), 40 µm (D).

View larger version (67K): [in a new window]   Fig. 3. mhAT3F organoids are functional. (A) Western-blot analysis of albumin secretion by mhAT3F cells grown under the indicated conditions. Albumin accumulation was allowed to proceed for 24 hours and the amount of medium assayed was normalized to the number of cells present in the culture, estimated by their GAPDH content. A representative blot is shown, the numbers represent mean fold increase compared with monolayer cultures, as calculated after scanning of three western blots. (B) Monolayer grown mhAT3F show no detectable bilirubin (a), whereas the green Hall's staining of the whole organoids (b) indicates the accumulation of bilirubin. A section of a cholestatic human liver (c) is shown as a positive control. Bars. 20 µm.

View larger version (57K): [in a new window]   Fig. 4. MhAT3F organoids are sensitized to Fas-induced apoptosis. (A-F) Monolayers (2D) or 3D organoids (3D) were treated with the anti-Fas antibody (Jo2) in the presence or absence of cycloheximide, as indicated. (A) Phase-contrast microscopy. Arrows indicate organoids enlarged in inserts. (B) Pooled floating and adherent monolayer cells deposited on microscope slides by cytospin (top) or sections of Matrigel (bottom) were analysed by immunofluorescent staining of active caspase 3. Nuclei are shown in blue (Hoechst 33258), caspase 3 in green (fluorescein isothiocyanate). (C,D) Quantification of caspase-3-positive cells in monolayer cultures (C; 450 cells counted) and in 3D cultures (D; 40 organoids counted). (E) Monolayer cultures treated with TNF with or without actinomycin D, as indicated, were collected and analysed as in C. (F) 3D organoids were treated with TNF with or without actinomycin D and analysed as in D. (G) Cells grown in collagen sandwich were treated as indicated and analysed by phase-contrast microscopy (top) or by immunofluorescent staining of the entire collagen plug with antibody against active caspase 3 (green). Nuclei were stained with Hoechst 33258 (blue). Bars, 20 µm (A,B), 40 µm (G).

View larger version (29K): [in a new window]   Fig. 5. NF-B regulates Fas-induced apoptosis. (A) Monolayer-grown mhAT3F were co-transfected with vectors encoding a truncated rat CD2 and IBAA and treated with the Jo2 anti-Fas antibody. Floating and adherent cells were pooled, deposited on a slide by cytospin and analysed (nuclei are blue, active caspase 3 is green and CD2 is red). (B) Quantification of cells containing active caspase 3 within the subpopulation of transfected cells. (C) Western blot of NF-B p65 subunit in total cell extracts of cells grown in monolayer (2D) or as organoids in Matrigel (3D). Actin serves as a loading control. (D) Monolayer cultures of mhAT3F cells were co-transfected with NF-B/firefly-luciferase and constitutive Renilla-luciferase reporter constructs. 18 hours later, cells were replated and grown either as monolayer cultures (open bars) or 3D organoids (hatched bars) and stimulated by the anti-Fas antibody Jo2 or TNF, as indicated, for 7 hours. Results are shown as means±s.e.m. Bars, 20 µm.

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