Ultra-structural analysis reveals a bulkhead-like pattern of transversal structures sealed by T-shaped TJs in the BC lumen. (a) EM image of a BC branched between three cells. Longitudinal section of in vitro differentiated hepatocytes. GG, glycogen granules. The outlined region is shown in b. Scale bar: 5 µm. (b) Section of BC formed by two cells with membrane connections highlighted in rectangles c and d and shown in detail in corresponding panels c and d. The sections were acquired in a series of longitudinal 90-nm sections shown in b, I–IV. Note that the membrane connections are not visible in every section. Scale bar: 1 µm. (c and d) Detailed view on the membrane connections in rectangles c and d from panel b. Membrane connections are formed by apical surfaces of both cells lining the BC and include the TJs (arrows). Vesicles (V) are often observed to accumulate in the vicinity of the connections. Scale bar: 1 µm. (e) 3D reconstruction of serial sections in b (I–IV) based on apical plasma membranes and TJs rendering. The cytoplasm of the lumen-forming cells is in green and blue, and the TJs are highlighted in red. See also Video 5. (f) Simplified model of BC based on the 3D reconstruction in e with periodic bulkhead-like membrane connections formed from the top or the bottom of the lumen (arrowheads). The TJs (red) have a T-shape, with the junctions longitudinal along the tube connected with the junctions extending along the ridgeline within each bulkhead. Uninterrupted flow within the lumen between bulkheads is shown with a dotted line. See also Video 6.