Figure 1. Transient opening of epithelial junctions by JO-1.

A) Structure of Ad3 viral particles. Left panel: complete, infectious Ad3 particle. The capsid proteins fiber and penton base are shown in green and blue, respectively. The trimeric fiber knob is shown in red. Middle panel: Ad3 penton-dodecahedra (PtDd) formed by spontaneous assembly of 12 recombinant pentons (fiber + penton base). Right panel: dimeric Ad3 fiber (JO-1). B) Schematic structure of JO-1 containing an N-terminal His-tag, a dimerization domain [K-coil (32)], a flexible linker, one fiber shaft motif, and the homotrimeric Ad3 fiber knob domain. C) Left panel: simplified structure of epithelial junctions with tight junctions, desmosomes, and adherens junctions. Confocal immunofluorescence microscopy of T84 cells. Shown are stacked XZ images. Cells were treated with JO-1 (5 μg/ml) for 1 h on ice. After removal of JO-1, cells were incubated at 37°C and analyzed 0, 30, and 60 min later. Upper panel: DSG2 (green) appears at the apical site of baso-lateral junctions marked by claudin 7 (red). Middle panel: within 30 min after adding JO-1, claudin 7 staining increases and DSG2 staining becomes visible along the upper part of the lateral membrane (yellow signals). Lower panel: By 60 minutes, lateral junctions resemble those of time point “0 min”. The scale bar is 40 μm. D) Transmission electron microscopy of junctional areas of polarized colon cancer T84 cells. Cells were either treated with PBS (left panel) or JO-1 (right panel) for 1 h on ice, washed, and then incubated for 1 h at 37 °C. At this time, the electron-dense dye ruthenium red (33) was added together with the fixative. The scale bar is 1μm. E) ¹⁴C-PEG-4,000 diffusion through monolayers of T84 cells at different time points after adding JO-1 or anti-DSG2 antibody 6D8 (directed against ECD3/4). F) Effect of various DSG2 ligands on the transepithelial electrical resistance (TEER) of polarized T84 epithelial cells. Cells were either treated as described in E).