Fig. 5. CNF-induced internalization of occludin is via caveolae and early-/recycling endosomes but not late endosomes. T84 epithelial monolayers were incubated with vehicle or CNF-1 (1 nM) for 24 hours, and the internalization of occludin monitored in relation to markers of endosomal/caveolar pathways by immunofluorescence/confocal microscopy. Occludin in the en face plane of control cells (a1, c1, e1, g1, i1) was characteristically distributed in discrete TJ rings at cell-cell borders. Caveolin-1 staining in control cells (a2) consisted of a large pool of apical staining, together with a sub-pool of caveolin-1 localizing in a ring pattern at TJs (arrowhead). This was observed to co-localize with occludin (a3, #). Occludin internalization in CNF-treated cells (b1, arrows) was seen to correspond closely with a pool of internalized caveolin-1 in the same cells (b2, arrowheads) and in a composite image (B3, #). The transferrin receptor in control cells (c2) localized mainly to the apical surface and underneath the membrane, showing no overlap (c3) with the staining pattern for occludin. Following CNF-1 incubation, internalized occludin (d1, arrow) and internalized transferrin receptor (d2, arrowhead) did not appear to co-localize with each other (d3, #). Another early endosomal marker, EEA-1, localized to sub-membranous structures with some additional staining at the TJ membrane (E2, arrowhead). This, like caveolin-1 staining in control cells, overlapped with occludin (e3, #). Occludin internalization upon CNF-1 incubation (F1, arrows) overlapped with EEA-1 distribution in the same cells (F2, arrowheads). This overlap in the merged image (f3, #) suggests internalization of occludin in EEA-1-positive early endosomes. Distribution of the recycling endosomal marker Rab11 was primarily submembranous in control cells (g2), showing no co-localization with occludin (g3). However, occludin (h1, arrow) and Rab11 (h2, arrowhead) internalization in CNF-treated monolayers were observed to overlap significantly (h3, #). The late endosomal marker LAMP-1 localized to the membrane at the level of intercellular junctions in control cells (i2), in a pattern that resembled but did not overlap with (i3, #) that of occludin. Occludin internalization after CNF-1 treatment (j1) did not co-localize with LAMP-1-positive late endosomes in the same cells (j3, #). Results are representative of 6 experiments. Bar10 µm.