Vinculin and α18 focus at tricellular and multicellular junctions in ZO KD. (A–C and F–Q) 3-µm confocal (A–C) or 2-µm SIM (F–Q) apical projections. (B, D, and E) ZO KD. Vinculin and α18 are elevated at BCJs (A vs. B, arrows) and especially at TCJs (A vs. B [arrowheads]; D and E, mean line scan intensities). Error bars are SDs of >30 tricellular contacts (n = 3). au, arbitrary unit. (C) ZO/afadin KD. Vinculin and α18 are elevated but variable at TCJ/MCJs (arrowheads). At some BCJs, staining is fragmented (yellow vs. red arrows and inset). (F–J, N, and O) ZO KD. Vinculin and α18 enrichment at TCJs (yellow arrows) and MCJs (magenta arrows) is membrane proximal to actin (H and O) or myosin (I). (K–M, P, and Q) ZO/afadin KD. Vinculin and α18 line membrane-proximal edges of actin cables (L and Q, magenta arrows). In regions of ZA discontinuity (K and P, white arrows), they surround the region or align along actin filaments (M). Bars: (A–C, F, G, K–N, and P) 5 µm; (H–J, O, and Q) 1 µm.