Desmosomal cadherins require MTs for rapid accumulation at intercellular junctions. (A) Dual-label immunofluorescence revealed that Dsg2 and Dsc2 colocalize at cell–cell junctions but are present in separate cytoplasmic vesicles. Boxes indicate areas of magnification on the right. (B) Cells coexpressing Dsg2-GFP and tubulin-mCherry (blue) were imaged at 5-s intervals (Video 2). Dsg2-containing vesicles move along MTs toward the plasma membrane. (C) Contacting cells expressing Dsg2-GFP and tubulin-mCherry were imaged at 5-s intervals. Video 3 shows the cropped area in the left image. White brackets indicate cell–cell contact. Green track shows the path taken by a Dsg2-containing vesicle to the contact site. (D) Scc9s were switched to low-calcium medium (LCM) for 2 h before incubation with nocodazole for 1 h in low-calcium medium and then switched to normal Ca²⁺ (with or without nocodazole) for 30 min to trigger junction assembly. Cytoplasmic Dsg2 and Dsc2 vesicles are present in control (Cntrl) and nocodazole (noc)-treated cells in low-calcium medium. Disruption of MT delays Dsg2 and Dsc2 assembly at newly forming cell–cell interfaces. Bars: (A [left] and D) 20 µm; (A [right] and C) 10 µm; (B) 5 µm.