Fat2 and WRC accumulate at tricellular junctions. (A–C) Structured illumination microscopy (SIM) images from the basal surface of stage 3 (A), stage 5/6 (B), and stage 7 (C) egg chambers expressing a Fat2-GFP transgene. Fat2 (green), endogenous Abi (red), and actin-rich protrusions (F-actin, white) colocalize at tricellular junctions. Bars, 5 µm. (D) Quantification of the relative Fat2-GFP signals (costained with an anti-GFP antibody) at tricellular contacts and along the membrane in fixed follicle cells confirms a significant accumulation of Fat2 at tricellular junctions. The strongest localization of Fat2 at tricellular junctions is found at stage 7. ***, P < 0.0001 (ANOVA). Error bars represent mean ± SD. (E) Colocalization analysis of Fat2 and Abi based on Pearson's colocalization coefficient. Fat2 and Abi shows the strongest overlap at tricellular contacts at stage 7. Triangles, circles, and squares represent single measurements of the Pearson's colocalization coefficient at indicated developmental stages. (F and G) Quantification of the relative Fat2-GFP fluorescence signals at tricellular contacts and along the membrane in living follicle cells as depicted in (F) confirms a significant accumulation of Fat2 at tricellular junctions from stages 5–9. Again, the strongest localization of Fat2 at tricellular junctions is found at stage 7. The fluorescence signal intensities were substracted from the background signal. ***, P < 0.0001 (ANOVA). Bars represent mean ± SD. Bars, 5 µm.