Fat2 is a novel WRC ligand. (A) Spinning disc time-lapse series of an egg chamber stage 5/6 coexpressing a LifeAct-RFP transgene under the da-Ga4 driver in Fat2-GFP background. The arrowheads mark the enrichment of Fat2-GFP at dynamic cell protrusions emerging from tricellular junctions over time. Red arrowheads mark beginning, white arrowheads mark position after 120 s. Bars, 5 µm. (B) Quantification of the relative Fat2-GFP fluorescence signals at the tips versus the base of LifeAct-RFP-marked protrusions in migrating follicle cells. The fluorescence signal intensities were subtracted from the background signal. ***, P < 0.0001 (t test). Bars represent mean ± SD. (C) Schematic diagram of the molecular domain structure of the cadherin Fat2. Fat2 is a single-pass transmembrane cell adhesion receptor. The large extracellular region contains 34 cadherin repeats arrayed in tandem and in proximity to the membrane a laminin A subunit G domain and five EGF motifs. The sequence of the small intracellular C terminus, consisting of 383 amino acids (aa), contains three putative WIRS motifs, highlighted in yellow. (D) Coomassie blue–stained SDS-PAGE gel shows that immobilized Drosophila wild-type (WT) WRC, but not a mutant with a disrupted WIRS binding surface (AW for R118A/G122W-dAbi), selectively retained distinct GST-tagged fusions with the C terminus of Drosophila Fat2 (GST-dFat2 CT) containing distinct mutations of the three WIRS motifs, as highlighted in E. The relative position of the molecular weight markers in D are marked by black lines, based on their position in E (lane 1), which shows the same protein band pattern. (E, right) Loading control of the distinct GST-dFat2 CT fusion proteins.