The Gα_i interactive domain is not required for Daple-mediated MT organization. (A) Immunofluorescence images of α-tubulin and Daple in WT MTECs treated with DMSO and 6.6 µM nocodazole for 2 h. Red arrows indicate the localization of Daple and Fzd6 in tracheal MCCs. (B) Representative immunofluorescence images of the mEGFP–Daple FL GBA motif mutant (GBA mut; green), Fzd6 (magenta), and α-tubulin (blue) in Daple-KO MTECs. The mEGFP–Daple FL GBA motif mutant (GBA mut) was expressed using a lentivirus system. Yellow arrows indicate mEGFP–Daple FL (GBA mut) and Fzd6 localization in tracheal MCCs. (C) Representative images of mEGFP–Daple FL (GBA mut; magenta) and α-tubulin (green) in Daple-KO MTECs expressing mEGFP–Daple FL (GBA mut). The intensity of the α-tubulin signal is represented by the color map (red indicates high intensity; blue indicates low intensity). (D and E) Intensity profiles of α-tubulin in mEGFP–Daple FL (GBA mut)-positive MCCs (orange) and -negative MCCs (blue) in Daple-KO MTECs. (F) Statistical analysis of the slope coefficient (Coef.) acquired from the α-tubulin intensity profiles in mEGFP–Daple FL-, mEGFP–Daple FL (GBA mut)-, and mEGFP–Daple FL ΔPBM-expressing MCCs (orange) and Daple-KO MCCs (n = 6 cells). Significant differences were analyzed using Kruskal-Wallis tests with Steel-Dwass multiple-comparison tests. *, P < 0.05. (G) Schematic illustration of the proposed mechanism for MT accumulation specifically at the Fzd side of the AJC in a Gα_i- and Dvl-independent manner through Daple. Scale bars represent 5 µm.