Figure 6.
Daple regulates MT dynamics at the Fzd side of the AJC in tracheal MCCs. (A) Schematic drawing of ALI culture of MTECs for the lentivirus infection and live imaging. (B) Representative fluorescence images of EB1-mRuby3 (green) and centrin2 (blue) in GFP-centrin2–positive WT MTECs expressing EB1-mRuby3 via the lentivirus system. (C and D) Representative image for EB1-mRuby3 (red) in GFP-centrin2–positive WT MTECs expressing EB1-mRuby3 due to the lentivirus system (C) and time-lapse images of the boxed regions in C for EB1-mRuby3 at the Fzd and the other side of the AJC in tracheal MCCs (D). Red arrows indicate the EB1 comets that contact the cell membrane, whereas yellow arrows indicate the EB1 comets in the cytoplasm. (E) Representative fluorescence image for EB1-mRuby3 in GFP-centrin2–positive WT MTECs with boxes indicating the I and P regions. (F) EB1 speed at the I and P regions in MCCs of WT MTECs. Speed of EB1 comets showing spatial differences (n = 10). Two-tailed Mann-Whitney U test; **, P < 0.01. (G) EB1 speeds in the I and P regions around the Fzd and the other sides of the AJC in MCCs of WT MTECs (n = 10 cells). Kruskal-Wallis test with the Steel-Dwass multiple-comparison test; **, P < 0.01; n.s., P ≥ 0.05. (H) Schematic illustration of differences in EB1 dynamics around the Fzd and other side of the AJC in MCCs of GFP-centrin2–positive WT MTECs. The EB1 comets display a long residence time at the Fzd side of the AJC. Inter, I region; Peri, P region. (I) Representative image for mEGFP–α-tubulin (mEGFP-αtub; red) expressed in MCCs of WT MTECs (upper panel) and kymograph showing the motility of MTs in MCCs of WT MTECs (lower panel). Note the high motility of MTs as they move near the Vangl side of the AJC, as shown in the boxed region (yellow dashed lines). (J) Representative images of mEGFP–α-tubulin in MCCs of WT and Daple-KO MTECs (left panels) and analyses of MT live imaging using the LPX flow plugin (right panels). The movement speed of the α-tubulin signal is repressented by colors (red indicates high speed; blue indicates low speed). The arrow indicates a decrease in speed at the Fzd side of the AJC in WT MCCs. (K) Statistical analysis of the speed of MTs in WT MCCs (n = 4 cells). ROIs were set at the Fzd and other side of the AJC. Two-tailed Mann-Whitney U test; *, P < 0.05. Scale bars represent 5 µm in B, C, E, I, and J and 1 µm in D.

Daple regulates MT dynamics at the Fzd side of the AJC in tracheal MCCs. (A) Schematic drawing of ALI culture of MTECs for the lentivirus infection and live imaging. (B) Representative fluorescence images of EB1-mRuby3 (green) and centrin2 (blue) in GFP-centrin2–positive WT MTECs expressing EB1-mRuby3 via the lentivirus system. (C and D) Representative image for EB1-mRuby3 (red) in GFP-centrin2–positive WT MTECs expressing EB1-mRuby3 due to the lentivirus system (C) and time-lapse images of the boxed regions in C for EB1-mRuby3 at the Fzd and the other side of the AJC in tracheal MCCs (D). Red arrows indicate the EB1 comets that contact the cell membrane, whereas yellow arrows indicate the EB1 comets in the cytoplasm. (E) Representative fluorescence image for EB1-mRuby3 in GFP-centrin2–positive WT MTECs with boxes indicating the I and P regions. (F) EB1 speed at the I and P regions in MCCs of WT MTECs. Speed of EB1 comets showing spatial differences (n = 10). Two-tailed Mann-Whitney U test; **, P < 0.01. (G) EB1 speeds in the I and P regions around the Fzd and the other sides of the AJC in MCCs of WT MTECs (n = 10 cells). Kruskal-Wallis test with the Steel-Dwass multiple-comparison test; **, P < 0.01; n.s., P ≥ 0.05. (H) Schematic illustration of differences in EB1 dynamics around the Fzd and other side of the AJC in MCCs of GFP-centrin2–positive WT MTECs. The EB1 comets display a long residence time at the Fzd side of the AJC. Inter, I region; Peri, P region. (I) Representative image for mEGFP–α-tubulin (mEGFP-αtub; red) expressed in MCCs of WT MTECs (upper panel) and kymograph showing the motility of MTs in MCCs of WT MTECs (lower panel). Note the high motility of MTs as they move near the Vangl side of the AJC, as shown in the boxed region (yellow dashed lines). (J) Representative images of mEGFP–α-tubulin in MCCs of WT and Daple-KO MTECs (left panels) and analyses of MT live imaging using the LPX flow plugin (right panels). The movement speed of the α-tubulin signal is repressented by colors (red indicates high speed; blue indicates low speed). The arrow indicates a decrease in speed at the Fzd side of the AJC in WT MCCs. (K) Statistical analysis of the speed of MTs in WT MCCs (n = 4 cells). ROIs were set at the Fzd and other side of the AJC. Two-tailed Mann-Whitney U test; *, P < 0.05. Scale bars represent 5 µm in B, C, E, I, and J and 1 µm in D.

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