FIGURE 2:

p115RhoGEF is required for Rho flares and local TJ remodeling. (A-B) Time-lapse montage of active RhoA (mCherry-2xrGBD, Fire LUT) and GFP-occludin (Fire LUT) in control embryos (A) and p115.S KD embryos (B). p115.S KD flares exhibit decreased Rho activity (white arrowheads) and occludin reinforcement (white dashed boxes, enlarged below) in comparison to controls. Time 0 represents the start of the Rho flare increase. (C-E) Graphs of mean normalized intensity of active RhoA (C), occludin (E), and change in junction length (D) in control (black lines) and p115.S KD Rho flares (green lines) over time. Shading represents SEM. Control: n = 10 flares, 3 embryos, 3 experiments; p115.S KD: n = 8 flares, 3 embryos, 3 experiments. (C’–E’) Scatter plot of AUC for active RhoA (C’) was calculated from C; p = 0.0047(**). Scatter plot of change in length (D’) was calculated from D. L₁ and L₂ represent average length of junctions from individual traces from time –20–30 s and 400–450 s, respectively; p = 0.0059(**). Scatter plot of change in occludin intensity (E’) was calculated from E. I₁ and I₂ represent average intensity of occludin from individual traces from time –20–30 s and 300-350 s, respectively; p = 0.0004(***). Error bars represent mean ± SEM; significance was calculated using unpaired t tests. (F–G) Kymographs of occludin (green), active RhoA (magenta), and merged image from representative junctions projected from vertex-to-vertex over time in control (F) and p115.S KD (G) embryos. Kymographs highlight repeated Rho flares, lack of occludin reinforcement, and lack of junction contraction in p115.S KD embryos (note that p115.S KD kymographs were enhanced for brightness and contrast, as p115RhoGEF KD reduces signal intensity of occludin and active RhoA [see Supplemental Figure S3 for raw data]).