Spatiotemporal recruitment of PACSIN2, BIN1, and SNX9 correlates with specific stages of junction remodeling. (a) Quantification of residency time of overexpressed GFP-tagged PACSIN2, BIN1, and SNX9 at AAJs in collectively migrating BOECs following scratch wounding, from widefield time-lapse recordings acquired at 6-s intervals. n = 71, 124, and 73 AAJs positive for the different proteins, respectively, from three independent experiments. (b) Widefield fluorescence images of BAR protein distribution along AAJs from collectively migrating BOECs expressing VE-cadherin–mScarlet (magenta) and GFP-tagged BAR proteins (green). Schematic drawings show how subjunctional distribution was classified into three categories based on the localization of BAR proteins relative to VE-cadherin: mid, over, or trailing end. (c) Quantification of the proportion of AAJs in which the indicated GFP-tagged BAR proteins localize to each of the defined subjunctional regions. n = 71, 124, and 73 AAJs positive for the different proteins, respectively, from three independent experiments. (d) Widefield time-lapse sequence depicting stages of AAJ remodeling from collectively migrating BOECs expressing VE-cadherin–mScarlet (magenta) and GFP-tagged BAR proteins (green). Merged and VE-cadherin channels are shown to highlight the progression of remodeling events associated with the presence of each BAR protein. Orange arrows indicate VE-cadherin internalizing events at AAJ trailing ends. (e) Bar graphs showing the distribution of remodeling stages among AAJs positive for the indicated GFP-tagged BAR proteins. n = 71, 124, and 73 AAJs positive for the different proteins, respectively, from three independent experiments. (f) Widefield images of collectively migrating BOECs expressing BIN1-GFP or GFP-SNX9 (green), immunostained for VE-cadherin (magenta) and PACSIN2 (yellow). White boxes indicate ROIs that are magnified in the right panels, highlighting AAJs positive for both PACSIN2 and the indicated BAR protein. Green arrowheads indicate regions of BIN1 or SNX9 recruitment, and yellow arrowheads indicate regions of PACSIN2 recruitment along AAJs. (g) Dot plots showing the percentage of BIN1- or SNX9-positive AAJs per cell that are positive for PACSIN2. n = 29 cells for BIN1-GFP and 28 for GFP-SNX9, from three independent experiments. (h) Dot plots showing the percentage of PACSIN2-positive AAJs per cell that are positive for BIN1 or SNX9. n = 29 cells for BIN1-GFP and 28 for GFP-SNX9, from three independent experiments. (i) Dot plots of Mander’s split colocalization coefficient of BIN1-GFP and GFP-SNX9 with PACSIN2 at AAJs. n = 70 and 42 junctions, respectively, from three independent experiments. (j and k) Widefield time-lapse images of collectively migrating BOECs expressing VE-cadherin–mTurquoise (blue), PACSIN2-GFP (green), and BIN1-mScarlet (magenta) (j) or mScarlet-SNX9 (magenta) (k) imaged at 6-s time resolution (see Videos 7 and 8). Arrowheads indicate signal of junctional BAR proteins (green for PACSIN2, magenta for BIN1 or SNX9). (l) Dot plots showing differences in recruitment timing of BIN1 and SNX9 following junctional PACSIN2 recruitment. n = 10 AAJs per condition, from at least three independent live-imaging recordings. (m) Representative widefield time-lapse images of mosaic AAJs between HUVECs expressing lentiviral GFP-SNX9 (green) or mScarlet-SNX9 (magenta) from three independent experiments. Arrowheads indicate junctional recruitment of SNX9-GFP (green) or SNX9-mScarlet (magenta) (See Video 9). Data are presented as median in a (box plots show interquartile range 25–75 percentiles, and whiskers representing the 5–95 percentiles); as mean ± SEM in (c and e) and mean ± SD of experiment means in g, h, i, and l, circles indicate individual data points and triangles indicate means of individual experiments with colors corresponding to the experimental replicates. Statistical analysis was performed using a Kruskal–Wallis test followed by Dunn’s multiple comparisons test in a, Mann–Whitney test in g and h, and a two-sided Student’s t test in i. P values are indicated on the graphs. Scale bars = 30, 5 and 3 μm.