Panel a: The cross-sectional microscopy images of three asymmetric adhesion junctions (AAJs) in endothelial cells, showing the localization of VE-cadherin-mScarlet in magenta and PACSIN2-GFP in green. Panel b: Magnified microscopy views of individual AAJs with orthoslices in XY, YZ, and XZ planes. Arrowheads indicate narrow concave curvature at the junction front, while arrows point to ring-shaped structures and spiral-shaped membranes. Panel c: A representative FIB-SEM slice of three endothelial AAJs overlaid with plasma membrane segmentations in cyan. The right images show plasma membrane surface renderings of individual AAJs. Panel d: Violin and box plots depicting the average membrane curvature per pixel from the follower cell sides perspective. Panel e: Heatmaps of subjunctional curvature values with radius of curvature within AAJs.
Ultrastructural characterization of the asymmetric junctional membrane with Cryo-SIM and FIB-SEM. (a) Representative FIB-SEM slice of three AAJs in BOECs acquired at 4 nm isotropic voxel size overlaid with Cryo-SIM–acquired localization of VE-cadherin–mScarlet (magenta) and PACSIN2-GFP (green). (b) Magnification of FIB-SEM images of individual AAJs with orthoslices in XY, YZ, and XZ, with dotted lines indicating axes along which the volume was sliced. Arrowheads indicate narrow concave curvature at the junction front; arrows indicate ring-shaped structures and spiral-shaped membranes. (c) Left image is a representative FIB-SEM slice of three endothelial AAJs acquired at 4 nm isotropic voxel size overlaid with plasma membrane segmentations in cyan. Right images show plasma membrane surface rendering of individual AAJs. See Video 3. (d) Violin and box plots depicting the average membrane curvature per pixel from a 60 nm search radius from the follower cell side’s perspective. (e) Heatmaps of subjunctional curvature values (k) with radius of curvature (nm) within AAJs. k = 0–0.133, radius = ∼30-infinity nm. Scale bars = 0.3 and 0.5 μm.