(A) 3D view still from two endothelial cells showing a turquoise expressing EC and a NeonGreen expressing EC. White box indicates zoom on the right. Arrowhead indicates shows that the turquoise EC displays a membrane protrusion at the junction region, hence, an asymmetric JMP. Bar, 4 μm. (B) Scanning electron microscopy image of asymmetric JMPs. Black box indicates zoom region, displayed on the right. Bar, 5 μm. (C) Ratio of membrane dynamics in EC with JMP and the other EC at a junction region. Open dots are individual data points from three independent experiments, represented by three different colors. Filled dots are means from three experiments. Median with 95% confidence interval (CI) is shown. One-sample T-test: *p=0.344. (D) Immunofluorescent staining for HA (green), F-actin (red), VE-cadherin (white) and DNA (blue) on HUVECs transfected with Tiam1-C1199-HA after overnight TNFα stimulation. Filled white arrowheads indicate the F-actin present at the cell-cell junction between a Tiam1-C1199-HA expressing cell and a control cell, where the open white arrowheads indicate the F-actin present at the junction between two control cells. Panel shows Z-stack from basal (0.0 µm) towards apical (2.5 µm) from left to right, respectively. Arrowheads indicate presence of F-actin rich membrane ruffles in the different focal planes. Scale bar, 30 µm. (E–G) Quantification of diapedesis direction of PMN upon TEM under flow. Majority of the PMNs cross form a wt EC underneath a (E) TIAM1-C1199, (F) Rac1-Q61L, (G) Photoactivatable (PA)-Rac1 EC (dark bar) (H) Quantification of PMN TEM under flow in cells expressing PA-Rac1 either not illuminated (Ctrl) or blue-light illuminated (activated) HUVEC. Data is normalized to control conditions. Upon Rac1 activation, increased number of neutrophils crossed the EC monolayer. Open dots are individual data points from three independent experiments, represented by three different colors. Filled dots are means from three experiments. Median with 95% CI is shown. T-test: p=0.0483. (I) Schematic overview of neutrophil transmigrating from ‘inactive’ EC (bottom left, light) underneath an ‘active’ EC (upper right, dark) showing presence of a JMP. JMP displays Rac1 activity, Arp3, and expression of PECAM-1, ICAM-1, and ACKR1.