Figure 9.
Figure 9. Rac trafficking is essential for endothelial barrier recovery after thrombin stimulation. (A) Inhibitors of intracellular trafficking delay barrier recovery. TNF-pretreated HUVECs were stimulated with thrombin to induce acute contraction, and after 20 min, 10 µM/l monensin and 10 µg/ml brefeldin A or 60 µM/l of the dynamin inhibitor dynasore was added when indicated. Changes in TEER were recorded and quantified at the indicated times. Graph shows the mean + SEM from four independent experiments. *, P < 0.031. (B) TNF-pretreated HUVECs stimulated with thrombin for 72 min in the presence or absence of the indicated inhibitors and stained for β−catenin and F-actin. Automated image analysis was applied to detect empty areas (mask). Bar, 20 µm. (C) HGF accelerates endothelial barrier reformation in TNF-pretreated endothelial cells. Relative contraction levels after thrombin stimulation at the indicated times in the presence or absence of 30 ng/ml HGF added 20 min after stimulation. Graph shows the mean + SEM of eight different experiments. **, P = 0.006. (D) Cells were treated as in C, fixed 72 min after thrombin stimulation, and stained for the indicated proteins. Automated image analysis was applied to detect empty areas in the cell monolayer (mask). Bar, 20 µm. (E) Effect of dynasore and HGF on mCherry-Rac1 localization. EA.hy.926 cells were transfected with mCherry-Rac1, pretreated with TNF, and stimulated with thrombin alone or in combination with dynasore and HGF as in B and D, respectively. Cells were fixed and stained for F-actin. Bar, 20 µm. (F) TNF increases endothelial sensitivity to Rac inhibition during endothelial barrier reformation. HUVECs were treated with or without TNF, and the effect of NSC23766 on endothelial barrier recovery upon acute contraction was measured by electric cell substrate impedance sensing; T, thrombin. The mean + SEM from three independent experiments is shown. *, P = 0.011. (G) A model for the endocytic control of Rac1 translocation to the plasma membrane through RhoB during barrier restoration. Rac1 shuttles between early endosomal compartment and the plasma membrane but is also delivered to a RhoB-positive late endosomal compartment. High RhoB levels displace Rac1 to endosomes and prevent Rac1 recycling during barrier recovery. Low RhoB activity favors Rac1 recycling to the cell border, accelerating barrier recovery after contraction.

Rac trafficking is essential for endothelial barrier recovery after thrombin stimulation. (A) Inhibitors of intracellular trafficking delay barrier recovery. TNF-pretreated HUVECs were stimulated with thrombin to induce acute contraction, and after 20 min, 10 µM/l monensin and 10 µg/ml brefeldin A or 60 µM/l of the dynamin inhibitor dynasore was added when indicated. Changes in TEER were recorded and quantified at the indicated times. Graph shows the mean + SEM from four independent experiments. *, P < 0.031. (B) TNF-pretreated HUVECs stimulated with thrombin for 72 min in the presence or absence of the indicated inhibitors and stained for β−catenin and F-actin. Automated image analysis was applied to detect empty areas (mask). Bar, 20 µm. (C) HGF accelerates endothelial barrier reformation in TNF-pretreated endothelial cells. Relative contraction levels after thrombin stimulation at the indicated times in the presence or absence of 30 ng/ml HGF added 20 min after stimulation. Graph shows the mean + SEM of eight different experiments. **, P = 0.006. (D) Cells were treated as in C, fixed 72 min after thrombin stimulation, and stained for the indicated proteins. Automated image analysis was applied to detect empty areas in the cell monolayer (mask). Bar, 20 µm. (E) Effect of dynasore and HGF on mCherry-Rac1 localization. EA.hy.926 cells were transfected with mCherry-Rac1, pretreated with TNF, and stimulated with thrombin alone or in combination with dynasore and HGF as in B and D, respectively. Cells were fixed and stained for F-actin. Bar, 20 µm. (F) TNF increases endothelial sensitivity to Rac inhibition during endothelial barrier reformation. HUVECs were treated with or without TNF, and the effect of NSC23766 on endothelial barrier recovery upon acute contraction was measured by electric cell substrate impedance sensing; T, thrombin. The mean + SEM from three independent experiments is shown. *, P = 0.011. (G) A model for the endocytic control of Rac1 translocation to the plasma membrane through RhoB during barrier restoration. Rac1 shuttles between early endosomal compartment and the plasma membrane but is also delivered to a RhoB-positive late endosomal compartment. High RhoB levels displace Rac1 to endosomes and prevent Rac1 recycling during barrier recovery. Low RhoB activity favors Rac1 recycling to the cell border, accelerating barrier recovery after contraction.

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