RhoB regulates endothelial Rac1 localization and trafficking. (A) mCherry-Rac1 and HA-RhoBV14 were expressed in EA.hy.926 cells and stained for HA epitope and F-actin. Right graph quantifies relative mCherry-Rac1 localization at cell border and shows the mean + SEM of at least 10 cells per experiment quantified in three different experiments. Arrowheads point to mCherry-Rac1 at the plasma membrane. RhoBV14 reduces Rac1 border localization even in cells correctly spread on the substrate. ***, P = 5.30 × 10−5. (B) HUVECs were transfected with the indicated siRNAs and then mCherry-Rac1 expressed during the last 24 h. TNF-pretreated cells were stimulated with thrombin for 60 min and fixed, and mCherry-Rac1 was quantified as in A. Graph shows the mean + SEM of four experiments. *, P = 0.026; ***, P = 5 × 104. (C) mCherry-Rac1 colocalizes with active RhoB in intracellular compartments. mCherry-Rac1 and HA-RhoBV14 were expressed in EA.hy.926 cells. Significant colocalization was observed in a perinuclear compartment (1) and in a dispersed vesicular pattern (2; arrowheads). (D and E) HA-RhoBV14 and mCherry-Rac1 co localize in Rab5-GFP (D) and Rab7-FGP–positive (F) compartments. Right images show an enlargement of the squared area. (F) Endogenous RhoB and mCherry-Rac1 partly colocalize in a vesicular compartment in confluent, TNF-pretreated HUVECs stimulated or not with thrombin for 30 and 72 min (arrowheads). mCherry-Rac1 is also localized in nascent membrane protrusions (arrowhead). Bars: (A–F) 20 µm; (D–F, enlarged areas) 5 µm. (G) STED confocal images of Rac1 clusters, which appear different to those of RhoB in the same vesicle (arrowhead). Bar, 4 µm (H) BioID assay with BirA-RhoB. HEK293 cells were transfected with BirA-RhoB and GFP-Rac1 when indicated. GFP-Rac1 was immunoprecipitated with anti-GFP antibodies. Biotinylated proteins were detected by blotting with streptavidin peroxidase (asterisks). IgG, isotype-specific antibody control. Coexpression of BirA-RhoB reduces GFP-Rac1 detection in the cell lysates. (I) mCherry-Rac1 was expressed for 24 h in siRNA-transfected HUVECs. TNF-pretreated cells were stimulated with thrombin between 60 and 100 min, and Rac1 vesicular movement was recorded at 15-s intervals during a minimum of 5 min. Arrowheads point to mCherry-Rac–positive vesicles. In the top row of images, one vesicle divides in two and merges again. Bars: 20 µm; (zoom) 2 µm. (J) Each graph represents the tracks of 15 vesicles per cell, in eight cells, from three different experiments. Vesicle tracks were plotted with a common origin.
