Migratory cells were stained and counted under a light microscope. dystroglycan promoted endothelial cell migration and organization into capillary-like structures. CD93 proved to be phosphorylated on tyrosine 628 and 644 following cell adhesion on laminin through dystroglycan. This phosphorylation was shown to be necessary for a proper endothelial migratory phenotype. Moreover, we Rabbit Polyclonal to PECAM-1 showed that during cell spreading phosphorylated CD93 recruited the signaling protein Cbl, which in turn was phosphorylated on tyrosine 774. Altogether, our results identify a new signaling pathway which is activated by the cooperation between CD93 and dystroglycan and involved in the control of endothelial cell function. and M= 3). Scale bar, 12 m. In the inset white dots show CD93 and -DG colocalization at the cell margin. Scale bar of the inset is 3 m. (B) CD93-YFP and -DG-CFP were cotransfected AR-C155858 into ECs. Fully spread cells on laminin-coated surfaces were fixed and subjected to immunofluorescence. Immunofluorescence shows CD93 and -DG colocalization both at the plasma membrane and within intracellular vesicles. Scale bar, 8 m. (C) Cells treated as in B were subjected to FRET analyses. The mean value of the FRET efficiency between acceptor (CD93-YFP) and donor (-DG-CFP) was 9.11 0.84%, after subtraction of the background. FRET data represent the means SD of three independent experiments, carried out on different days and with different cell preparations. (D) Representative confocal images of CD93/-DG protein interaction detected by Duolink stain. HUVEC exponentially growing on laminin-coated surfaces were fixed and treated at the same time with mouse anti-CD93 and rabbit anti–DG antibodies (CD93–DG). Close proximity of the primary antibodies was revealed by localized amplification. Protein-protein interactions were visualized as individual spots by red fluorescence. Background was assayed by removing one of the two primary antibodies from the reaction (anti–DG antibodies removed, neg. contr. CD93; anti-CD93 removed, neg. contr. -DG). DIC images of stained cells are shown. The corresponding cell boundary is indicated by white dotted lines. Experiment was performed three times. Scale bars represent 18 m. To assess if the interaction was direct as suggested by the FRET analyses, we performed a proximity ligation assay, which allows localization of protein-protein interactions at single-molecule resolution . In exponentially growing ECs treated simultaneously with anti-CD93 and anti–DG primary antibodies, we observed the presence of fluorescent spots due to localized amplification of the probes bound in close proximity, whereas we did not observe any fluorescent signal when the primary antibodies were used alone (Figure ?(Figure2D).2D). Altogether, these results support the idea that in ECs CD93 and -DG are in close association. CD93 or DG silencing impairs EC function Previously, we demonstrated that proliferation, migration, and differentiation of human primary ECs were strongly decreased when the function of CD93 was neutralized . Therefore, to assess whether CD93/-DG convergence had functional consequences in ECs during angiogenesis, we first analyzed changes in cell number and viability in DG-silenced HUVEC at different time points of cell growth. ECs infected with lentiviruses expressing either DG shRNA showed a decrease in cell viability, as well as in cell number when compared to cells not infected or infected with an unrelated shRNA (Figure 3A and 3B). Importantly, the same extent of reduction in cell number and viability was observed also in CD93-silenced cells (Figure 3A and 3B). Moreover, analysis of cell migration showed that ECs silenced for DG exhibited AR-C155858 a significant decrease in VEGF-stimulated migration compared to control cells (Figure ?(Figure3C),3C), similar to that previously observed in CD93-silenced ECs . Since in a wound healing assay the open gap is sealed through a combination of proliferation and migration , we asked whether CD93? or DG-silenced cells were able to heal a wound. As expected, HUVEC expressing either CD93 or DG shRNAs were unable to heal the wound in 8 hours of cell growth, in contrast to cells infected with an unrelated shRNA that filled the open gap in the same period of time (Figure 3D and 3E). Interestingly, proliferation and migration of CD93/DG double-silenced cells decreased in comparison to control cells and the extent of reduction was equal or higher to that observed for individual-silenced cells (Figure S4), suggesting that CD93 and -DG exert unidirectional effects on downstream effector(s). Finally, we performed a tube formation assay on AR-C155858 Matrigel, a substrate that allows attachment and differentiation of AR-C155858 ECs. HUVEC infected with an unrelated shRNA formed a complete network of tubular-like structures, whereas only a small number of tubes were formed by DG-silenced ECs (Figure 3F and 3G). The same impaired tubulogenesis was previously described for HUVEC grown on Matrigel in the presence of an anti-CD93 neutralizing antibody . Open in a separate window Figure 3 CD93 or DG knockdown impairs EC functionHUVEC were infected with a lentiviral vector expressing unrelated (unr), or CD93 (clones 85 or 86), or DG (clones C7 or C10) shRNAs. Not infected ECs were also.