Fig. 4

Fig. 4 BMPR2 promotes CDC42 activity at the plasma membrane of ECs via the PI3K-CDC42 signaling axis.

a Immunofluorescence staining of BMPR2^wt ECs overexpressing HA-tagged BMPR2 (BMPR2-HA). Phalloidin (white), DAPI (blue), BMPR2-HA (green), CDC42 (magenta). Insets show regions of interest. Arrows indicate sites of colocalization between BMPR2-HA and CDC42 at cell cortex or in filopodia compartment base and shaft. Scale bar: 10 µm. b Heat-map showing FRET-measured CDC42 activity across whole areas of BMPR2^wt ECs (left) and BMPR2^+/- ECs (right). Insets show regions of interest. Quantification of FRET signal from depicted TFP-WASP-Venus-CDC42 construct in BMPR2^wt ECs and BMPR2^+/- ECs (right). ***p < 0.001. c Immunofluorescence staining of BMPR2^+/- ECs overexpressing GFP or a constitutively active CDC42 in fusion with GFP (C.A-CDC42-GFP) and quantification of the number of filopodia per 100 µm of cell edge (right). Phalloidin (magenta), DAPI (blue), GFP (green). Insets show regions of interest. **p < 0.01. Scale bar: 20 µm. d Immunoblot against CDC42 and GAPDH from BMPR2wt ECs and BMPR2+/- ECs cultured in EC activation medium containing 20% Serum and pro-angiogenic growth factors. e Immunoblot against pAKT-Ser473 (S473), pAKT-Thr308 (T308), total AKT (tAKT) and GAPDH from BMPR2^wt ECs or BMPR2^+/– ECs treated with either DMSO, 10 µM LY294002 or 10 µM UCL-TRO-1938 for 60 min in EC activation medium containing 20 % Serum and pro-angiogenic growth factors. f Quantifications of the number of filopodia per 100 µm cell edge of BMPR2^wt ECs or BMPR2^+/– ECs treated with either DMSO, 10 µM LY294002 or 10 µM UCL-TRO-1938 for 60 min in EC activation medium. *p < 0.05, ***p < 0.001. g Spinning disk microscopy images of BMPR2^wt ECs or BMPR2^+/– ECs expressing dTomato-WASp(CRIB) active CDC42 biosensor, and co-expressing either GFP or BMPR2-GFP for BMPR2^+/– ECs. Scheme depicts the mechanism of action of the CDC42 activity biosensor: Upon activation by PIP₃-anchored GEFs, CDC42 is enriched at the plasma membrane and can be bound by dTomato-WASp(CRIB) biosensor, promoting relocation and local enrichment of the fluorescent biosensor. Relocation of the dTomato–WASp(CRIB) biosensor was measured by quantifying the ratio of the sensor membranous intensity over its cytosolic intensity. Arrowheads indicate sites of enrichment of the biosensor at the plasma membrane. Scale bar: 20 µm. *p < 0.05, ****p < 0.0001.