Table 1 Key resource table referring to individual key resources used in this study

 Human EAhy926

BMPR2^wt; WT ECs; WT

⁴⁸

See supporting information⁴⁸ Figure S1 and corresponding Materials and Methods for further information

 Human EAhy926 BMPR2 KO1

BMPR2^+/-; BMPR2 deficient ECs; ECs with BMPR2 deficiency

⁴⁸

Haploinsufficiency model for BMPR2, created by CRISPR/Cas9 in Hiepen et al. Plos Biol. 2019; see supporting information⁴⁸ Figure S1 and corresponding Materials and Methods for further information

 Human umbilical vein endothelial cells (female)

HUVEC

¹¹³

Single donor cells were sex-specifically genotyped prior to application in this work

 African green monkey kidney fibroblasts (SV40 T antigen)

COS7

ATCC

CRL-1651

 Tg(kdrl:EGFP) ^s843

Tg(kdrl:EGFP)

¹⁰⁴

ZFIN:ZDB-ALT-050916-14

 pcDNA3.1 BMPR2-HA- LF

BMPR2-HA

⁴⁸

Long form of BMPR2 amino- terminally tagged with HA

 pcDNA3.1 BMPR2-HA- SF

BMPR2-HA-SF

⁴⁸

Short form of BMPR2 amino- terminally tagged with HA

 pcDNA3.1 BMPR2-MYC-K230R

BMPR2-K230R-Myc

 pcDNA3.1 ALK1-meGFP

ALK1-GFP

 pcDNA3.1 ALK2-meGFP

ALK2-GFP

 pcDNA3.1 ALK3-meGFP

ALK3-GFP

 pcDNA3.1 Endoglin-meGFP

Endoglin -GFP

 BMPR2 c-term myc

BMPR2-myc

⁴⁸

The coding sequence of human BMPR2 was C-terminally myc-tagged and subcloned into pcDNA3.1 from HA-BMPR2-LF plasmid

 BMPR2- n-term EGFP

BMPR2-GFP

this paper

Monomeric enhanced GFP cloned after signal peptide. Gly5 linker between megfp and mature LF BMPRII sequence.

 pRK5 meGFP

GFP

Addgene #18696

 CDC42-Q61L-eGFP

C.A. CDC42-GFP

¹¹⁴

Activating mutation at position 61

 pTriEx4-Cdc42-2G

CDC42- biosensor; CDC42 activity sensor; CDC42 2 G activity sensor

¹¹⁵

Addgene #68814

Second generation CDC42 activity sensor based on original sensor design from Klaus Hahn lab but with modified domain orientation and permutated fluorescent proteins for better dynamic range

 Lifeact-TagGFP2

LifeAct-GFP

Ibidi USA; Fisher scientific

#50-114-9044

 CMVdel-dimericTomato-WASp(CRIB)

Active endogenous CDC42 biosensor

⁸⁹

Addgene #191450

 bmpr2b E2 MO

5ʹAGCTGCCGACACACAAAATGAGAAA-3ʹ

GENE Tools

 gRNA exon 1

5ʹ- TGAGAGTGCGGATCCTAAAC – 3ʹ

Integrated DNA Technologies

 gRNA exon 3

5ʹ-GCGTTGTGGTAACCACGCAA -3ʹ

Integrated DNA Technologies

 Accell siRNA SMARTPOOL targeting BMPR2

siBMPR2

Dharmacon

E-005309-00-0010

 Accell FITC-labeled non-targeting siRNA

si-FITC

Dharmacon

D-001950-01-20

 Accell ONTARGETplus non-targeting siRNA

siscr

Dharmacon

D-001920-01-50

 Accell siRNA delivery media

Dharmacon

B-005000

 ZF-Bmpr2b_E2_rev1

5ʹ-CACGATAGCCAAAGAACAGCG-3ʹ

 ZF-b-actin_fwd

5ʹ-TGTTTTCCCCTCCATTGTTGG-3ʹ

 ZF-b-actin_rev

5ʹ-TTCTCCTTGATGTCACGGAC-3ʹ

 ZF-bmpr2b-Exon1_fwd

5ʹ-ATGAGCTTGAACTTCGGTCGAT-3ʹ

 ZF-bmpr2b-Exon1_rev

5ʹ- GACACCCTTTAAGACATTGCAGG-3ʹ

 ZF-bmpr2b-Exon3_fwd

5ʹ- GTATCTCAGTGTTTCACTCCACC-3ʹ

 ZF-bmpr2b-Exon3_rev

5ʹ- ATGACATCACAACATGCTGATACA-3ʹ

 anti-phospho SMAD1/5 (Ser463/465) (41D10)

Cell Signaling Technology

#9516

Dilution WB: 1:1000

 Anti-Smad1 XP

Cell Signaling Technology

#6944

Dilution WB: 1:1000

 anti-phospho-AKT (Ser473)

Cell Signaling Technology

#9271

Dilution WB: 1:1000

 anti-phospho-AKT (Thr308)

Cell Signaling Technology

#9275

Dilution WB: 1:1000

 anti-AKT

Cell Signaling Technology

#9272

Dilution WB: 1:1000

 anti-phospho MLC2 (Thr18/Ser19)

Cell Signaling Technology

#3674

Dilution WB: 1:1000

 anti-GAPDH

Cell Signaling Technology

#14C10

Dilution WB: 1:1000

 anti-Myc

Cell Signaling Technology

#9B11

Dilution IF: 1:10.000

Dilution

PLA: 1:5000

 anti-GFP

Cell Signaling Technology

#2956

Dilution PLA: 1:500; IF: 1:750

 anti-HA (HA-7)

Sigma-Aldrich

Dilution SPTM: 1 µg/ml;

IF: 1:1000

 anti- vinculin

Sigma-Aldrich

#V9131

Dilution

IF: 1:400

 anti- Beta-Catenin

BD Bioscience

#610153

Dilution IF: 1:200

 anti-BMPR2

BD Bioscience

#612292

Dilution

WB: 1:1000

 anti-CDC42

Abcam

#155940

Dilution IF: 1:750

 anti-CD34

Abcam

#81289

 Anti-Mouse Alexa Fluor 488 (goat)

Thermofisher Scientifics

A10684

 Anti-Mouse Alexa Fluor 594 (goat)

Thermofisher Scientifics

A11020

 Anti-Rabbit Alexa Fluor 488 (chicken)

Thermofisher Scientifics

A21441

 Anti-Rabbit Alexa Fluor 594 (goat)

Thermofisher Scientifics

A11072

 Goat anti rabbit 2ndary HRP-linked antibody

Dianova

111-035-144

Pan BMP-Smad inhibition

 Goat anti mouse 2ndary HRP-linked antibody

Dianova

115-035-068

Pan PI3K inhibition

 Critical commercial assays

Rho Kinase inhibition

 Proximity Ligation Assay (PLA) DUOLINK kit

Sigma-Aldrich

#DUO92101

 DAPI

Sigma-Aldrich

D954

 Phalloidin CruzFluor 594 conjugate

Santa Cruz Bio

sc-363795

 Phalloidin CruzFluor 647 Conjugate

SantaCruz

Sc-363797

 Vybrant™ DiI Cell Labeling Solution

Thermo- Fisher Scientific

V22885

Vybrant™ DiI Cell Labeling Solution

 Vybrant™ DiO Cell Labeling Solution

Thermo- Fisher Scientific

V22886

Vybrant™ DiO Cell Labeling Solution

 Celltracker Red CMTPX Dye

Thermo- Fisher Scientific

C34552

 Celltracker Green CMFDA Dye

Thermo- Fisher Scientific

C2925

 LDN-193189

LDN

Tocris Bioscience

# 6053

 LY294002

LY

Cell Signaling

# 9901

 UCL-TRO-1938

TRO1938

MCE

HY-154848

 Y-27632

Rocki, Rock inhibitor

Stemcell Technologies

# 72304

 CytochalasinD

CytoD

Thermofisher Scientifics

PHZ1063

 Dimethyl sulfoxide (DMSO)

Sigma-Aldrich

D2650

 Protease Inhibitor Cocktail

Sigma-Aldrich

P6148

 Paraformaldehyde 4% (PFA)

VWR

VWRK4186

 Thrombin

Sigma-Aldrich

T4648 1KU

 Fibrinogen type 1

Sigma-Aldrich

F8630

 Endothelial cell growth supplement (ECGS)

ECGS

Corning, NY

#356006

 Heparin

Merck

375095-100KU

 Fibronectin from bovine plasma

Sigma-Aldrich

F1141

 Normal goat serum

Sigma-Aldrich

NS02L

 Aprotinin

Sigma-Aldrich

A1153

 Recombinant hBMP6

BMP6

¹⁰²

Human mature BMP-6 produced in Chinese hamster ovary cells

 Fluospheres Dark Red

Thermofisher Scientifics

F8807

 Quantum dot labeled F(ab’)2-Goat anti-Mouse IgG (H + L) Secondary Antibody, Qdot 655 nm

Qdot antibody

Thermo- Fisher Scientific

#Q-11021MP

Further information on QDot labeling and SPTM ca be found in Zelman-Femiak M and Harms GS et al.: Biotechniques 2010, Guzman A. And Knaus P. Et al. J Biol Chem. 2012

 96 well plate heating insert for life cell microscopy

PECON

M96 2000 EC

 Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix

Corning

354230

 Cytodex 3 beads

GE Healthcare

GE17-0485-01

 Scratch wound assay- Culture-Insert 2 Well in µ-Dish 35 mm

Ibidi

#81176

 µ-Slide 8 Well Glass Bottom

Ibidi

#80827

 Fiji imagej

¹¹¹

 3D TFM analysis

TFMLAB

¹⁰⁸

Code freely available at: https://gitlab.kuleuven.be/MAtrix/Jorge/tfmlab_public

 MATLAB

MathWorks

 Paraview

Kitware

 ZEN Blue

Carl Zeiss

 ZEN Black

Carl Zeiss

 Prism

Graphpad

 Biorender

Biorender.com