Fig 2: Summary schematic.Internalised NRP-α5 integrin complexes undergo Rab11+ long-loop recycling via p120RasGAP, promoting polarised nascent adhesion assembly and directional migration in endothelial cells. In the absence of NRP1 and NRP2, α5 integrin arrests in early endosomes and Rab7+ late endosomes, before being recycled via retrograde transport and the dynein–dynactin complex. In vivo, endothelial deletion of NRP1 and NRP2 severely impairs polarised sprouting and fibronectin deposition. Created with BioRender.com.

Fig 3: FAK phosphorylation mediates p120RasGAP interactions with NRP - α5 integrin complexes.a Immunoprecipitation from Ctrl and siRNA-depleted EC lysates of NRP1, NRP2 and p120RasGAP followed by SDS-PAGE and Western blotting with anti-p120RasGAP antibody. b Total cell lysate input confirming siRNA depletion of NRP1, NRP2 and p120RasGAP by Western blotting. c Representative immunoprecipitation from Ctrl and siNRP1-depleted EC lysates of NRP2 followed by SDS-PAGE and Western blotting with anti-p120RasGAP antibody. d Quantification of relative p120RasGAP expression in Ctrl and siNRP1 lysates following immunoprecipitation of NRP2, (N = 3 independent experiments) one-sample t-test, *p < 0.05. e Total cell lysate input confirming inhibition of FAK phosphorylation following pre-treatment with 2 µM PF562271. Below shows the chemical structure of PF562271 compound. f Immunoprecipitation of NRP1 and NRP2 from EC lysates followed by SDS-PAGE and Western blotting with anti-p120RasGAP antibody showing loss of association following pre-treatment with 2 µM PF562271. g Total cell lysate input showing NRP1, NRP2, α5 integrin, FAK and p120RasGAP expression by Western blotting following pre-treatment with 2 µM PF562271. h Immunoprecipitation of NRP2 from Ctrl and siNRP1 EC lysates followed by SDS-PAGE and Western blotting with anti-p120RasGAP antibody showing loss of association following pre-treatment with 2 µM PF562271. i Representative confocal microscopy images showing α5 integrin colocalisation with Rab11, EEA1, Rab4 or Rab7 in Ctrl ECs ± pre-treatment with PF562271. j Quantification of α5 integrin+ Rab11, EEA1, Rab4 or Rab7 endosomes/cell in Ctrl and siRNA-depleted ECs, ±pre-treatment with PF562271 (n = 30 cells from three independent biological replicates), Student’s t-test, ****p < 0.0001.

Fig 4: Development of α5 integrin+ fibrillar adhesions and subsequent EDA-FN fibrillogenesis is lost in siNRP1/2 ECs.a Representative confocal microscopy images showing α5 integrin+ and tensin-1+ fibrillar adhesions in Ctrl, siNRP1, siNRP2 and siNRP1/2 ECs following 24 h adhesion to FN. b Quantification of mean α5 integrin+ and tensin-1+ adhesion length (µm), (n = 30 cells from three independent biological replicates), one-way ANOVA + post hoc multiple comparisons tests, *p < 0.05, **p < 0.01, ****p < 0.0001. c Representative confocal microscopy images showing endogenous EDA-FN in Ctrl, siNRP1, siNRP2 and siNRP1/2 ECs following 24 h adhesion to FN. Middle panels show rendered EDA-FN in Ctrl and siNRP1/2 ECs with XZ-ratio. d Quantification of mean EDA-FN intensity and mean EDA-FN fibre density from confluent ECs, (n = 30 ROIs from three independent biological replicates), one-way ANOVA + post hoc multiple comparisons tests, **p < 0.01, ****p < 0.0001. e Relative adhesion to FN by NRP1, NRP2 and NRP1/2 siRNA-treated ECs at 24 h, (N = 3 independent experiments), one-way ANOVA + post hoc multiple comparisons tests, ****p < 0.0001. f Representative confocal microscopy images showing colocalisation between EDA-FN, α5 integrin and EEA1 in Ctrl and siNRP1/2 ECs following 24 h adhesion to FN. g Quantification of α5 integrin+ and EDA-FN+ EEA1 early endosomes/cell, (n = 30 cells from three independent biological replicates), Student’s t-test, ****p < 0.0001. h Representative confocal microscopy images showing colocalisation between EDA-FN, α5 integrin and Rab7 in Ctrl and siNRP1/2 ECs following 24 h adhesion to FN. i Quantification of α5 integrin+ and EDA-FN+ Rab7 late endosomes/cell, (n = 30 cells from three independent biological replicates), Student’s t-test, ****p < 0.0001.

Fig 5: Endothelial NRPs and p120RasGAP promote cell polarity during directional cell migration.a Representative Western blotting of Ctrl and siRNA-depleted EC lysates ± 5 or 30 mins VEGF165 stimulation, showing expression changes in total FAK, pFAKTyr397 and pFAKTyr407. b Supporting densitometric timecourse analysis of pFAKTyr397 and pFAKTyr407 expression shown in (a), relative to total FAK expression, (N ≥ 3 independent experiments), one-way ANOVA + post hoc multiple comparisons tests, *p < 0.05, **p < 0.01. c Densitometric analysis of unstimulated pFAKTyr397 and pFAKTyr407 expression shown in (a), relative to β-actin loading control, (N = 3 independent experiments), one-sample t-test, *p < 0.05. d Immunolabelling of endogenous pFAKTyr407+ leading vs trailing edge focal adhesions the scratch-wound edge. D’ and D” panels show the process of skeletonising focal adhesions prior to analysis. e Ratio quantification of pFAKTyr407+ leading vs trailing edge adhesions in Ctrl and siRNA-depleted ECs at the scratch-wound edge, (n = 30). f Representative confocal microscopy images quantified in (e). g Representative confocal microscopy images showing GM130+ Golgi body positioning and F-actin protrusions in Ctrl and siRNA-depleted ECs at the scratch-wound edge fixed 8-h post scratch initiation. h Polar plots showing Golgi body orientation in Ctrl and siRNA-depleted ECs, (n = 100). i Total cell lysate input confirming siRNA depletion of Rab11 by Western blotting. j Representative confocal microscopy images showing α5 integrin colocalisation with pFAKTyr407 in Ctrl and siRab11a + b ECs following 180 min adhesion to FN. k Quantification of α5 integrin+ and pFAKTyr407+ adhesions/cell in Ctrl and siRab11a + b ECs, (n = 30 cells from three independent biological replicates), Student’s t-test, ****p < 0.0001. l Quantification of α5 integrin+ EEA1 endosomes/cell, (n = 30 cells from three independent biological replicates), Student’s t-test, ****p < 0.0001. m Quantification of α5 integrin+ Rab7 endosomes/cell, (n = 30 cells from three independent biological replicates), Student’s t-test, ****p < 0.0001. n Polar plot showing Golgi body orientation in Ctrl and siRab11 ECs, (n = 100).