Extended Data Fig. 7: Rho GTPase localization and interaction with GS.

a, Co-IP assays showing no detectable interaction between GS and RHOA or RHOC (red asterisk indicates a non-specific band (also present in the IgG controls and unaffected by silencing of RHOA or RHOC)). Image shown is representative of 3 independent experiments. b, Co-IP of overexpressed GLUL and RHOJ-eGFP or ΔN20-RHOJ-eGFP in ECs. Quantifications are mean ± s.e.m.; n = 4 independent experiments; *P < 0.05, one-sample t-test. In some of the experiments, the expression of ΔN20-RHOJ–eGFP was lower than the expression of RHOJ–eGFP. To correct for this, densitometric quantification was performed and signals in immunoprecipitation lanes were normalized to input signals. c, Immunoblotting for RHOA and RHOC on cytosolic (c) and membrane (m) fractions of ECs with NaK as membrane marker and GAPDH as cytosolic marker. Image shown is representative of 3 independent experiments. d, BiFC assay with GS coupled to the N-terminal half of eGFP, and RHOJ coupled to the C-terminal half of eGFP. Only when GS and RHOJ are in close proximity do the two eGFP half-sites complement each other and form a functional eGFP. e, Percentage of ECs displaying BiFC upon overexpression of GLUL-eGFP^1/2 and RHOJ-eGFP^2/2 or GLUL-eGFP^1/2 and ΔN20-RHOJ-eGFP^2/2. Data are mean ± s.e.m.; n = 3 independent experiments; *P < 0.05; Student’s t-test. f, Schematic of SPT-PALM imaging under TIRF illumination with the plasma membrane depicted at the top. The TIRF region is bright (whereas the part outside the TIRF region is greyed out) and contains the plasma membrane and its immediately adjacent space (not shown at exact relative dimensions). Weight and number of arrowheads represent the velocity of single particles (the photoswitchable fluorescent protein (PSFP) or the PSFP coupled to the protein of interest (here GS)). The PSFP is activated upon entry into the TIRF region and is colour-coded differently inside and outside of the TIRF region. PSFP–GS displays reduced velocity in the TIRF region, presumably because of palmitoylation and membrane association of GS. g, Scheme for the in-cell labelling of proteins with clickable alkyne-containing palmitoylation probes and subsequent biotin-azide clicking. X represents a palmitoylated protein, N₃ is the biotin-coupled azide. h, i, Rate of CoA release from palmitoyl-CoA as a readout for recombinant human GS autopalmitoylation while varying either the doses of palmitoyl-CoA (h) or the amounts of recombinant GS (i) (mean ± s.e.m.; n = 4 independent experiments for h and n = 5 for i; *P < 0.05, one-way ANOVA with Dunnett’s multiple comparisons versus 0 µM palmitoyl-CoA or versus 0.5 µg recombinant GS). j, Representative GS immunoblot (of 3 independent experiments) for the binding of recombinant human GS to palmitoyl-CoA agarose. IF, input fraction; FT, flow through; W8, wash fraction 8; SDS is the eluate. k–m, Representative images of RHOJ–eGFP localization in ECs under vehicle-treatment (k) or treatment with 2BP (a pan-palmitoylation inhibitor) (l). Red arrowheads indicate eGFP signal at membrane ruffles, which was quantified as the percentage of the total cellular area (m) (mean ± s.e.m.; n = 4 independent experiments; *P < 0.05 versus vehicle-treated, paired Student’s t-test). n–p, Representative images of ECs overexpressing wild-type RHOJ-eGFP (n), RHOJ-eGFP encoding the point mutation C3A (o) or RHOJ-eGFP encoding the point mutation C11A (p). Red arrowheads indicate RHOJ at the plasma membrane. ECs that are not completely in the field of view have been masked out in blue. q, Quantification of the RHOJ–eGFP positive area at the plasma membrane as a percentage of the total cell area. Data are mean ± s.e.m.; n = 5 independent experiments; *P < 0.05; one-way ANOVA with Dunnett’s comparison versus wild-type RHOJ. r, RHOJ immunoblotting on membrane versus cytosolic fractions from ECs overexpressing wild-type RHOJ-eGFP (RHOJ^WT), RHOJ-eGFP encoding the point mutation C3A (RHOJ^C3A) or RHOJ-eGFP encoding the point mutation C11A (RHOJ^C11A), with NaK as membrane marker and GAPDH and α-tubulin as cytosolic markers. s, Densitometric quantification of RHOJ/NaK as determined in r. Data are mean ± s.e.m.; n = 6 independent experiments; *P < 0.05; one-sample t-test. t, RHOJ activity in ECs under treatment with vehicle or 2BP (blots are representative of 3 independent experiments; densitometric quantification in arbitrary units is mean ± s.e.m.; *P < 0.05, paired Student’s t-test versus vehicle-treated). u, RHOJ immunoblotting of control and GLUL^KD ECs overexpressing RHOJ (RHOJ^OE) subjected to acyl-resin-assisted capture. The cleaved bound fraction (cBF) represents palmitoylated RHOJ. IF is the input fraction, whereas the cleaved unbound fraction (cUF) and the preserved bound fraction (pBF) are controls showing the depletion of RHOJ from the thioester-cleaving reagent and the near absence of non-specific binding of RHOJ to the resin (see Methods). Densitometric quantification of cBF/IF is shown (mean ± s.e.m.; n = 3 independent experiments; *P < 0.05, one-sample t-test versus control). v, Left, autopalmitoylation enables endothelial GS to interact directly (or indirectly) with the Rho GTPase RHOJ and to sustain the palmitoylation, membrane localization and activity of RHOJ (reflected by GTP binding). RHOJ activity then sustains normal EC migration and lamellipodia formation, and keeps actin stress-fibre formation at levels that promote normal EC migration and vessel branching in vivo. Through mechanisms that are not completely understood, active RHOJ inhibits signalling of the RHOA/B/C–ROCK–(p)MLC pathway (itself known to promote stress-fibre formation). The relative contribution of a direct effect of RHOJ on migration versus the indirect effect through RHOA/B/C–ROCK–(p)MLC is yet to be determined. Reduced opacity of RHOA/B/C, ROCK and (p)MLC indicates reduced signalling of this pathway. Right, loss of endothelial GS renders RHOJ less active (visually reflected by fewer palmitoylated, membrane-bound RHOJ proteins), and reduces the inhibition of the RHOA/B/C–ROCK–(p)MLC pathway. The resulting excessive stress-fibre formation causes ECs to lose migratory capacity and reduces vessel branching in vivo. Dashed lines indicate reduced activity; the red cross indicates GS blockade; the question mark indicates unknown mechanisms. Exact P values are as follows: 0.0153 (b); 0.0334 (e); 2 versus 0 μM: 0.6327; 5 versus 0 μM: 0.2841; 10 versus 0 μM: 0.1090; 20 versus 0 μM: 0.0339; 40 versus 0 μM: 0.0034 (h); 1 versus 0.5 μg: 0.5806; 2 versus 0.5 μg: 0.0319; 4 versus 0.5 μg: 0.0037; 8 versus 0.5 μg: 0.0001; 16 versus 0.5 μg: 0.0001 (i); 0.0313 (m); RHOJ C3A versus RHOJ WT: 0.0001; RHOJ C11A versus RHOJ WT: 0.0001 (q); RHOJ C3A versus RHOJ WT: 0.0015; RHOJ C11A versus RHOJ WT: 0.0007 (s); 0.0051 (t); 0.0461 (u). Scale bar, 200 μm (k, l, n–p). For gel source images, see Supplementary Fig. 1.