Fig 2: Multiple guidance cue receptors interact with ribosomes.(A) Volcano plots showing statistically enriched proteins in DCC-IP and Nrp1-IP samples identified by permutation-based FDR-corrected t-test based on three biological replicates. The LFQ intensity of the DCC or Nrp1 pulldowns over IgG pulldowns are plotted against the -log10 p-value. FDR < 0.05; S0 = 2. (B) Gene enrichment analysis of statistically enriched proteins in the DCC and Nrp1 pulldown samples. The values in each circle denotes protein count. (C–F) Western blot validation of RP co-immunoprecipitation with DCC, Nrp1 and Robo2 but not with EphB2. Each Western blot was repeated 2 to 4 times, representative images are shown. (G–J) Relative 18S and 28S ribosomal RNA abundance after control (IgG) pulldown or receptors pulldowns shows enrichment of rRNA in DCC, Nrp1, and Robo2 but not EphB2 pulldowns (unpaired two-tailed t-test; three biological replicates). Bars indicate means, error bars indicate standard deviation; *p<0.05.

Fig 3: Cdc42 is activated by the VEGF/NRP1 axis, which is associated with invadopodia formation in the cell front.a Immunofluorescence analysis showing F-actin (red) and cortactin (green) colocalisation in SW480 and LS174T cells. Arrows indicate colocalization of F-actin and cortactin in the cytoplasm. Scale bar, 5 µm. b The percentage of SW480 and LS174T cells with colocalized F-actin/cortactin was quantified. Data were derived from eight random fields and are expressed as the mean ± SD, *P < 0.001. c Treated cells were seeded onto Oregon Green 488-conjugated gelatin-coated coverslips for 24 h and then stained with an F-actin antibody (red). Arrows indicate the black region of gelatine degradation. Scale bar, 5 µm. d The percentage of gelatine degradation was quantitatively analysed. Data were derived from six random fields and are expressed as the mean ± SD, *P < 0.001. e The level of MMP9 in CRC cells cultured in different conditions.

Fig 4: DCC and Nrp1 are in close proximity to ribosomes in axonal growth cones in a cue-dependent manner.(A) Expansion imaging shows partial co-localization of DCC and (B) Nrp1 with ribosomal proteins (Scale bars, 5 µm). (C) Representative proximity ligation assay signal in axonal growth cones between DCC and RPL5/uL18, RPS4X/eS4 or IgG control (Scale bars, 5 µm). (D) Representative proximity ligation assay signal in axonal growth cones between Nrp1 and RPS3A/eS1, RPS23/uS12 or IgG control (Scale bars, 5 µm). (E) Representative PLA signal in axonal growth cones between EphB2 and RPL5/uL18 (left) and quantification of PLA signal in axonal growth cones compared to DCC-RPL5/uL18 or Nrp1-RPS23/uS12 (right) (Mann-Whitney test; three biological replicates; bars indicate mean, error bars indicate SEM, ***p<0.0001; Scale bars, 5 µm). (F) EM image of an unstimulated axonal growth cone showing ribosomes aligned in a row (red arrows) under plasma membrane (PM). Inset shows the growth cone at lower magnification; the red box indicates the area shown in higher magnification. The section glances through the extreme surface of growth cone, where it attaches to the culture dish, giving rise to areas that lack subcellular structure. (G) Distribution frequency of the inter-ribosome distance in nm of ribosomes in axonal growth cones (n = 20) or in RGC soma (n = 5). All distances larger than 100 nm were pooled together. (H, I, J, K) Quantification of PLA signal in cue-stimulated axonal growth cones relative to control (unpaired two-tailed t-test; bars indicate mean, error bars indicate SEM; ***p<0.0001; *p=0.0423; for n.s. in J p=0.3522; for n.s. in K, p=0.885). (L) Relative PLA quantification of DCC and RPL5/uL18 compared to control after Dynasore pre-treatment (50 µM for 20 min), Netrin-1, or Netrin-1 + Dynasore (one-way ANOVA with Bonferroni’s multiple comparisons test; bars indicate mean, error bars indicate SEM; p=0.001027 for Control vs. Netrin-1, p=0.000402 for Netrin-1 vs Netrin-1 + Dynasore, p=0.590377 for Control vs. Dynasore, p=0.384848 for Control vs Netrin + Dynasore). For all PLA experiments, numbers in bars indicate total number of growth cones quantified from at least three independent experiments.Figure 3—source data 1.Spreadsheet containing PLA counts and relative comparisons from each axonal growth cone in Figure 3E, all inter-ribosome distances and distribution shown in Figure 3G, and all normalized PLA count values for each axonal growth cone in Figure 3H–L.

Fig 5: NRP1 is required for Cdc42 activation, and Cdc42 knockdown or A7R blockade impairs Cdc42 activation and relocation in CRC cells.a Cdc42 and NRP1 have dual staining in CRC cells. Scale bar: 5 µm. b NRP1 and Cdc42 are expressed in CRC cells with (+) or without (-) VEGF (50 ng/mL) treatment in the coimmunoprecipitation assay. c Western blot analysis of NRP1 in control (si-Ctrl) and NRP1 knockdown (si-NRP1) cells. d SW480/LS174T cells were transfected with control (-) or NRP1 siRNA (+) and then cultured with 50 ng/mL VEGF. SW480/LS174T cells were cultured with 50 ng/mL VEGF and either treated (+) or not (-) with A7R for 24 h. The lysates were obtained, and Cdc42-GTP levels were assessed using the PAK1 pull-down assay. e Cdc42/CD44-stained SW480/LS174T cells. SW480/LS174T cells were cultured with 50 ng/mL VEGF (VEGF/Blank); SW480/LS174T cells were cultured with 50 ng/mL VEGF mixed with A7R (VEGF/A7R or A7R); or SW480/LS174T cells were transfected with NRP1 siRNA and then cultured with 50 ng/mL VEGF (VEGF/siNRP1 or siNRP1). Scale bar: 5 µm. f Cell membrane fractions were isolated and probed for Cdc42. g, h SW480/LS174T cells were treated as follows: SW480/LS174T cells were cultured to confluency on glass coverslips, starved, scratched, and cultured with 50 ng/mL VEGF (VEGF/Blank) or with 50 ng/mL VEGF mixed with A7R (VEGF/A7R or A7R). Alternatively, SW480/LS174T cells were transfected with NRP1 siRNA and were cultured to confluency on glass coverslips, starved, scratched, and then cultured with 50 ng/mL VEGF (VEGF/siNRP1 or siNRP1). g shows Cdc42 localisation of the front in live CRC cells, scale bar, 10 µm. Frames from typical time-lapse sequences show SW480 and LS174T cells with unrecognisable protrusions even at 24 min. h shows fluorescent immunostaining of fixed Cdc42 (in red) and nuclei in blue (DAPI). White arrows point to Cdc42 in cells that are oriented towards the front. The white reference line is parallel to the scratched edge to determine individual cell polarisation vs. cells polarised towards the scratched edge. Scale bar, 20 µm. i Quantitative analysis shows the percentage of cells with Cdc42 oriented towards the front. Error bars represent the mean ± SD of triplicate experiments, *P < 0.001.