Fig 2: Exosomes stimulate actin-based non-directional migration in a centrosome and MT-independent manner. a Experimental schematic. MDA-MB-231 cells were treated with centrinone for 5 days to induce centrosome loss during division. Centrosome-less cells were treated with ACM. The protrusive activity and motility of individual cells were imaged and tracked. b MDA-MB-231 cells were treated with DMSO or centrinone for 5 days. CETN1 (centrioles) and CEP192 (centrosomes) staining is shown. Bar = 20 µm. c Percentage of cells from (b) with > 4 centrioles, 1 ≤ centrioles ≤ 4 and 0 centrioles (n = 3 independent experiments analysing at least 100 cells per each replicate). d MDA-MB-231 cells were treated with DMSO or centrinone for 5 days, followed by overnight incubation with the DMEM or ACM. Cell motility was measured as described in Methods and plotted as box and whiskers. Boxes represent median and 25th to 75th percentiles, whiskers the minimum and maximum values with each individual cell value superimposed. The data were compared with one-way ANOVA Kruskal–Wallis test and post-tested with Dunn’s multiple comparison test. Arrow indicates the control bar used for comparison (****p < 0.0001; n = 4, at least 50 cells were tracked per condition). e MDA-MB-231 cells from (d) were stained for α-tubulin, actin (phalloidin) and centrosomes (PCNT). Arrowhead indicates cell protrusion. Bar = 20 µm, inset box 3.88 × 3.88 μm (n = 3). f Mean-squared displacement (MSD) of cells stimulated with PBS (light blue) or purified exosomes (light red). Linear regression analysis of MSD was preformed (dark blue line is PBS, dark red line is purified exosomes). MSD R2 = 1 indicates a total random movement (n = 3, at least 40 cells were tracked per condition). g Quantitative data from wound-healing assay on MDA-MB-231 cells treated overnight with DMEM or ACM are shown in Supplementary Fig. 1e. Graph shows mean area recovered. The data were analysed with two-way ANOVA post-tested with Bonferroni test (*p < 0.05; n = 3, at least 30 regions were measured per condition). h MDA-MB-231 cells were stimulated with DMEM or ACM overnight. Cell motility was tracked for 6 h. Then, the same cells were further tracked after being treated with DMSO or Nocodazole for 12 h. Running average speed was plotted (n = 3, at least 40 cells were tracked per condition). i MDA-MB-231 cells were stimulated with ACM overnight. DMSO or NOC were added for the last 2 h of stimulation. Actin (red) and α-tubulin (green) staining are shown. Region of interest (ROI) shows the enlarged area of the selected cell protrusion. Bar = 20 µm (n = 3) j MDA-MB-231 cells were stimulated with DMEM or ACM overnight. Cell motility was tracked for 3 h, then cells were treated with DMSO, cytochalasin B or cytochalasin D and further tracked for 15 h. Running average speed was plotted (n = 3, at least 40 cells were tracked per condition). The data from c, f, g, h and j are plotted as mean ± s.e.m.

Fig 3: The effects of CEP192 on the function of PLVAP+ ECs. (A) UMAP was colored by 14 subpopulations of PLVAP+ ECs. (B) Expression of CEP192 and EC-specific marker genes in PLVAP+ EC subpopulations. Red boxes highlighted PLVAP+ EC clusters of high CEP192 expression. (C–E) Single-cell trajectory of pseudotime (C), state (D), and CEP192 expression (E). (F) Box plot showing the GO-BP enrichment results of PEC(7). (G) Chord diagram exhibiting the GO-BP enrichment results of PLVAP+ ECs in state 3.

Fig 4: Nonhydroxylatable Cep192 Interferes with Centrosome Function(A) Cells expressing nonhydroxylatable Cep192 accumulate in prometaphase. HeLa cells stably expressing the indicated constructs were transfected with the indicated siRNAs and the mitotic distribution was determined. Values represent averages of three different experiments (n = 100; error bars indicate ± 1 SD).(B) Nonhydroxylatable Cep192 disrupts the mitotic spindle. Quantification of spindle morphology phenotypes observed in cells stably expressing the indicated constructs and depleted with the indicated siRNAs.(C) Cep192 mutated on proline 1717 accumulates at the centrosome in interphase. HeLa cells stably expressing wild-type or the LAA mutant of Cep192-GFP (green) were treated with the indicated siRNAs and probed for pericentrin (red) (top panels). The scale bars represent 1 μm. The relative fluorescence intensity of Cep192-GFP, pericentrin, and γ-tubulin in interphase cells was determined (bottom panel) (n = 20; error bars indicate ± 1 SEM; p values are significant according to the Student’s t test; ∗∗p < 0.01, ∗∗∗p < 0.0001).(D) The LAA mutant of Cep192 does not accumulate at the centrosome in mitosis. Panels as in (C). The relative fluorescence intensity of Cep192-GFP, pericentrin, and γ-tubulin in mitotic cells was determined (bottom panel) (n = 20; error bars indicate ± 1 SEM; p values are significant according to the Student’s t test; ∗∗p < 0.01, ∗∗∗p < 0.0001).

Fig 5: DVL2 orchestrates the recruitment of CEP192 at protrusions. a, c MDA-MB-231 cells were transfected with control, CEP192 or DVL2 siRNAs for 72 h, and then stimulated overnight with DMEM or ACM. Cells were stained for (a) CEP192 and actin, or (c) CEP192 and PCNT. Bar = 20 µm. b, d, e Intensity of CEP192 at cell cortex (b), at centrosome (d), and PCNT signal at centrosome (e) was measured as described in Methods and plotted as box and whiskers. Boxes represent median and 25th to 75th percentiles, whiskers the minimum and maximum values with each individual value superimposed. The data were compared with one-way ANOVA Kruskal–Wallis test and post-tested with Dunn’s multiple comparison test. Arrow indicates the control bar used for comparison. (*p < 0.05, ****p < 0.0001; n = 3, at least 60 cells were measured per condition). Light blue is CEP192, light green is PCNT. f Model for regulation of centrosomal and cortical CEP192 pools by DVL2