Figure 7: Exosomes promote adhesion assembly

(a) Image from TIRF movie of pHLuorin-CD63 (green) and mCherry-paxillin (red)-expressing HT1080 cell on FN-coated (1 μg ml⁻¹) MatTek dishes. Time series shows bursts of pHLuorin-CD63 fluorescence (arrowheads) preceding adhesion formation (indicated by *, red paxillin fluorescence visible at that time). Two adhesion formation events are, respectively, indicated with white and yellow markers. Representative of n=7 movies. See also Supplementary Movie 8. (b) Quantitation of per cent of adhesions with a pHLuorin-CD63 fluorescence burst before or coincident with their formation. (c) Quantitation of the number of minutes between pHLuorin-CD63 appearance and adhesion formation. (d) Representative time series of GFP-paxillin-marked adhesion assembly and disassembly for control (Sc) and Rab27a-KD HT1080 cells on FN-coated (1 μg ml⁻¹) MatTek dishes. Magnifications of boxed area are shown at right by heat map coloration to show intensity. 0 min indicates start of adhesion formation. White arrows indicate example adhesions and yellow arrows indicate the peak adhesion intensity. Asterisk indicates a newly forming adhesion. Note that a longer period of time is required for adhesion assembly in KD cells. (e,f) Box-and-whisker plots of adhesion assembly (e) and disassembly (f) rates. Each box shows the 25–75th percentile and whiskers indicate the 5–95th percentile and the black line indicates the median. Analysis from >30 adhesions for e and >40 adhesions for f from three to eight cells per condition. See also Supplementary Movie 9. (g) Western blot analysis of FN association with exosomes. GRGESP (100 μM; RGE, control) or GRGDSP (100 μM; RGD, integrin-binding) peptides were added to cultured HT1080 control cells (Sc) for 48 h before exosome isolation. Equal exosome numbers were loaded on western blots. Quantitations show FN intensity normalized to flotillin intensity as a loading control from n≥4 western blots. α5-KD, integrin α5-knockdown-HT1080. *P<0.05; **P<0.01; ***P<0.001 compared with Sc using Mann–Whitney U-test. Scale bars in a and d represent 30 μm.