Figure 5: Density gradient-purified exosomes have bound FN and rescue Rab27a-KD cell motility defects
From: Directional cell movement through tissues is controlled by exosome secretion
Extracellular vesicles were purified by ultracentrifugation. Microvesicle (Microvesicles) and exosome (Ultracentrifuged exosomes) fractions were analysed by NanoSight (a). Part of the Ultracentrifuged exosomes fraction was further purified by Optiprep density gradient centrifugation. (b) Western blot analysis of the fractions (Density gradient fractions 1–12) for exosome markers HSP70 and flotillin identified Fraction 6 as the major peak containing exosomes. NanoSight analysis of this fraction ((a): Density gradient fraction 6) showed a typical exosome size profile similar to the Ultracentrifuged exosomes profile, whereas Microvesicles fraction contained larger vesicles. Both FN and its receptor α5 integrin were present in the Fraction 6 exosome peak. FN was also present separate from exosome markers in Fraction 9. Conditioned media (CM) contained FN but the microvesicle (MV) fraction did not. T=Total cell lysate. Representative blots from n=2. (c) Western blot analysis of MVs, ultracentrifuge-purified exosomes (UC-Exo) and exosomes present in Fraction 6 density gradients (DG-Exo) from control cells cultured in normal media (Regular) or in FN-depleted media for 10 days. Equal numbers of vesicles were loaded in the gel lanes. Note that FN, HSP70 and CD63 were uniquely present in the exosome samples. Although the lanes are demarcated from each other due to intervening lanes not being shown, the regular and FN-depleted Exo lanes came from the same blot and are directly comparable for all antibodies. (d) The ability of various extracellular vesicles to rescue motility of Rab27a-KD cells was tested by coating 10 μg ml−1 UC-Exo, microvesicles purified from an equivalent number of cells that produced 10 μg ml−1 UC-Exo or 10 or 20 μg ml−1 DG-Exo. Similar to results with UC-Exo (Fig. 4d,h), twice as many DG-Exo purified from FN-depleted cells were required to rescue Rab27a-KD speed as Regular DG-Exo. Data from ≥30 cells from ≥3 independent experiments for each condition. Error bars indicate s.e.m. ***P<0.001 compared with Sc by Student’s t-test. #P<0.05; ##P<0.01; ###P<0.001 compared with the same cell line on Exo 0 by Student’s t-test.
