Background/Objectives
Exosomes, a subclass of small extracellular vesicles (sEVs), derive from the endosomal system. Syntenin is a cen- tral regulator of their biogenesis, linking proteoglycans of the Syndecan family to the ESCRT machinery, thereby supporting their budding at late endosomal membranes and export onto exosomes. The adapter protein CD2AP, which interacts with the ESCRT components Alix and Tsg101, was found enriched on exosomes. However, if CD2AP itself is involved in exosome biogenesis, has not been investigated yet.
Methods
Small EVs were isolated by differential ultracentrifugation (143,000 g) from human MCF-7 breast cancer cells and characterized by NTA, immunoblots and electron microscopy. CD2AP or Syntenin overexpression/knockdown effects on sEV number and cargo were analyzed by NTA and immunoblots. Immunofluorescence and co-immunoprecipitations were used to study protein-protein interactions. Endosomal budding was visualized in enlarged endosomes induced by overexpressing constitutively active mutant Rab5(Q79L).
Results
The expression level of CD2AP, but not its paralogue CIN85, on sEVs was controlled by Syntenin. Conversely, CD2AP regulated the exosomal export of Syntenin. CD2AP and Syntenin co-localized in Rab5(Q79L)-positive endosomes and at late endosomal mem- branes. The interaction of CD2AP with Syntenin as well as Alix was confirmed by co-immunoprecipitation. Mechanistically, CD2AP regu- lated the endosomal budding of Syntenin dependent on Alix, Arf6 and its downstream effector Pld2. CD2AP contains three N-terminal SH3 domains, followed by a proline-rich region, four C-terminal actin binding sites and a coiled-coil domain. In line with its function as an adaptor protein, deletion of the SH3 domains and the C-terminal part of CD2AP antagonized its effect on Syntenin endosomal budding and exosomal export.
Conclusion
In summary, our results have identified CD2AP as a novel regulator of exosome biogenesis, controlling the endosomal bud- ding and exosomal export of Syntenin.
Keywords
CD2AP, Syntenin, exosomes, biogenesis, endosomal budding
Funding/Acknowledgments
The project was funded by the DFG (projects 424252458, ME 4573/1-1), the Else Kröner-Fresenius-Stif- tung (project 2019_A162), the innovative medical research of the Medical Faculty, University of Münster (project ME 12 19 14) and the Heidenreich-von-Siebold programme of the University Medical Center Göttingen.
