The adaptor protein CD2AP controls the biogenesis of endosomal-derived small extracellular vesicles

Menck, K; Irmer, B; Imjeti, NS; David, G; Zimmermann, P

Abstract

Introduction: Extracellular vesicles (EVs) can be divided into several subpopulations that differ in size, cargo, biogenesis, and function. The main EV populations recognized at present are large EVs (diameter 150-1000 nm) and small EVs (sEVs) (diameter50-150 nm). While large EVs are directly secreted from the plasma membrane, sEVs bud into the lumen of late endosomalcompartments and are released into the extracellular space upon fusion of the endosomes with the cell membrane. Recently, the intracellular adaptor protein CD2AP was found to be involved in the early steps of sEV biogenesis by regulating the endocytosisof the exosomal cargo protein GPRC5B. However, whether CD2AP also influences other steps of the sEV biogenesis pathway iscurrently unknown. Methods: We modulated CD2AP levels in human MCF-7 breast cancer cells by knockdown/overexpression and analysed the different steps of the endosomal pathway by confocal microscopy. Corresponding sEVs were isolated by differential ultracentrifugation and characterized by nanoparticle tracking analysis and immunoblot. Results: CD2AP knockdown reduced the expression of two well-known marker proteins, Syntenin and CD63, on sEVs. Consis-tently, overexpression of CD2AP increased exosomal Syntenin and CD63 levels. In order to understand the molecular mechanism underlying these observations, we analysed the endosomal trafficking of CD63. Confocal microscopy revealed that upon CD2AP knockdown, CD63 showed an increased co-localisation with the late endosomal marker LAMP2 indicating that CD2AP is important for CD63 trafficking through this compartment. Using overexpression of mutant Rab5Q79L, which induces the formation of enlarged endosomes, we showed that CD2AP regulates the budding of CD63 and Syntenin at endosomal membranes. UsingRNAi and different inhibitors, we placed the function of CD2AP downstream of SRC, and upstream of ARF6 and its effector PLD2. CD2AP has been described as a direct interactor of ALIX, which could link CD2AP to the Syntenin pathway that is crucial for the biogenesis of sEVs. Indeed, the knockdown of ALIX antagonized the effects of CD2AP on endosomal budding and sEV cargo. Summary/Conclusion: Taken together, our results identified CD2AP as a novel regulator of trafficking and budding processes at late endosomal compartments and shed further light on the process of sEV biogenesis.