Comparative characterization of mitochondrial components across size-defined extracellular vesicle populations derived from HaCaT keratinocytes.

The transfer of mitochondrial cargo through extracellular vesicles (EVs) has been reported in several model systems and cell types, yet a detailed characterization of mitochondrial components across different EV populations remains limited. Here, we characterized the mitochondrial cargo of small EVs (sEVs) and large EVs (lEVs) released by human HaCaT keratinocytes and examined the impact of oxidative stress on their content composition. EVs were isolated from keratinocyte-conditioned medium and characterized using complementary analytical approaches. Their mitochondrial cargo was assessed using super-resolution microscopy, RT-qPCR, nano-flow cytometry, and mass spectrometry. We found that mitochondrial proteins and transcripts were predominantly associated with lEVs, whereas sEVs contained low levels of selected mitochondrial proteins and RNA. Comparative proteomic analysis revealed distinct mitochondrial-associated signatures between EV populations. lEVs were enriched in proteins with predominantly mitochondrial localization and proteins associated with antioxidant-related functions, whereas sEVs were enriched in proteins associated with RNA- and tRNA-related processes. The preferential association of mitochondrial cargo with lEVs was increased upon oxidative stress. Together, these findings highlight EV heterogeneity as an important determinant of mitochondrial cargo transfer and reveal that mitochondrial cargo is preferentially associated with larger EV populations.