Dottorato in MEDICINA SPERIMENTALE

Titolo della tesi: Evidence for Raft Components within Extracellular Vesicles Secreted by Human Fibrosarcoma Cells following Autophagy Triggering

Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes to the release of cytosolic proteins through an unconventional secretion pathway. Recently, also lipid microdomains, i.e. lipid rafts, have been identified as further actors of the autophagic process. In the present work, we first observed an enrichment of GD3 in MAMs associated lipid raft by using in HT1080-derived human fibrosarcoma 2FTGH cells. This association seems thus to take place in the early phases of autophagic process in which MAMs have been hypothesized to play a key role. Next, we analyzed autophagy-induced extracellular vesicles (EVs) in 2FTGH cells using transmission electron microscopy and atomic force microscopy (AFM). We preliminary observed that autophagy induces the formation of a subset of large heterogeneous intracellular vesicular structures. Moreover, AFM showed that autophagy triggering led to a more visible smooth cell surface with a reduced amount of plasma membrane protrusions. Next, we characterized EVs secreted by cells following autophagy induction, demonstrating that cells release both plasma membrane derived microvesicles and exosomes. A self-forming iodixanol gradient was performed for cell subfractionation. Western blot analysis showed that endogenous LC3-II co-fractionated with CD63 and CD81. Then, we analyzed whether raft components are enriched within EV cargoes following autophagy triggering. We observed that the raft marker GD3 and ER marker ERLIN1 co-fractionated with LC3-II; dual staining by immunogold electron microscopy and coimmunoprecipitation revealed GD3-LC3-II association, indicating that autophagy promotes enrichment of raft components within EVs. Introducing a new brick in the crosstalk between autophagy and the endolysosomal system may have important implications for the knowledge of pathogenic mechanisms, suggesting alternative raft target therapies in diseases in which the generation of EV is active.