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. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 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 membranederived 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.

Autophagy promotes enrichment of raft components within extracellular vesicles secreted by human 2FTGH cells / Manganelli, Valeria; Dini, Luciana; Tacconi, Stefano; Dinarelli, Simone; Capozzi, Antonella; Riitano, Gloria; Recalchi, Serena; Caglar, TUBA RANA; Fratini, Federica; Misasi, Roberta; Sorice, Maurizio; Garofalo, Tina. - In: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. - ISSN 1422-0067. - 25:(2024), pp. 1-19. [10.3390/ijms25116175]

Autophagy promotes enrichment of raft components within extracellular vesicles secreted by human 2FTGH cells

Valeria Manganelli
Primo
;
Luciana Dini
Secondo
;
Stefano Tacconi;Simone Dinarelli;Antonella Capozzi;Gloria Riitano;Serena Recalchi;Tuba Rana Caglar;Federica Fratini;Roberta Misasi;Maurizio Sorice
Penultimo
;
Tina Garofalo
Ultimo
2024

Abstract

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. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 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 membranederived 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.
2024
autophagy; extracellular vesicles; exosomes; lipid rafts; ERLIN1
01 Pubblicazione su rivista::01a Articolo in rivista
Autophagy promotes enrichment of raft components within extracellular vesicles secreted by human 2FTGH cells / Manganelli, Valeria; Dini, Luciana; Tacconi, Stefano; Dinarelli, Simone; Capozzi, Antonella; Riitano, Gloria; Recalchi, Serena; Caglar, TUBA RANA; Fratini, Federica; Misasi, Roberta; Sorice, Maurizio; Garofalo, Tina. - In: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. - ISSN 1422-0067. - 25:(2024), pp. 1-19. [10.3390/ijms25116175]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/1711680
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