Generation of the heterogeneity of extracellular vesicles by membrane organization and sorting machineries

• Published in: Biochimica et biophysica acta. General subjects Vol. 1863; no. 4; pp. 681 - 691
• Main Authors: Harada, Yoichiro, Suzuki, Takehiro, Fukushige, Tomoko, Kizuka, Yasuhiko, Yagi, Hirokazu, Yamamoto, Mika, Kondo, Kiyotaka, Inoue, Hiromasa, Kato, Koichi, Taniguchi, Naoyuki, Kanekura, Takuro, Dohmae, Naoshi, Maruyama, Ikuro
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2019
• Subjects: biogenesis; detergents; ESCRT; Exosomes; Extracellular vesicles; hepatocyte growth factor receptor; Heterogeneity; Membrane microdomains; metalloproteinases; precipitin tests; proteolysis; RNA interference; secretion; solubilization; sucrose; ultracentrifugation; Extracellular vesicles; Heterogeneity; ESCRT; Membrane microdomains; Exosomes
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2019.01.015

Cells secrete heterogeneous populations of extracellular vesicles (EVs) via unknown mechanisms. EV biogenesis has been postulated to involve lipid-protein clusters, also known as membrane microdomains. Membrane properties and heterogeneity of melanoma-derived EVs were analyzed by a detergent solubilization assay, sucrose density gradient ultracentrifugation and immunoprecipitation. EV secretion was modulated by RNA interference and pharmacological treatments. We identified two EV membranes (low-density exosomal detergent-insoluble membranes [EV-DIMs]; EV detergent-soluble membranes [EV-DSMs]) and discovered an abundant, novel type of high-density EV-DIMs. The high-density EV-DIMs accumulated the microdomain-resident protein flotillin-1, as well as a disintegrin and metalloproteinase domain containing protein 10 (Adam10), the hepatocyte growth factor receptor Met and its proteolytic fragments. Low-density EV-DIMs also contained flotillin-1. EV-DSMs were enriched with tetraspanin CD81, melanogenic enzymes and proteolytic fragments of Adam10. Intact and fragmented forms of Adam10, which resided in distinct membrane types, were secreted by different EVs. The fragmented form of Met was associated with DIMs much more efficiently than the intact form and they were secreted by distinct EVs. We identified that the endosomal sorting complexes required for transport machinery was indispensable for EV secretion of both mature and fragmented forms of Adam10 and Met. The findings of this study reveal the role of the interplay between membrane organization and sorting machineries in generating the heterogeneity of EVs. This study provides novel insights into important aspects of EV biogenesis. •Membrane properties and subtypes of melanoma-derived EVs were analyzed.•Three distinct membrane types with unique protein signatures were identified.•These membrane types separated intact and damaged forms of membrane proteins.•The two forms of membrane proteins were secreted by distinct EVs.•The ESCRT machinery was required for the differential EV secretion.