Yoichiro Harada1, Takehiro Suzuki2, Tomoko Fukushige3, Yasuhiko Kizuka4, Hirokazu Yagi5, Mika Yamamoto6, Kiyotaka Kondo7, Hiromasa Inoue7, Koichi Kato8, Naoyuki Taniguchi9, Takuro Kanekura3, Naoshi Dohmae2, Ikuro Maruyama6. 1. Department of Systems Biology in Thromboregulation, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. Electronic address: yoharada@m.kufm.kagoshima-u.ac.jp. 2. Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. 3. Department of Dermatology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. 4. Disease Glycomics Team, Systems Glycobiology Research Group, RIKEN-Max Planck Joint Research Center, Global Research Cluster, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, Gifu 501-1193, Japan. 5. Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan. 6. Department of Systems Biology in Thromboregulation, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. 7. Department of Pulmonary Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. 8. Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan; Exploratory Research Center on Life and Living Systems (ExCELLS), Institute for Molecular Science (IMS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan. 9. Department of Glyco-Oncology, Osaka International Cancer Institute, 3-1-69 Otemae, Chuoku, Osaka 541-8567, Japan.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The findings of this study reveal the role of the interplay between membrane organization and sorting machineries in generating the heterogeneity of EVs. GENERAL SIGNIFICANCE: This study provides novel insights into important aspects of EV biogenesis.
BACKGROUND: 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. METHODS: 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. RESULTS: 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 receptorMet 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. CONCLUSION: The findings of this study reveal the role of the interplay between membrane organization and sorting machineries in generating the heterogeneity of EVs. GENERAL SIGNIFICANCE: This study provides novel insights into important aspects of EV biogenesis.
Authors: Courtney F Pierce; Vienna R Brown; Steven C Olsen; Paola Boggiatto; Kerri Pedersen; Ryan S Miller; Scott E Speidel; Timothy J Smyser Journal: Front Vet Sci Date: 2020-11-25
Authors: Nan He; Sirisha Thippabhotla; Cuncong Zhong; Zachary Greenberg; Liang Xu; Ziyan Pessetto; Andrew K Godwin; Yong Zeng; Mei He Journal: Commun Biol Date: 2022-07-04