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      Current knowledge on exosome biogenesis and release

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          Abstract

          Exosomes are nanosized membrane vesicles released by fusion of an organelle of the endocytic pathway, the multivesicular body, with the plasma membrane. This process was discovered more than 30 years ago, and during these years, exosomes have gone from being considered as cellular waste disposal to mediate a novel mechanism of cell-to-cell communication. The exponential interest in exosomes experienced during recent years is due to their important roles in health and disease and to their potential clinical application in therapy and diagnosis. However, important aspects of the biology of exosomes remain unknown. To explore the use of exosomes in the clinic, it is essential that the basic molecular mechanisms behind the transport and function of these vesicles are better understood. We have here summarized what is presently known about how exosomes are formed and released by cells. Moreover, other cellular processes related to exosome biogenesis and release, such as autophagy and lysosomal exocytosis are presented. Finally, methodological aspects related to exosome release studies are discussed.

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          Most cited references 54

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          The mechanisms of vesicle budding and fusion.

          Genetic and biochemical analyses of the secretory pathway have produced a detailed picture of the molecular mechanisms involved in selective cargo transport between organelles. This transport occurs by means of vesicular intermediates that bud from a donor compartment and fuse with an acceptor compartment. Vesicle budding and cargo selection are mediated by protein coats, while vesicle targeting and fusion depend on a machinery that includes the SNARE proteins. Precise regulation of these two aspects of vesicular transport ensures efficient cargo transfer while preserving organelle identity.
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            Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes.

            Exosomes are 40-100nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of oncogenic proteins as well as mRNA and miRNA. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. However, all preparations invariably contain varying proportions of other membranous vesicles that co-purify with exosomes such as shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, in this study we performed a comprehensive evaluation of current methods used for exosome isolation including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM coated magnetic beads (IAC-Exos). Notably, all isolations contained 40-100nm vesicles, and were positive for exosome markers (Alix, TSG101, HSP70) based on electron microscopy and Western blotting. We employed a proteomic approach to profile the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, we found IAC-Exos to be the most effective method to isolate exosomes. For example, Alix, TSG101, CD9 and CD81 were significantly higher (at least 2-fold) in IAC-Exos, compared to UG-Exos and DG-Exos. Application of immunoaffinity capture has enabled the identification of proteins including the ESCRT-III component VPS32C/CHMP4C, and the SNARE synaptobrevin 2 (VAMP2) in exosomes for the first time. Additionally, several cancer-related proteins were identified in IAC-Exos including various ephrins (EFNB1, EFNB2) and Eph receptors (EPHA2-8, EPHB1-4), and components involved in Wnt (CTNNB1, TNIK) and Ras (CRK, GRB2) signalling. Copyright © 2012 Elsevier Inc. All rights reserved.
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              Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes

              Using ferritin-labeled protein A and colloidal gold-labeled anti-rabbit IgG, the fate of the sheep transferrin receptor has been followed microscopically during reticulocyte maturation in vitro. After a few minutes of incubation at 37 degrees C, the receptor is found on the cell surface or in simple vesicles of 100-200 nm, in which the receptor appears to line the limiting membrane of the vesicles. With time (60 min or longer), large multivesicular elements (MVEs) appear whose diameter may reach 1-1.5 micron. Inside these large MVEs are round bodies of approximately 50-nm diam that bear the receptor at their external surfaces. The limiting membrane of the large MVEs is relatively free from receptor. When the large MVEs fuse with the plasma membrane, their contents, the 50-nm bodies, are released into the medium. The 50-nm bodies appear to arise by budding from the limiting membrane of the intracellular vesicles. Removal of surface receptor with pronase does not prevent exocytosis of internalized receptor. It is proposed that the exocytosis of the approximately 50-nm bodies represents the mechanism by which the transferrin receptor is shed during reticulocyte maturation.
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                Author and article information

                Contributors
                +4722781825 , Alicia.Martinez.Llorente@rr-research.no
                Journal
                Cell Mol Life Sci
                Cell. Mol. Life Sci
                Cellular and Molecular Life Sciences
                Springer International Publishing (Cham )
                1420-682X
                1420-9071
                21 July 2017
                21 July 2017
                2018
                : 75
                : 2
                : 193-208
                Affiliations
                [1 ]ISNI 0000 0004 0389 8485, GRID grid.55325.34, Department of Molecular Cell Biology, Institute for Cancer Research, , Oslo University Hospital, The Norwegian Radium Hospital, ; 0379 Oslo, Norway
                [2 ]ISNI 0000 0004 1936 8921, GRID grid.5510.1, Centre for Cancer Biomedicine, , University of Oslo, ; 0379 Oslo, Norway
                Article
                2595
                10.1007/s00018-017-2595-9
                5756260
                28733901
                © The Author(s) 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100008730, Kreftforeningen;
                Funded by: The Research Council of Norway
                Award ID: 179571
                Award Recipient :
                Funded by: The Norwegian Financial Mechanism
                Award ID: NFI/R/2014/045
                Award Recipient :
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                Review
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                © Springer International Publishing AG, part of Springer Nature 2018

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