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      Exosomes purified from a single cell type have diverse morphology

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          ABSTRACT

          Extracellular vesicles (EVs) are produced by all known organisms and are important for cell communication and physiology. Great morphological diversity has been described regarding EVs found in body fluids such as blood plasma, breast milk, and ejaculate. However, a detailed morphological analysis has never been performed on exosomes when purified from a single cell type.

          In this study we analysed and quantified, via multiple electron microscopy techniques, the morphology of exosomes purified from the human mast cell line HMC-1. The results revealed a wide diversity in exosome morphology, suggesting that subpopulations of exosomes with different and specific functions may exist. Our findings imply that a new, more efficient way of defining exosome subpopulations is necessary. A system was proposed where exosomes were classified into nine different categories according to their size and shape. Three additional morphological features were also found in exosomes regardless of their morphological classification.

          These findings show that exosomes purified from a single cell line are also morphologically diverse, similar to previous observations for EVs in body fluids. This knowledge can help to improve the interpretation of experimental results and widen our general understanding of the biological functions of exosomes.

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          Structural basis of membrane invagination by F-BAR domains.

          BAR superfamily domains shape membranes through poorly understood mechanisms. We solved structures of F-BAR modules bound to flat and curved bilayers using electron (cryo)microscopy. We show that membrane tubules form when F-BARs polymerize into helical coats that are held together by lateral and tip-to-tip interactions. On gel-state membranes or after mutation of residues along the lateral interaction surface, F-BARs adsorb onto bilayers via surfaces other than their concave face. We conclude that membrane binding is separable from membrane bending, and that imposition of the module's concave surface forces fluid-phase bilayers to bend locally. Furthermore, exposure of the domain's lateral interaction surface through a change in orientation serves as the crucial trigger for assembly of the helical coat and propagation of bilayer bending. The geometric constraints and sequential assembly of the helical lattice explain how F-BAR and classical BAR domains segregate into distinct microdomains, and provide insight into the spatial regulation of membrane invagination.
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            A negative staining method for high resolution electron microscopy of viruses.

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              Recovery of extracellular vesicles from human breast milk is influenced by sample collection and vesicle isolation procedures

              Extracellular vesicles (EV) in breast milk carry immune relevant proteins and could play an important role in the instruction of the neonatal immune system. To further analyze these EV and to elucidate their function it is important that native populations of EV can be recovered from (stored) breast milk samples in a reproducible fashion. However, the impact of isolation and storage procedures on recovery of breast milk EV has remained underexposed. Here, we aimed to define parameters important for EV recovery from fresh and stored breast milk. To compare various protocols across different donors, breast milk was spiked with a well-defined murine EV population. We found that centrifugation of EV down into density gradients largely improved density-based separation and isolation of EV, compared to floatation up into gradients after high-force pelleting of EV. Using cryo-electron microscopy, we identified different subpopulations of human breast milk EV and a not previously described population of lipid tubules. Additionally, the impact of cold storage on breast milk EV was investigated. We determined that storing unprocessed breast milk at −80°C or 4°C caused death of cells present in breast milk, leading to contamination of the breast milk EV population with storage-induced EV. Here, an alternative method is proposed to store breast milk samples for EV analysis at later time points. The proposed adaptations to the breast milk storage and EV isolation procedures can be applied for EV-based biomarker profiling of breast milk and functional analysis of the role of breast milk EV in the development of the neonatal immune system.
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                Author and article information

                Journal
                J Extracell Vesicles
                J Extracell Vesicles
                ZJEV
                zjev20
                Journal of Extracellular Vesicles
                Taylor & Francis
                2001-3078
                2017
                20 June 2017
                : 6
                : 1
                : 1329476
                Affiliations
                [ a ] Department of Chemistry and Molecular Biology, University of Gothenburg , Gothenburg, Sweden
                [ b ] Krefting Research Center, University of Gothenburg , Gothenburg, Sweden
                [ c ] Electron Microscopy Core Facility, European Molecular Biology Laboratories , Heidelberg, Germany
                Author notes
                CONTACT Johanna L Höög johanna.hoog@ 123456gu.se ; Jan Lötvall jan.lotvall@ 123456gu.se
                [*]

                These authors contributed equally to this work.

                Author information
                http://orcid.org/0000-0002-5912-4601
                http://orcid.org/0000-0001-9195-9249
                http://orcid.org/0000-0003-2162-3816
                Article
                1329476
                10.1080/20013078.2017.1329476
                5505001
                28717422
                8ced7437-0502-4357-bce6-a69565f7353e
                © 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 22 December 2016
                Page count
                Figures: 5, References: 55, Pages: 12
                Funding
                Funded by: Vetenskapsrådet 10.13039/501100004359
                Award ID: 2015-05427
                This work was supported by the Vetenskapsrådet [2015-05427]; and the Göran Gustafsson Foundation for Research in Natural Sciences and Medicine. JLH was supported by a VR young investigator grant and the Göran Gustafsson Foundation for Research in Natural Sciences and Medicine. This work was funded by the Swedish Research Council (K2014-85x-22504-01-3), the VBG Group Herman Krefting Foundation for Asthma and Allergy Research (20141209), the Swedish Heart and Lung Foundation (20150588), and the Swedish Cancer Foundation (CAN2014/844).
                Categories
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                Transferred Article

                dolores di vizio, usa,cryo-electron microscopy,extracellular vesicles,multivesicular bodies,hmc-1,density gradient,spikes

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