Real-time imaging of multivesicular body-plasma membrane fusion to quantify exosome release from single cells

Autor: Stephan Huveneers, Guillaume van Niel, Philippe Bun, Frederik J. Verweij, Maarten P. Bebelman, D. Michiel Pegtel
Přispěvatelé: Pathology, Amsterdam Neuroscience - Cellular & Molecular Mechanisms, CCA - Imaging and biomarkers, Medicinal chemistry, AIMMS, Department of Pathology [Amsterdam, The Netherlands] (Amsterdam Neuroscience ), VU University Medical Center [Amsterdam], Division of Medicinal Chemistry [Amsterdam, The Netherlands], Vrije Universiteit Amsterdam [Amsterdam] (VU)-Amsterdam Institute for Molecules Medicines and Systems (AIMMS), Institut de psychiatrie et neurosciences de Paris (IPNP - U1266 Inserm - Paris Descartes), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Vrije Universiteit Medical Centre (VUMC), Vrije Universiteit Amsterdam [Amsterdam] (VU), Dupuis, Christine, Medical Biochemistry, ACS - Atherosclerosis & ischemic syndromes, ACS - Microcirculation, AII - Inflammatory diseases
Jazyk: angličtina
Rok vydání: 2020
Předmět:
Zdroj: Nature Protocols, 15(1), 102-121. Nature Publishing Group
Nature Protocols
Nature Protocols, Nature Publishing Group, 2019, Epub ahead of print. ⟨10.1038/s41596-019-0245-4⟩
Bebelman, M P, Bun, P, Huveneers, S, van Niel, G, Pegtel, D M & Verweij, F J 2020, ' Real-time imaging of multivesicular body–plasma membrane fusion to quantify exosome release from single cells ', Nature Protocols, vol. 15, no. 1, pp. 102-121 . https://doi.org/10.1038/s41596-019-0245-4
Nature Protocols, Nature Publishing Group, 2020, 15 (1), pp.102-121. ⟨10.1038/s41596-019-0245-4⟩
Nature protocols, 15(1), 102
Bebelman, M P, Bun, P, Huveneers, S, van Niel, G, Pegtel, D M & Verweij, F J 2020, ' Real-time imaging of multivesicular body-plasma membrane fusion to quantify exosome release from single cells ', Nature Protocols, vol. 15, no. 1, pp. 102-121 . https://doi.org/10.1038/s41596-019-0245-4
Nature protocols, 15(1). Nature Publishing Group
ISSN: 1754-2189
1750-2799
Popis: International audience; Exosomes are small extracellular vesicles with a diameter of 40-150 nm, and are implicated in cellular homeostasis and cell-cell communication. They can be secreted in bulk in response to cell-extrinsic and cell-intrinsic signals that cause multivesicular body (MVB) fusion with the plasma membrane (PM). However, research on the regulation of exosome release is hampered by the failure of current methods to capture the dynamics of exosome release. Here we describe how live imaging with tetraspanin-based pH-sensitive fluorescent reporters can quantify the MVB-PM fusion rate of single cells. Our approach enables identification of exogenous stimuli, signaling pathways, and fusion complexes, and can map subcellular sites of fusion events. In addition, dual-color imaging can be used to assess simultaneous release of different cargo by MVB exocytosis. This protocol describes the complete imaging experiment, consisting of transient expression of tetraspanin reporters (2 d), live-cell (dual-color) total internal reflection fluorescence microscopy (30-60 min per condition), and semiautomatic image analysis by using a newly developed ImageJ macro (±30 min per condition).
Databáze: OpenAIRE
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