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).
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The data that support this study are available from the corresponding author upon reasonable request.
The AMvBE macro, a Readme file, and example data can be found in Supplementary Software 1. The software in this protocol has been peer reviewed.
Huotari, J. & Helenius, A. Endosome maturation. EMBO J. 30, 3481–3500 (2011).
van Niel, G., D’Angelo, G. & Raposo, G. Shedding light on the cell biology of extracellular vesicles. Nat. Rev. Mol. Cell Biol. 19, 213–228 (2018).
Bebelman, M. P., Smit, M. J., Pegtel, D. M. & Baglio, S. R. Biogenesis and function of extracellular vesicles in cancer. Pharmacol Ther. 188, 1–11 (2018).
Costa-Silva, B. et al. Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat. Cell Biol. 17, 816–826 (2015).
Peinado, H. et al. Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat. Med. 18, 883–891 (2012).
Bobrie, A., Colombo, M., Raposo, G. & Thery, C. Exosome secretion: molecular mechanisms and roles in immune responses. Traffic 12, 1659–1668 (2011).
Bian, S. et al. Extracellular vesicles derived from human bone marrow mesenchymal stem cells promote angiogenesis in a rat myocardial infarction model. J. Mol. Med. 92, 387–397 (2014).
Tkach, M. & Thery, C. Communication by extracellular vesicles: where we are and where we need to go. Cell 164, 1226–1232 (2016).
Kowal, J. et al. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc. Natl Acad. Sci. USA 113, E968–E977 (2016).
van der Vlist, E. J., Nolte-‘t Hoen, E. N., Stoorvogel, W., Arkesteijn, G. J. & Wauben, M. H. Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry. Nat. Protoc. 7, 1311–1326 (2012).
Zhang, H. & Lyden, D. Asymmetric-flow field-flow fractionation technology for exomere and small extracellular vesicle separation and characterization. Nat. Protoc. 14, 1027–1053 (2019).
Gardiner, C. et al. Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey. J. Extracell. Vesicles 5, 32945 (2016).
Pegtel, D. M. & Gould, S. J. Exosomes. Annu. Rev. Biochem 88, 487–514 (2019).
Miesenbock, G., De Angelis, D. A. & Rothman, J. E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).
Sankaranarayanan, S., De Angelis, D., Rothman, J. E. & Ryan, T. A. The use of pHluorins for optical measurements of presynaptic activity. Biophys. J. 79, 2199–2208 (2000).
Sung, B. H., Ketova, T., Hoshino, D., Zijlstra, A. & Weaver, A. M. Directional cell movement through tissues is controlled by exosome secretion. Nat. Commun. 6, 7164 (2015).
Verweij, F. J. et al. Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling. J. Cell Biol. 217, 1129–1142 (2018).
Lu, A. et al. Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs. eLife 7, e41460 (2018).
Gutknecht, J. Proton/hydroxide conductance and permeability through phospholipid bilayer membranes. Proc. Natl Acad. Sci. USA 84, 6443–6446 (1987).
Paula, S., Volkov, A. G., Van Hoek, A. N., Haines, T. H. & Deamer, D. W. Permeation of protons, potassium ions, and small polar molecules through phospholipid bilayers as a function of membrane thickness. Biophys. J. 70, 339–348 (1996).
Raven, J. A. & Beardall, J. The intrinsic permeability of biological membranes to H+: Significance for the efficiency of low rates of energy transformation. FEMS Microbiol. Lett. 10, 1–5 (1981).
Verweij, F. J. et al. LMP1 association with CD63 in endosomes and secretion via exosomes limits constitutive NF-κB activation. EMBO J. 30, 2115–2129 (2011).
Sung, B. H., Pelletier, R. & Weaver, A. M. pHluo_M153R-CD63, a bright, versatile live cell reporter of exosome secretion and uptake, reveals pathfinding behavior of migrating cells. Preprint at https://www.biorxiv.org/content/10.1101/577346v1 (2019).
Mittelbrunn, M. et al. Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells. Nat. Commun. 2, 282 (2011).
van Niel, G. et al. Intestinal epithelial cells secrete exosome-like vesicles. Gastroenterology 121, 337–349 (2001).
Hoshino, D. et al. Exosome secretion is enhanced by invadopodia and drives invasive behavior. Cell Rep. 5, 1159–1168 (2013).
Verweij, F. J. et al. Live tracking of inter-organ communication by endogenous exosomes in vivo. Dev. Cell 48, 573–589.e4 (2019).
Van Deun, J. et al. EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research. Nat. Methods 14, 228–232 (2017).
van der Pol, E. et al. Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J. Thromb. Haemost. 12, 1182–1192 (2014).
Yuana, Y., Levels, J., Grootemaat, A., Sturk, A. & Nieuwland, R. Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation. J. Extracell. Vesicles 3, https://doi.org/10.3402/jev.v3.23262 (2014).
Coumans, F. A. W. et al. Methodological Guidelines to Study Extracellular Vesicles. Circ. Res. 120, 1632–1648 (2017).
Linares, R., Tan, S., Gounou, C., Arraud, N. & Brisson, A. R. High-speed centrifugation induces aggregation of extracellular vesicles. J. Extracell. Vesicles 4, 29509 (2015).
Jeppesen, D. K. et al. Reassessment of exosome composition. Cell 177, 428–445.e18 (2019).
Zhang, H. et al. Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat. Cell Biol. 20, 332–343 (2018).
Messenger, S. W., Woo, S. S., Sun, Z. & Martin, T. F. J. A Ca2+-stimulated exosome release pathway in cancer cells is regulated by Munc13-4. J. Cell Biol. 217, 2877–2890 (2018).
Nabhan, J. F., Hu, R., Oh, R. S., Cohen, S. N. & Lu, Q. Formation and release of arrestin domain-containing protein 1-mediated microvesicles (ARMMs) at plasma membrane by recruitment of TSG101 protein. Proc. Natl Acad. Sci. USA 109, 4146–4151 (2012).
Colombo, M., Raposo, G. & Thery, C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu. Rev. Cell Dev. Biol. 30, 255–289 (2014).
Shen, Y., Rosendale, M., Campbell, R. E. & Perrais, D. pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis. J. Cell Biol. 207, 419–432 (2014).
de Wit, J., Toonen, R. F. & Verhage, M. Matrix-dependent local retention of secretory vesicle cargo in cortical neurons. J. Neurosci. 29, 23–37 (2009).
Urbina, F. L., Gomez, S. M. & Gupton, S. L. Spatiotemporal organization of exocytosis emerges during neuronal shape change. J. Cell Biol. 217, 1113–1128 (2018).
Yuan, T., Lu, J., Zhang, J., Zhang, Y. & Chen, L. Spatiotemporal detection and analysis of exocytosis reveal fusion “hotspots” organized by the cytoskeleton in endocrine cells. Biophys. J. 108, 251–260 (2015).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Thevenaz, P., Ruttimann, U. E. & Unser, M. A pyramid approach to subpixel registration based on intensity. IEEE Trans. Image Process. 7, 27–41 (1998).
Mattheyses, A. L., Simon, S. M. & Rappoport, J. Z. Imaging with total internal reflection fluorescence microscopy for the cell biologist. J. Cell Sci. 123, 3621–3628 (2010).
We acknowledge the NeurImag facility of the Institute of Psychiatry and Neuroscience of Paris where the AMvBE macro has been developed. This Protocol was funded by a Dutch Organizations for Scientific Research–Amsterdam Institute for Molecules, Medicines, and Systems STAR Graduate Program grant (022.005.031) to M.P.B., a Dutch Cancer Fund (KWF-5510) and a Cancer Center Amsterdam–VU University Medical Center grant to D.M.P., and a European Molecular Biology Organization grant (EMBO ALTF 1383-2014) and a Fondation ARC pour la Recherché sur le Cancer fellowship (PJA 20161204808) to F.J.V.
The authors declare no competing interests.
Peer review information Nature Protocols thanks David Perrais and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key references using this protocol
Verweij, F. J. et al. J Cell Biol, 217 (3) 1129–1142 (2018): http://jcb.rupress.org/content/217/3/1129/
AMvBE macro, Readme file and example data
TIRF microscopy of a CD63-pHluorin expressing HeLa cell at 8× speed. Scale bar, 20 μm
Dual-color TIRF microscopy of a CD63-pHluorin (green) and CD81-pHuji (red) expressing HeLa cell at 8× speed. Scale bar, 20 μm
Dual-color TIRF microscopy of a CD63-pHluorin (green) and CD63-mRFP (red) expressing HeLa cell at 8× speed. Scale bar, 20 μm. White arrows highlight MVBs containing CD63-pHluorin and CD63-mRFP that are visible in red before fusion
Dual-color TIRF microscopy of a CD63-C-term-pHluorin (green) and CD63- pHuji (red) expressing HeLa cell at 8× speed. Scale bar, 20 μm. Original source: ref. 17
TIRF microscopy of HeLa cells stably expressing CD63-pHluorin 6 weeks post-transduction at 8× speed. Scale bar, 20 μm
TIRF microscopy of a CD63-pHluorin expressing HUVEC cell at 8× speed. Scale bar, 20 μm
TIRF microscopy of a CD63-pHluorin expressing HEK293T cell at 8× speed. Scale bar, 20 μm
Example of the summary TIFF file as created by the AMvBE macro upon analysis of an MVB-PM fusion event (highlighted by a green circle) or a neutral CD63-pHluorin-positive vesicle moving in the TIRF plane (highlighted by a red circle). Scale bar, 5 μm
About this article
Cite this article
Bebelman, M.P., Bun, P., Huveneers, S. et al. Real-time imaging of multivesicular body–plasma membrane fusion to quantify exosome release from single cells. Nat Protoc 15, 102–121 (2020). https://doi.org/10.1038/s41596-019-0245-4
Tumor Exosomes Reprogrammed by Low pH Are Efficient Targeting Vehicles for Smart Drug Delivery and Personalized Therapy against their Homologous Tumor
Advanced Science (2021)
Cell Communication and Signaling (2021)
Frontiers in Pharmacology (2021)
Cell Reports (2021)
Frontiers in Oncology (2021)