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. 2020 Apr 29;11:2092. doi: 10.1038/s41467-020-15747-2

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as 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. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Sequence of pHluo_M153R-CD63. pHluorin sequence is in green color. Highlighted regions in gray represent small (underlined) and large extracellular loops. M153R mutation is marked in red. b Diagram of pHluo_M153R-CD63 construct. Notice pHluo_M153R tag has bright fluorescence upon fusion of the multivesicular body (MVB) with the plasma membrane due to the exposure to neutral pH. Otherwise, it is nonfluorescent in the acidic condition of the MVB lumen. ILV intraluminal vesicle, PM plasma membrane. c Representative trace from nanoparticle tracking analysis of large EVs and small EVs. d Western blot analysis of cells and EVs with anti-CD63, anti-GFP, EV markers (TSG101, Flotillin) and Golgi marker (GM130). TCL total cell lysate, lEV large EV, sEV small EV, P parental cells, pH pHluo_M153R-CD63-expressing cells. Black arrows indicate full-length pHluo_M153R-tagged CD63, which is shifted due to the GFP moiety of 27 kDa, while white arrows indicate potential cleaved form of CD63 tagged with pHluo_M153R. Asterisk (*) indicates cellular CD63, which has a broad range due to glycosylation. e Still images from Supplementary Movie 1 (upper panel) showing a migrating HT1080 cell stably expressing mCherry-CaaX (magenta) and pHluo_M153R-CD63 (green). Colocalization of magenta and green is white. Notice that the deposits left behind the migrating cell are only labeled with CD63 not with CaaX. Scale bar, 50 µm. Movie representative of 38 movies. f Still images from Supplementary Movie 1 (lower panel) showing a migrating HT1080 cell transiently expressing pHluo-CD63. Scale bar, 50 µm. Movie representative of 12 movies. Source data are provided as a Source Data file.