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. 2018 May 22;9:2013. doi: 10.1038/s41467-018-04419-x

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© The Author(s) 2018

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. 5 — VAMP7 vesicles track between microclusters on microtubules at later times after activation. Jurkat cells transfected with indicated constructs were dropped onto stimulatory coverslips and imaged by TIRF-SIM 5 min after initial stimulation. a–e Jurkat cells were transfected with ZAP-70-apple and emerald-VAMP7. a Image of the entire cell. Scale bar equals 2 μm. b, c Zoomed-in images focusing on a single VAMP7 vesicle and a few ZAP-70 microclusters. b shows a vesicle tracking on a microcluster; c shows a vesicle tracking between multiple microclusters (n = 7 cells, three independent experiments). Scale bars for b and c equal 0.2 μm. d Zoomed-in area of a region of interest of a cell in which vesicles (red) and microclusters (green) have been segmented and show two tracks of VAMP7 vesicles moving between ZAP70 clusters. To the right are time plots of the tracks shown in d indicating when colocalization (coloc) or no colocalization (no coloc) between VAMP7 and ZAP-70 was observed. e Spatiotemporal map of an entire cell with ZAP-70 microclusters in gray and VAMP7 tracks in yellow (when they colocalized with ZAP-70) or purple (when no colocalization was observed). f Graph of the percentage of vesicles per cell associated with microclusters and the frames that a vesicle was associated with a microcluster in the lifetime of its appearance in the TIRF field. g Jurkat cells were transfected with EMTB-GFP, Grb2-apple, and Halo-VAMP7. Image of the entire cell. Scale bar equals 2 μm. h, i Zoomed-in image focusing on a single tracked VAMP7 vesicle. h shows a vesicle tracking on a microtubule; i shows a vesicle tracking on a microtubule between two Grb2 microclusters indicated by indented arrowheads (n = 5 cells, three independent experiments). Scale bars for h and i equal 0.2 μm. In b, c, h, and i, the tracked vesicle is indicated as a gray sphere. The entire track is shown and color-coded to indicate time (earliest time point in blue and the latest time point in red). In the first panel, the entire track is shown. The remaining panels show four time points, each with a track that displays the previous five time points in b and c or previous 20 time points in h and i