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. 2018 Jul 26;9:2932. doi: 10.1038/s41467-018-05345-8

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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. 1 — ESCRT proteins localize to “late” early endocytic compartments. a Colocalization analysis of unstimulated fixed HeLa cells stably expressing CHMP4B-GFP shows good overlap of CHMP4B with early endocytic compartments. Manders’ colocalization coefficient (MCC): overlap of CHMP4B with endocytic markers and overlap of EEA1 with early or late endocytic markers as positive and negative control for colocalization. Statistics CHMP4B: One-way ANOVA, p = 0.0004. Tukey’s post hoc test; **p < 0.01; n.s. not statistically significant; Statistics EEA1: t-test, ***p < 0.001; n = 2–6 confocal experiments with 3-4 images (each 4–5 cells) per condition. Shown are details. Arrows: CHMP4B-GFP on endosomes. Data are mean ± SD. b Pulse-chase experiment: HeLa cells were stimulated for 2 min with 50 ng ml⁻¹ EGF-Al647 (white) and after removing unbound ligand chased for the indicated amount of time. Immunofluorescence staining and colocalization analysis establishes the endocytic trafficking of EGF ligand through the degradative pathway from early (EEA1 and RAB5 compartments) to late (RAB7 and LAMP1 compartments) within 45 min. Green and red arrows: EGF-Al647 colocalizing with early or late markers, respectively. MCC: Overlap of EGF with endocytic markers. One-way ANOVA EEA1: p = 0.0056; RAB5: n.s; RAB7: n.s.; LAMP1:p = 0.0014; n = 3–4 confocal experiments with 3–4 images (each 4–5 cells) per condition. Shown are details. Data are mean ± SEM. c Pulse-chase experiment as in b, but analyzing ESCRT colocalization with EGF-Al647. ESCRT-0 (HRS), ESCRT-III (CHMP4B) and the Bro1 domain protein HD-PTP all show a maximum overlap with EGF at 15 min after stimulation, indicating a concerted recruitment to endosomes. Arrows indicate colocalization between EGF and ESCRTs. One-way ANOVA HRS: p < 0.0001; HD-PTP: p = 0.0072; CHMP4B: n.s.; n = 4–6 confocal experiments with 3–4 images (each 4–5 cells) per condition. Shown are details from those images. Data are mean ± SEM. d Pulse-chase experiment as in b, but analyzing the very early time points after EGF stimulation. HRS colocalization with EGF increases from 2 min onward, and CHMP4B is clearly detected 5 min after the EGF pulse. t-test, **p < 0.01; n = 3–4 experiments with 3–4 images (each 4–5 cells) per condition. Data are mean ± SEM. All scale bars, 5 µm