Fig. 2.

ESCRT proteins act concertedly 5 to 15 min after EGF stimulation. a Experimental setup and representative images from a movie showing the uptake of EGF-Al647 into endocytic vesicles. Scale bars, 5 µm (left) and 1 µm (right). b Quantification of endosomal localization was done by segmenting spots in all three fluorescence channels by semi-automated thresholding on complete frames which typically contain one or two HeLa cells. Spots below a threshold distance of <5 pixels (400 nm) were counted as co-occurring with EGF and this is visualized on a gray scale with white displaying minimal and black maximal co-occurrence. As expected, early endocytic markers showed maximal co-occurrence with EGF at early time points after starting the live-cell imaging, and late markers at later time points. Representatives of the ESCRT subcomplexes are color-coded according to the schematic. RAB5: 6 exp; SNX15: 5 exp.; EEA1: 7 exp.; RAB7: 6 exp; HRS: 55 exp.; HD-PTP: 7 exp.; TSG101: 7 exp.; CHMP4B: 32 exp.; CHMP3: 14 exp.; VPS4A: 13 exp. c Tracking of individual EGF-positive endosomes shows recruitment and dissociation of mCherry-HRS and CHMP4B-GFP and their normalized fluorescence intensity over time. Images of one representative endosome at indicated time points and corresponding fluorescence intensity measurement over time (in min). Representative example from >30 tracks from 9 independent experiments. Scale bar, 0.5 µm. d Tracking of individual EGF-positive endosomes in cell lines stably expressing combinations of ESCRT-0 (HRS) or ESCRT-III (CHMP4B) with other ESCRT proteins. Normalized fluorescence intensities over time of one representative track out of≥25 tracks from ≥4 independent experiments per combination. Tracks are displayed until the endosome went out of focus. Note the coordinated recruitment and dissociation of ESCRT proteins and that they show either slow and gradual waves (HRS, HD-PTP, TSG101) or rapid and transient kinetics (CHMP4B, CHMP3, VPS4A). e A total of 23 isolated fluorescence profiles of coordinated HRS and CHMP4B recruitment were individually normalized between 0 and 100 and then averaged. Mean intensity profiles of ESCRT waves ± SD. Data from 14 tracked endosomes, 6 independent live-cell imaging experiments