Fig 2: ESCRT structures surround actin-independent integrin clusters. (A) Confocal micrographs of monocyte-derived dendritic cells (moDCs) incubated for 40 min and immunolabeled for IST1 (magenta in merge), integrin aM or integrin ß2 (cyan), and phalloidin (yellow). Blue: DAPI. Bar graphs show cells with integrin-wrapping IST1 structures as a percentage of all cells forming IST1 structures, at the indicated time points, for integrin ß2 and integrin aM. Line graphs show fluorescence intensity profiles as indicated by the white line. (n = 3 donors; two-sided paired t tests; *: P < 0.05; **: P < 0.01; NS: not significant). Scale bars: 10 µm. (B) Immunofluorescence labeling of a temporal artery biopsy (same donor 1 as in Fig. 5 A) for DAPI (blue), IST1 (magenta), integrin ß2 (yellow), and macrophage marker CD68 or fibroblast marker vimentin (cyan). (C) Confocal micrographs of moDCs seeded in the presence or absence of 5 mM EDTA, and immunostained for IST1 (magenta), integrin ß2 (green), and phalloidin (yellow). Arrows: IST1-positive structures. Graph: average number of IST1-positive structured per cell (n = 3 donors). For the statistical analysis of A and C, data distribution was assumed to be normal, but this was not formally tested. Scale bars: 10 µm.

Fig 3: IST1 structures are located at the plasma membrane of cells cultured on glass support and in collagen matrices. (A) moDCs seeded on top of collagen matrix. Cells were allowed to migrate into the matrix for 5 h. Shown are confocal z-stacks. Cells are labeled for IST1 (magenta). Green: phalloidin. Blue: DAPI. The cells depicted in panels I-III are shown in Videos 2, 3, and 4, respectively. Scale bars: 10 µm. (B) Same as A, but now with FITC-labeled collagen (green in merge) and phalloidin in yellow. Scale bars, 10 µm. (C) STED imaging of moDCs cultured for the indicated times and immunostained for IST1. Scale bars: 1 µm. (D) Additional images of the cell shown in main Fig. 1 F. Top images: Confocal laser scanning microscopy (CLSM) images of a moDC cultured on glass support labeled with fluoronanogold (FNG) for CHMP1B (magenta). Green: phalloidin. Bottom images: CLEM using fluoronanogold labeling of CHMP1B (magenta hot) showing the area marked by the dotted lines in the top images. A tomography series of the 100-nm-thick bottom section (closest to the glass support) was generated. The bottom slice of this tomogram of the bottom section, and the 3D-rendered volume of all tomographic slices of this section are shown. Silver enhanced gold particles are indicated with an arrow. Scale bars: 2 µm. (E) Abberior STAR 635 dye was mounted on a coverslip and measured with identical 3D STED settings as used for measuring IST1 structures in main Fig. 1 G. A FWHM of 79 ± 13 nm (±1 STD; total of 40 measurements; n = 3) was obtained. The FWHM of the IST1 structure is thus ~24 nm thicker than just plain dye on a coverslip. Scale bar: 1 µm. (F) Confocal micrograph with orthogonal views of moDC immunolabeled for IST1 (magenta in merge) and the fluorescent lipophilic membrane marker Memglow (green). Line profiles of the cross-section indicated by the yellow line are shown. Scale bar: 10 µm. (G) Western blot showing IST1 expression in moDCS cultured on glass in the presence or absence of fetal bovine serum over time. The graph shows the mean ± SD of IST1 levels normalized to tubulin and to 0 min incubation. N = 2 donors. O/N: overnight. Source data are available for this figure: SourceData FS1.

Fig 4: IST1 structures do not colocalize with caveolin-1 and the ALIX?PRR mutant. (A) More examples for the GPI-anchored RFP shown in main Fig. 6 B. TIRF microscopy of moDCs co-expressing GFP-labeled IST (magenta in merge) with GPI-anchored RFP (green). (B) TIRF microscopy of moDCs co-expressing GFP-labeled IST (magenta in merge) with mCherry-tagged caveolin 1 (green). (C) TIRF microscopy of moDCs co-expressing GFP-labeled IST (magenta) with mCherry-tagged ALIX?PRR mutant (green) or only mCherry (control). Graph shows the average number of IST1-positive structures for cells with detectable expression of both constructs for three different donors (paired two-sided t test). For the statistical analysis, data distribution was assumed to be normal, but this was not formally tested. Scale bars: 10 µm, unless indicated otherwise.

Fig 5: IST1 and CHMP1B form clusters of flat ring- and tube-shaped structures at the plasma membrane of dendritic cells. (A) Confocal micrograph of monocyte-derived dendritic cell (moDC) immunostained for IST1 (magenta in merge). Green: phalloidin. Blue: DAPI. The graph shows the percentage of cells showing IST1 structures (n = 4 donors). Scale bars: 10 µm. (B) Confocal z-stack of moDC cultured in collagen matrix. The graph shows the percentage of cells showing IST1 structures (n = 4 donors). Scale bar: 10 µm. This image is part of Video 1. (C) Confocal micrograph of moDC overexpressing IST1-GFP. Scale bars: 10 µm. (D) Side-by-side comparison of conventional epi-fluorescence microscopy and total internal resonance fluorescence (TIRF) microscopy of moDC expressing IST1-GFP (green) and stained with lysotracker (magenta). Note the improved signal-to-noise of IST1 at the plasma membrane in TIRF. This image is part of Video 6. Scale bars: 10 µm. (E) STED microscopy of monocyte-derived dendritic cells (moDCs) cultured on glass supports immunostained for IST1 (magenta in merge) and CHMP1B (green). Scale bar: 1 µm. (F) Correlative light electron microscopy (CLEM) using fluoronanogold labeling of CHMP1B (magenta hot). TEM: transmission electron microscopy. Scale bars: 1 µm. (G) Top-left: 2D STED image of immunolabeled IST1 and CHMP1B structures in a moDC. The dashed red lines indicate the positions [1–7] at which single-line xz-scans were acquired using 3D STED (lower-left seven images). Of each of these xz-scans, the intensity distribution along the z-axis was obtained (right plots, black lines), and a Gaussian fit was made (lower plots, red lines) to determine the local thickness of the IST1 structure through the FWHM intensity of the Gaussian fits. In total, 19 cells from three donors were measured to obtain 135 line profiles. These results are shown in the lower-middle histogram, which shows an average FWHM of 102.5 ± 21.8 nm (±1 STD). Scale bar: 1 µm.