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.