Figure S1.
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.

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.

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