Figure 1: Podosomes protrude perpendicularly to the substratum.
(a) Illustration of the method for podosome protrusion measurement: PFM. At least 2 h after macrophage adhesion, AFM topographical images and height oscillation measurements of podosome protrusions were obtained on the reverse side of the Formvar sheet (for details see text; Supplementary Fig. 1; Supplementary Methods; ‘Preparation of Formvar-coated grids’; and ‘AFM experiments’ sections). (b) Representative AFM topographical image of podosome protrusions of a living human macrophage plated on a Formvar sheet. Scale bar, 2 μm. Cross-section extracted from the topographical AFM image (across the white line) depicting the heights of three podosome protrusions. (c) Correlative AFM and immunofluorescence images. The AFM deflection image (upper left panel; scale bar, 5 μm) was acquired on a living macrophage plated on Formvar sheet. The dashed line indicates the edges of the grid and a magnified view of the cell edge is shown in the upper right panel (scale bar, 20 μm). After fixation, cells were stained for F-actin and vinculin (middle and lower panels). An overlay of deflection and fluorescence microscopy is shown in the lower left panel. (d) Distribution of the height values of protrusions generated by podosomes, with a mean of 16.5±9.5 nm (n=787 podosomes from 23 cells, 12 experiments and five donors). (e) Example of the experimental profile of a podosome protrusion fitted with the model (see Supplementary equation (4) in Supplementary Note 2) so as to determine the radius a and the maximal height hmax of the protrusion. (f) Distribution of the force values generated by podosomes, with a mean of 94±59 nN, n=166 podosomes, seven experiments. (g) Linear correlation between relative protrusion height h/ef and podosome radius a, n=144 podosomes. h is the protrusion height and ef the thickness of the Formvar sheet. (h) Linear evolution of podosome protrusive forces versus relative podosome protrusion height h/ef according to Supplementary equation (7) in Supplementary Note 2, for the lowest values of h/ef. n=144 protrusions, seven experiments.
