Fig. 2. Podosome loss and FA formation in DCs upon PGE₂ stimulation, visualized with IRM. Also see Movies 1, 2 and Fig. S1A,B in the supplementary material. (A) IRM images compared to fluorescent images (detected with anti-vinculin mAb, GaM-ATTO633 and phalloidin-Texas Red, to detect F-actin) identify adhesion structures as podosomes (seen as dark dots with IRM and as actin dots surrounded by vinculin rings in the fluorescent images) and FAs (seen as dark stripes with IRM and as vinculin stripes in the fluorescent images). (B) Images from an IRM movie (Movie 1 in the supplementary material) showing podosomes before stimulation (–1 min) and loss of podosomes in response to PGE₂. Note how the dissolution of podosomes is (transiently) preceded by the formation of bright rings surrounding the podosome core. FA formation in response to stimulation can be observed in the upper-right part of the images. (C) Overlay of the fluorescence intensity of the vinculin staining (broken line) and the actin staining (solid line) from straight-line selections with ImageJ through the middle of the podosome. The actin peak flanked by two vinculin peaks reflects the actin core surrounded by the vinculin-containing ring. (D-F) The size of the adhesive contacts observed by live-imaging IRM, the actin core and the vinculin ring were determined in 50 podosomes from ten different cells by making straight-line selections with ImageJ through the middle of the podosome. A representative curve of one podosome is shown and the average size is depicted in the upper-right corner of the curves with errors depicted as s.e.m. (D) The fluorescence intensity of the actin staining from a straight-line selection through the middle of a podosome. The width of the peak at half the height was used to determine the width of the actin core (0.78±0.01 µm). (E) The fluorescence intensity of the vinculin staining from a straight-line selection through the middle of a podosome. The distance from one peak to the other peak was measured to obtain the size of the vinculin ring (0.91±0.02 µm). (F) The light intensity in the IRM image from a straight-line selection through the middle of a podosome. The width of the peak at half the height was used to determine the width of the adhesive contact (0.75±0.02 µm).