Fig. 1. Confocal analysis of the 3D structure of smooth muscle podosomes reveals a ring-like arrangement. (A) Three-dimensional structure of podosomes in a GFP-transfected cell fixed in 4% formaldehyde after 1 hour in 1 µM PDBu. Overview in phase contrast. Boxed areas 1, 2 and 3 show Nipkow disk confocal images of single podosomes, with GFP as cytoplasmic marker. X-Y frames show bottom confocal section. X-Y top frames show the uppermost confocal sections for each podosome. X-Z and Y-Z frames show vertical sections of the central regions of each podosome. Note the GFP-free area in the center of the cones in boxed areas 1 and 2, and the lack of such a zone in all X-Y top frames. (B) A living cell co-transfected with GFP and DsRed-SM22. Phase dense podosomes in the left panel (phase contrast) correspond to the hollow regions in ring-shaped GFP staining (arrows in middle panel). SM22 accumulates at the GFP-free zones in the center of these patches (right panel). (C) Nipkow disk confocal images of X-Y plane overview and X-Z planes of individual podosomes (in boxed areas) of a DsRed-SM22-transfected cell fixed in 4% formaldehyde. SM22 is present along the entire length along the middle axis of the podosome (boxed areas 4 and 5). (D,E) X-Z planes of single podosomes in GFP-p20-transfected cell as seen in a Nipkow disk confocal microscope. Arp2/3 is distributed along the middle axis of the podosomes, with decreasing density towards the dorsal cell surface.