Microglia with podosomes degrade the extracellular matrix molecule fibronectin. (A) Development of F-actin superstructures (podonuts) with time after plating microglia on coverslips. Upper panels: microglia were labeled for the podosome core component F-actin (phalloidin, red). Podonuts in the lamellae were prevalent after 20 hours of culturing (examples shown by arrows), but not after 2.5 or 10 hours. Lower panels: higher-magnification grayscale images of the boxed regions. Scale bar: 40 μm (upper), 20 μm (lower). (B) Panels indicate the control condition at 20 hours without microglia (left), or 2.5 and 20 hours after adding microglia. Microglia were labeled for F-actin (phalloidin, red), and the substrate, fibronectin, was conjugated to AlexaFluor 488 (green). Representative images from three separate experiments. Scale bar: 40 μm. (C) Higher-magnification, color-separated and merged images showing microglia-sized areas of fibronectin degradation. Stains for F-actin and fibronectin were as in (b), and microglial nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar: 10 μm.