Figure 3

The F-actin-rich punctae are podosomes. (A) Microglia were stained for F-actin (phalloidin, green), the plaque protein talin (red), and the nuclear stain 4′,6-diamidino-2-phenylindole (DAPI; blue). A representative image of the large F-actin ring (podonut) in front of the nucleus shows co-localization with talin. Scale bar: 5 μm. Right: higher-magnification deconvolved images of the two boxed regions, color-separated and merged to show that the dense F-actin ring is comprised of many tiny punctae of F-actin and talin. Individual podosomes are seen as a core of F-actin, surrounded by a ring of talin (arrows show examples). Scale bar: 1 μm. (B) Microglia were immunostained for the plaque protein, talin (green), the actin nucleator actin-related protein 2 (Arp2; red), and labeled with the nuclear stain DAPI (blue). Arp2 is enriched in the podosome core, and the plaque protein talin identifies the ring. Lower image: under phase contrast, podosomes were often seen as dark punctae. (C) Prevalence of podosome expression in primary microglia (that is, percentage of cells with podosomes); >65 cells were analyzed per replicate from each of three cultures (mean ± standard error of the mean).