Calmodulin (CaM) is expressed in microglia podosomes. (A) A Western blot for CaM shows a strong band of the expected molecular weight (approximately 17 kDa) for the positive control (left lane; 50 ng recombinant human CaM protein) and in lysates from untreated cultured rat microglia (right lane). The (B) CaM is enriched in the podonut of lamella-bearing migrating cells. Non-deconvolved image shows a cell stained with the microglia marker, tomato lectin (TL, green), the nuclear marker, DAPI (blue), and CaM (red), which is isolated in the color-separated image at the right. Scale bar, 20 μm. (C) A deconvolved image of a podonut in a microglia lamellum, which shows enriched staining for CaM (red) and the podosome ring marker, talin (green). Scale bar, 5 μm. (D) A high-resolution, deconvolved image from a podonut ring shows CaM (red) within and surrounding the core, which is labeled with Arp2 (green). (Note: A mouse monoclonal anti-CaM antibody was used.) Scale bar, 2 μm. Lower panel: Two podosomes are shown at higher magnification; one with extensive, diffuse CaM staining surrounding Arp2. Scale bar, 1 μm. (E) CaM (green) and SK3 (red) co-localize in podonuts within the lamellae (nuclei are labeled with DAPI, blue). Color-separated images at the right. Scale bars, 40 μm.