The small-conductance Ca
channel, SK3, is expressed in podosomes of rat microglia. (A) Live imaging: Migrating microglia have a lamellum at the leading edge (see arrows). Scale bars: 40 μm in A, B, C, E. (B) SK3 is prevalent in a large ring within many lamellae (arrows). Microglia were labeled with tomato lectin (green), anti-SK3 antibody (red), nuclear stain, DAPI (blue throughout). (C) SK3-labeled rings (red) co-label for F-actin (green; Alexa Fluor™ 488-conjugated phalloidin). Color-separated images are at right. (D) The large SK3 ring comprises many small punctae containing the podosome ring component, talin (green). High-magnification, deconvolved images (right; boxed areas) show tiny SK3 punctae (<1 μm diameter) surrounded by talin. Scale bars, 5 μm (left), 1 μm (middle, right). (E) A cell-sized area of fibronectin degradation, seen as loss of Alexa Fluor™ 488 staining (green), co-localized with enriched SK3 staining (red). (F) Co-localization (magenta) of SK3 (red) and F-actin (Alexa Fluor™ 488-conjugated phalloidin, false-colored blue) in a ‘podonut’. Scale bar, 5 μm. Right: higher-magnification, merged and color-separated images of the boxed region showing SK3 + F-actin (merged; magenta), SK3 (red) and Alexa Fluor™ 488-conjugated fibronectin (green). Note the many tiny punctae of F-actin and SK3 staining (podosomes) and similar-sized punctae of fibronectin degradation (some examples shown by arrows). Scale bars, 1 μm.