Lamellae in microglia have a large F-actin ring, comprised of many small punctae. F-actin was labeled with phalloidin (green) in all images. (A) Low-magnification image of many migrating microglia on a glass coverslip. Most lamellae have a large, donut-shaped F-actin ring (* indicates examples), which we call a podonut. Scale bar: 40 μm. (B) Higher-magnification, deconvolved image shows a podonut in the front half of a migrating microglial cell. Scale bar: 10 μm. (C), (D) F-actin rings were predominantly associated with microglial lamellae. Values expressed as mean ± standard error of the mean from three separate cultures. Most microglia with a lamella had a large F-actin ring (a podonut) (87 ± 5% of such cells (c)). As further demonstrated in Figure 3, the large ring was made up of many podosomes. When podosomes were present, they (and the large F-actin ring) were located in the lamella (95 ± 2% of such cells (d)). (E) The large F-actin rings contain hundreds of tiny punctae of F-actin. A representative deconvolved image of a single podonut shows that the large ring is composed of many tiny (<1 μm diameter) F-actin-rich punctae. Scale bar: 5 μm. Inset: a deconvolved three-dimensional (3D) reconstruction shows that the punctae of F-actin were located near the substrate-contacting cell membrane. (F) Microglia formed similar large, F-actin rings in their lamellae; nuclei stained with 4′,6-diamidino-2-phenylindole, (DAPI, blue) when cultured on Ultra-Web™, which is a 3D mesh of polyamide nanofibers (upper image). Right: higher magnification image of several microglia with large F-actin rings. Scale bar: 100 μm (left), 20 μm (right).