GM1-enriched domains formed at the plasma membrane in nicotine-stimulated cells contain the AnxA2 tetramer, actin filaments, and SNARE proteins. (A and B) Double staining of AnxA2 (10 nm gold particles) and phalloidin (6 nm gold particles) on plasma membrane sheets prepared from unstimulated (A) and nicotine-stimulated cells (B). The right panel in B shows a higher magnification of the region delimited by the rectangle. Note that AnxA2 is also present on the docked granule surface (arrowhead). (C) Bivariate K-function analysis of double-labeled membranes (n = 44 images). Values of L(r)-r greater than the 95% CI indicated the significant colocalization of F-actin and AnxA2 in nicotine-stimulated chromaffin cells. (D) Spatial distribution of GM1 (6 nm gold particles, squares) and actin (10 nm gold particles) on the plasma membrane from a nicotine-stimulated cell. (E and F) Plasma membrane sheets from nicotine-stimulated cells stained for either GM1 (6 nm gold particles, squares)/S100A10 (10 nm gold particles, circles)/AnxA2 (15 nm gold particles) or GM1 (6 nm gold particles, squares)/actin (15 nm gold particles)/syntaxin (10 nm gold particles, circles). Bars, 100 nm.