Synergy between membrane topography and domains to control signaling protein localization in mast cells facilitates...

Similar to T cells and B cells, mast cell surfaces are dominated by microvilli, and like these other immune cells we showed with microvillar cartography (MVC) that key signaling proteins for RBL mast cells localize to these topographical features. Although stabilization of ordered lipid nanodomains around antigen-crosslinked IgE-FcεRI is known to facilitate necessary coupling with Lyn tyrosine kinase to initiate transmembrane signaling in these mast cells, the relationship of ordered-lipid nanodomains to membrane topography had not been determined. With nanoscale resolution provided by MVC, standard error of the mean (SEM), and colocalization probability (CP) analysis, we found that FcεRI and Lyn kinase are positioned primarily on the microvilli of resting mast cells in separate nano-assemblies. Upon antigen-activation, FcεRI and Lyn merge into overlapping populations together with the LAT scaffold protein, accompanied by merger of microvilli into ridge-like ruffles. With selective lipid probes, we further found that ordered-lipid nanodomains preferentially occupy microvillar membranes, contrasting with localization of disordered lipids to flatter regions. With this proximity of signaling proteins and ordered lipid nanodomains in microvilli, the mast cells are poised to respond sensitively and efficiently to antigen but only in the presence of this stimulus. Use of a short chain ceramide to disrupt ordered-lipid regions of the plasma membrane and evaluation with MVC, CP, and flow cytometry provided strong evidence that the microvillar selective localization of signaling proteins and membrane environments is facilitated by the interplay between ordered-lipid nanodomains and actin attachment proteins, ERM (ezrin, radixin, moesin), and cofilin.