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 (MC) 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 MC, SEM and co-localization probability (CP) analysis, we found that FcεRI and Lyn kinase are positioned exclusively on the microvilli of resting mast cells in separate nano-assemblies, and upon antigen-activation they merge into overlapping populations together with the LAT scaffold protein, accompanied by elongation and 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 MC, CP, and flow cytometry provided strong evidence that the microvillar selective localization of signaling proteins and lipid environments is facilitated by the interplay between ordered-lipid nanodomains and actin attachment proteins, ERM (ezrin, radixin, moesin) and cofilin.
Significance statement: Participation of ordered-lipid nanodomains (colloquially dubbed "rafts") to target and regulate immune signaling in the plasma membrane is well documented. Recent studies also demonstrated the role of membrane topography, specifically microvilli, in T-cell and B-cell immune signaling. Here, we show how these features are coordinated in RBL mast cells, a well-established model for mast cells involved in multiple antigen-activated immune responses that include allergies and inflammation mediated by IgE-receptors (IgE-FcεRI). We found that the receptors and a key signaling kinase, together with ordered-lipid nanodomains localize to microvilli in resting cells, forming separated nano-assemblies. Antigen-activation causes elongation and merger of microvilli into ruffles where receptors and kinase coalesce to initiate transmembrane signaling. Selective pre-organization of signaling proteins and targeting lipid domains in microvilli facilitates sensitive and efficient responses to antigenic stimulation. Overall, our results demonstrate complex interplay between membrane topography, ordered-lipid nanodomains, and cytoskeleton attachment proteins in controlling mast cell activation.