The Synergy between Topography and Lipid Domains in the Plasma Membrane of Mast Cells Controls the Localization of Signaling Proteins and Facilitates their Coordinated Activation

Abstract

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-Fc{varepsilon}RI 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, SEM and co-localization probability (CP) analysis, we found that Fc{varepsilon}RI and Lyn kinase are positioned primarily on the microvilli of resting mast cells in separate nano-assemblies. Upon antigen-activation, Fc{varepsilon}RI 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. Significance StatementParticipation of ordered-lipid nanodomains (aka "rafts") to target immune signaling in the plasma membrane has been established. Separately, membrane topography, specifically microvilli, has also emerged as a participant. Here, we show how these features are coordinated in mast cells that serve as gatekeepers for antigen-triggered, receptor-mediated immune responses, including allergies and inflammation. We found that these specific antigen receptors and a key kinase, together with ordered-lipid nanodomains, localize to microvilli in resting cells, forming separated nano-assemblies. Antigen causes the merger of microvilli into ruffles where receptors and kinases couple to initiate transmembrane signaling. Selective pre-organization of signaling proteins and targeting lipid domains in microvilli and their coordinated redistribution upon antigen stimulation facilitates sensitive and efficient immune responses.