Lipid Driven Inter-leaflet Coupling of Plasma Membrane Order Regulates FcεRI Signaling in Mast Cells
Authors: Yang Gil-Suk , Wagenknecht-Wiesner Alice , Yin Boyu , Suresh Pavana , London Erwin , Baird Barbara A. , Bag Nirmalya 
Published: November 2022

ABSTRACTEngagement of high affinity immunoglobulin E (IgE) receptor FcεRI with extracellular, multivalent antigen (Ag) stabilizes co-existing ordered and disordered phases in the inner leaflet of the plasma membrane. This optimally controls biochemical interactions between signaling components required for transmembrane (TM) signaling in mast cells. The biophysical organization of the resting inner leaflet is poised to respond appropriately to this extracellular stimulation. The resting inner leaflet is generally less ordered than the outer leaflet, with a lipid composition that does not spontaneously phase separate in model membranes. We proposed that coupling between the two leaflets mediates separation into different phase-like domains in the inner leaflet. To test this hypothesis in live cells, we first established a straightforward approach to evaluate changes in membrane order due to inter-leaflet coupling by measuring inner leaflet diffusion of phase-specific lipid probes with Imaging Fluorescence Correlation Spectroscopy (ImFCS) before and after methyl-α-cyclodextrin (mαCD)-catalyzed exchange of outer leaflet lipids (LEX) with exogenous order- or disorder-promoting phospholipids. We examined the functional impact of LEX by monitoring two Ag-stimulated cellular responses, namely early-stage recruitment of Syk kinase to the inner leaflet and late-stage exocytosis of secretory granules (degranulation). Based on changes in probe diffusion, we observed global increase or decrease of inner leaflet order when outer leaflet is exchanged with order or disorder promoting lipids, respectively, in unstimulated cells. Furthermore, the degree of stimulated Syk recruitment and degranulation correlates with the inner leaflet order of the resting cells, which was varied using LEX. Overall, combined LEX and ImFCS platform provides strong evidence of lipid-based control of stimulated TM signaling in live mast cells. In addition, our functional results imply that resting-state lipid composition and ordering of the outer leaflet sets the ordering of the inner leaflet, likely via interleaflet coupling, and correspondingly modulates TM signaling initiated by antigen-activated IgE-FcεRI.STATEMENT OF SIGNIFICANCECoupling between plasma membrane leaflets, which are biochemically and biophysically asymmetric, results in a steady-state membrane organization that is thought to play fundamental roles in cellular functions. Here, we present a straightforward assay built around mαCD-catalyzed lipid exchange (LEX) and Imaging Fluorescence Correlation Spectroscopy (ImFCS) to quantitatively characterize a novel, lipid-driven, interleaflet coupling mechanism and its functional impact in live mast cells. We showed that elevation of outer leaflet lipid order induces ordering throughout the inner leaflet in resting cells. This ordering enhances protein-based reactions during Ag-stimulated FcεRI signaling and consequent cellular response. Overall, we provide a compelling evidence of functional relevance of plasma membrane organizational heterogeneity driven by lipid-based interleaflet coupling.