(380j) Multiway Clustering of Ganglioside GM1 in Heterogeneous Lipid Membranes By Sphingomyelinase-Mediated Hydrolysis

Ceramides are bioactive lipids that regulate the curvature and functions of biological membranes by forming specialized membrane domains. In particular, ceramides formed by sphingomyelinase (SMase)-mediated hydrolysis of sphingomyelin (SM) selectively aggregate specific signaling biomolecules (e.g., receptors and ion channels) into plasma-membrane domains. This ceramide-induced aggregation is highly significant as it is intimately involved in cell signaling and various pathophysiological conditions. However, the mechanism by which SM-derived ceramides concentrate signaling biomolecules remains poorly understood. To elucidate this mechanism at a fundamental level, we describe how SM-derived ceramides localize ganglioside GM1, a glycolipid membrane receptor that modulates diverse cell functions. We used an ultra-stable freestanding planar lipid membrane array that we recently developed (Small, 2020, Vol. 16, 2002541) to directly visualize the dynamic GM1 redistribution in the heterogeneous lipid membranes during SMase-mediated hydrolysis. This enabled us to find two types of condensed GM1: (1) rapidly formed but short-lived GM1 clusters in ceramide-rich domains; and (2) late-onset yet long-lasting, high-density GM1 clusters in cholesterol-rich domains. These GM1 clusters are formed by discrete mechanisms that differ from previously accepted thought. Our findings highlight that the SMase-mediated hydrolysis can precipitate two distinct forms of GM1 clusters at multiple spatiotemporal scales, depending on the dynamicity and heterogeneity of the plasma membrane. Our study suggests that SMase-mediated multiple clustering pathways synergistically facilitate the amplification and persistence of signals.