Abstract
Gangliosides in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. How gangliosides are dynamically organized and how they respond to ligand binding is poorly understood. Using fluorescence anisotropy imaging of synthetic, fluorescently labeled GM1 gangliosides incorporated into the plasma membrane of living cells, we found that GM1 with a fully saturated C16:0 acyl chain, but not with unsaturated C16:1 acyl chain, is actively clustered into nanodomains, which depends on membrane cholesterol, phosphatidylserine and actin. The binding of cholera toxin B-subunit (CTxB) leads to enlarged membrane domains for both C16:0 and C16:1, owing to binding of multiple GM1 under a toxin, and clustering of CTxB. The structure of the ceramide acyl chain still affects these domains, as co-clustering with the glycosylphosphatidylinositol (GPI)-anchored protein CD59 occurs only when GM1 contains the fully saturated C16:0 acyl chain, and not C16:1. Thus, different ceramide species of GM1 gangliosides dictate their assembly into nanodomains and affect nanodomain structure and function, which likely underlies many endogenous cellular processes.
Highlights
Gangliosides in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions
The synthetic GM1 species were added exogenously to a GSL-deficient cell line, GM95, using a protocol previously shown to result in functional incorporation of synthetic lipid analogs into the plasma membrane, as evidenced by reconstitution of cholera toxin function[29], the expected phase behavior for the different GM1 species in giant plasma membrane vesicles (Supplementary Fig. 1), and the fact that cholera toxin B-subunit (CTxB) bound to fluorescently labeled GM1 species with the same sensitivity as to the unlabeled counterparts (Supplementary Fig. 2)
GSLs are essential for life by controlling many cellular processes, including endocytosis[53,54,55]
Summary
Gangliosides in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. The plasma membrane of eukaryotic cells is composed of a large number of lipid species that are asymmetrically distributed between the inner and outer leaflets[1] as well as phase segregated laterally into functional domains[2,3,4]. The acto-myosin cortex renders an additional layer of active organization that can drive the formation of nanoclusters, as exemplified by studies on GPI-anchored proteins[9,13] These nanoclusters assemble in a cholesterol-dependent manner and are coupled across the bilayer to the acto-myosin cortex by the abundant inner leaflet lipid phosphatidylserine[14]. Glycosphingolipids (GSLs) are composed of complex oligosaccharide headgroups linked to ceramide[15] Among their many functions, GSLs form the basis for the assembly of raft domains induced by the interaction with extracellular GSL-binding ligands. GSL-binding protein galectin-3, which drives the clathrinindependent endocytic uptake of cargo proteins such as CD44 and α5β1 integrin[23]
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