Glycolipid Crosslinking is Required for Cholera Toxin to Partition into and Stabilize Ordered Domains

Abstract

Current models of lipid rafts propose that lipid domains normally exist as nanoscale compositional fluctuations at steady state in cells, but can be stabilized to form functional entities. However, the mechanism behind how stabilized rafts assemble and function remains unclear. Here, we test the role of glycolipid crosslinking as a raft targeting and ordering mechanism using the well-studied raft marker cholera toxin B pentamer (CTxB) as a model. We show that when bound to cell-derived Giant Plasma Membrane Vesicles, a variant of CTxB containing only a single functional GM1 binding site exhibits significantly reduced partitioning in the ordered phase compared to wild type CTxB with five binding sites. Moreover, monovalent CTxB does not increase membrane heterogeneity, unlike wild type CTxB. These results support the long-held hypothesis that CTxB stabilizes raft domains via a crosslinking mechanism and uncover an unexpected role for crosslinking in the targeting of CTxB to ordered domains.