Clusters of Cholera Toxin B Subunit on the Outer Leaflet Stabilize Lipid Heterogeneity on the Inner Leaflet of B Cell Membranes

Abstract

Protein sorting based on liquid-ordered or liquid-disordered phase preference is readily observed in giant plasma membrane vesicles (GPMVs) at low temperatures but is not observed directly in the intact cells from which they are derived. Here we utilize two-color super-resolution microscopy to quantify the sorting of two minimal inner leaflet anchored-peptides proximal to clusters of cholera toxin B subunit (CTxB) in chemically fixed CH27 B cells. One peptide, called PM, is anchored through saturated palmitoyl and myristoyl acylations, giving it a strong preference for more ordered lipids, while the second peptide, called GG, is anchored through an unsaturated and branched geranylgeranyl modification that gives it a strong preference for more disordered lipids. We find that the local density of PM is increased in the vicinity of CTxB clusters while the local density of GG is reduced in the vicinity of CTxB clusters when compared to the average density of peptides on the cell surface. These results indicate that clustering of CTxB on the outer leaflet affects the lateral organization of proteins anchored to the inner leaflet in a way that depends on the anchoring structure. Ongoing work is aimed at probing the membrane's ability to sort proteins based on the phase preference of their membrane anchor in response to biochemical perturbations and changes in temperature, with the goal of distinguishing possible physical bases of this lipid-mediated membrane heterogeneity.