High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

Abstract

“Lipid rafts” enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.

Role of Src Family Kinases in Regulation of Intestinal Epithelial Homeostasis

“…S6A). Moreover, staining of the ganglioside GM1 as a marker for lipid rafts (29,30) with the B subunit of cholera toxin revealed a pattern similar to that apparent for the three SFKs in intestinal crypts (see Fig. S6B).…”

Lipid rafts: contentious only from simplistic standpoints

“…Moreover, the results of earlier FRET and electron paramagnetic resonance (EPR)-spin labelling experiments can only be rationalized if rafts are small and unstable, or raft proteins exchange rapidly with non-raft membrane on a timescale of <0.1 ms (REFS 27,28 ). Other FRET studies have shown cholesterol-dependent clustering of certain lipid-anchored proteins on the inner leaflet, but did not estimate domain lifetime or an upper limit of domain size 29 .…”

Lipid rafts, cholesterol, and the brain

“…Based on FRET criteria, some raft proteins show little evidence for clustering in FRET studies (Kenworthy and Edidin 1998;Kenworthy et al 2000;Glebov and Nichols 2004), Others appear to be clustered in sub-micron domains, as predicted by the lipid raft model (Varma and Mayor 1998;Zacharias et al 2002;Nichols 2003;Sharma et al 2004). Interestingly, detailed analysis of such FRET data has shown that only a small fraction of GPI-anchored proteins are found in clusters and that these clusters consist of only a few molecules (Sharma et al 2004).…”