Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol

Abstract

Controlled distribution of lipids across various cell membranes is crucial for cell homeostasis and regulation. We developed an imaging method that allows simultaneous in situ quantification of cholesterol in two leaflets of the plasma membrane (PM) using tunable orthogonal cholesterol sensors. Our imaging revealed marked transbilayer asymmetry of PM cholesterol (TAPMC) in various mammalian cells, with the concentration in the inner leaflet (IPM) being ~12-fold lower than that in the outer leaflet (OPM). The asymmetry was maintained by active transport of cholesterol from IPM to OPM and its chemical retention at OPM. Furthermore, the increase in the IPM cholesterol level was triggered in a stimulus-specific manner, allowing cholesterol to serve as a signaling lipid. We found excellent correlation between the IPM cholesterol level and cellular Wnt signaling activity, suggesting that TAPMC and stimulus-induced PM cholesterol redistribution are crucial for tight regulation of cellular processes under physiological conditions.

Domain 4 (D4) of Perfringolysin O to Visualize Cholesterol in Cellular Membranes—The Update

“…It is likely that cholesterol concentration in the cellular membranes which D4 cannot bind to is lower than 30 mol % and/or environments around cholesterol like phospholipids composition inhibit binding of D4 to cholesterol in the membranes. To solve this issue, a couple of novel and excellent cholesterol probes derived from D4 have been developed by introducing a variety of mutations which decrease the threshold for cholesterol-binding to D4 [66,72]. …”

“…To decrease still high threshold for cholesterol-binding to D4H (D4 D434S ), other point mutations were introduced in D4 in addition to D434 [72]. A recent study identified three amino acid residues (Y415, Q433, and A463) in D4, which determine the affinity of D4 to cholesterol [72].…”

“…A recent study identified three amino acid residues (Y415, Q433, and A463) in D4, which determine the affinity of D4 to cholesterol [72]. Liu et al, developed new D4 mutants, D434A, D434/A463W, Y415A/D434W/A463W (named YDA), and Y415A/Q433W/D434W/A463W (named YQDA) [72].…”

“…A recent study identified three amino acid residues (Y415, Q433, and A463) in D4, which determine the affinity of D4 to cholesterol [72]. Liu et al, developed new D4 mutants, D434A, D434/A463W, Y415A/D434W/A463W (named YDA), and Y415A/Q433W/D434W/A463W (named YQDA) [72]. Threshold for cholesterol-binding to D434/A463W was approximately 10 mol %, which is lower than D434A (20 mol %) and not-mutated D4 (30 mol %) [72].…”

“…Liu et al, developed new D4 mutants, D434A, D434/A463W, Y415A/D434W/A463W (named YDA), and Y415A/Q433W/D434W/A463W (named YQDA) [72]. Threshold for cholesterol-binding to D434/A463W was approximately 10 mol %, which is lower than D434A (20 mol %) and not-mutated D4 (30 mol %) [72]. Surprisingly, YDA and YQDA were shown to be able to bind to liposomes in which cholesterol concentration is more than 1 mol % [72], indicating that YDA and YQDA can theoretically label cholesterol in cellular membranes with the wide dynamic range.…”