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

Abstract

The cellular membrane of eukaryotes consists of phospholipids, sphingolipids, cholesterol and membrane proteins. Among them, cholesterol is crucial for various cellular events (e.g., signaling, viral/bacterial infection, and membrane trafficking) in addition to its essential role as an ingredient of steroid hormones, vitamin D, and bile acids. From a micro-perspective, at the plasma membrane, recent emerging evidence strongly suggests the existence of lipid nanodomains formed with cholesterol and phospholipids (e.g., sphingomyelin, phosphatidylserine). Thus, it is important to elucidate how cholesterol behaves in membranes and how the behavior of cholesterol is regulated at the molecular level. To elucidate the complexed characteristics of cholesterol in cellular membranes, a couple of useful biosensors that enable us to visualize cholesterol in cellular membranes have been recently developed by utilizing domain 4 (D4) of Perfringolysin O (PFO, theta toxin), a cholesterol-binding toxin. This review highlights the current progress on development of novel cholesterol biosensors that uncover new insights of cholesterol in cellular membranes.

Keywords: D4; Perfringolysin O; cholesterol probes; domain 4; microscopy; theta toxin; visualization.

Figure 1

Molecular structure of cholesterol.

Figure 2

Scheme of visualization of cholesterol in cellular membranes by fluorescent sterols (A) filipin (B) and fluorophore-labelled cholesterol (C) Methodology, benefits and limitations of each cholesterol biosensor are described. Threshold for cholesterol-binding to filipin, 5 mol %, is shown ([37]; B).

Figure 3

Scheme of detection of cholesterol in cellular membranes by PFO-derived cholesterol biosensors. (A) Domain structure of PFO. The image is reproduced from [62]. Copyright MDPI 2015. (B,C) Scheme, methodology, benefits, and limitations of I¹²⁵-PFO* (B) and BCtheta (C) are shown. The number is cholesterol concentration (mol %) threshold required for each cholesterol probe-binding.

Figure 4

Scheme of visualization of cholesterol in cellular membranes by D4-derived cholesterol biosensors. Scheme, methodology, benefits, and limitations of D4-derived cholesterol biosensors to visualize cholesterol in the exofacial leaflets of the PM (A) and cytosolic leaflets of cellular membranes (B) are shown. The number is cholesterol concentration (mol %) threshold required for each cholesterol probe-binding.

Figure 5

Confocal images of CHO cells labeled with His-GFP-D4 recombinant proteins (A) and expressing mCherry-D4H (B). Bar, 10 µm. The images are reproduced from [67]. Copyright Nature Publishing Group 2016.