Perfringolysin O Theta Toxin as a Tool to Monitor the Distribution and Inhomogeneity of Cholesterol in Cellular Membranes

Abstract

Cholesterol is an essential structural component of cellular membranes in eukaryotes. Cholesterol in the exofacial leaflet of the plasma membrane is thought to form membrane nanodomains with sphingolipids and specific proteins. Additionally, cholesterol is found in the intracellular membranes of endosomes and has crucial functions in membrane trafficking. Furthermore, cellular cholesterol homeostasis and regulation of de novo synthesis rely on transport via both vesicular and non-vesicular pathways. Thus, the ability to visualize and detect intracellular cholesterol, especially in the plasma membrane, is critical to understanding the complex biology associated with cholesterol and the nanodomains. Perfringolysin O (PFO) theta toxin is one of the toxins secreted by the anaerobic bacteria Clostridium perfringens and this toxin forms pores in the plasma membrane that causes cell lysis. It is well understood that PFO recognizes and binds to cholesterol in the exofacial leaflets of the plasma membrane, and domain 4 of PFO (D4) is sufficient for the binding of cholesterol. Recent studies have taken advantage of this high-affinity cholesterol-binding domain to create a variety of cholesterol biosensors by using a non-toxic PFO or the D4 in isolation. This review highlights the characteristics and usefulness of, and the principal findings related to, these PFO-derived cholesterol biosensors.

Keywords: biosensor; cholesterol; membranes; microscopy; perfringolysin O.

Figure 1

Cholesterol-rich membranes visualized by the Perfringolysin O (PFO)-derived cholesterol biosensors. Cholesterol-rich membranes (red lines) detected or visualized by the PFO-derived cholesterol biosensors are shown. Radioisotope-labeled PFO ^{(Y181A/C459A)} also refered to as PFO* detects cholesterol in the exofacial leaflets of the plasma membrane by incubation at 4 °C. Recombinant fluorophore labled-D4 proteins can visualize cholesterol in the exofacial leaflets of the plasma membrane by incubation at room temperature. BCθ are accumulated in the internal membranes of the multivesicular bodies (MVBs) and recycling compartments in the fixed cells. Cytosolic mCherry-D4H localizes in the inner leaflet of the plasma membrane.

Figure 2

Domain structure of PFO. (A) The PFO monomer with the four domains is indicated in orientation to the membrane. Domain 4 (D4) directly associates to the membrane through the recognition of cholesterol. The scheme is reproduced from [12], Copyright Nature Publishing Group, 2013. (B) The location of the two amino acid residues (Threonine-490 and Leucine-491) in D4 required for cholesterol binding are shown. Loops L1–L3 at the base of D4 insert into the membrane in the cholesterol-dependent manner. The scheme is reproduced from [57] Copyright PNAS, 2010. (C) The bottom view of D4 is shown. Substitution of Aspartic acid-434 to Serine increases the affinity of PFO to cholesterol. The schematic is reproduced from [20], Copyright American Chemical Society, 2012.

Figure 3

Examples of images visualized by the PFO-derived cholesterol biosensors. (A) Cholesterol-rich organelles in the human B-lymphocytes cells were visualized by BCθ using immunoelectron microscopy. (e; exosomes, 4; multivesicular bodies (MVBs)). Bar, 200 nm. The image is reproduced from [62] Copyright John Wiley & Sons, 2003. (B) Recombinant Dronpa-D4 protein visualized by photoactivated localization microscopy (PALM) reveals cholesterol clusters in the exofacial leaflets of the plasma membrane in HeLa cells. Bar, 500 nm. The image is reproduced with permission from [63], Copyright Royal Society of Chemistry, 2011. (C) Cholesterol in the cytosolic leaflets of the plasma membrane is visualized by expression of mCherry-D4H in the cytosol of Raw264.7 cells. GFP-PH (PLC) is used as a plasma membrane marker. Bar, 10 μm.