Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells

Shigeki Sugii,Patrick C Reid,Nobutaka Ohgami,Yukiko Shimada,Robert A Maue,Haruaki Ninomiya,Yoshiko Ohno-Iwashita,Ta-Yuan Chang

doi:10.1194/jlr.d200036-jlr200

Abstract

BCtheta is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (theta-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCtheta can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCtheta signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCtheta. Under this condition, BCtheta mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCtheta staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to beta-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCtheta staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin. Thus, BCtheta is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.

Highlights

BC␪ is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (␪-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM)
Due to its lack of hemolytic activity and its relatively large molecular weight, previous studies have focused on using BC␪ for labeling cholesterol-rich microdomains at the PM of intact cells [27, 43]
We used the RA cells and CT cells unfixed or fixed with low concentrations of PFA (1%), incubated them with BC␪ followed by secondary staining using a fluorescent streptavidin, viewed cells under a fluorescent microscope with confocal and differential interference contract (DIC) imaging capabilities

Summary

Introduction

BC␪ is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (␪-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). B M1 is defined as the region of fluorescence intensity that contains less than 0.1% of control cells (no BC␪ incubation, with streptavidin only) for each staining method.

Results

Conclusion