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

Abstract

BCθ is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin θ (θ-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, BCθ can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCθ 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 BCθ. Under this condition, BCθ mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCθ staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to θ-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCθ staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin. Thus, BCθ is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.