Immunoelectron Microscopic Localization of Cholesterol Using Biotinylated and Non-cytolytic Perfringolysin O

TLDR

The study aimed to develop a fixation and immunolabeling protocol that preserves membrane integrity and prevents cholesterol loss during thin‑section preparation. The authors employed a proteolytically modified, biotinylated perfringolysin O derivative to label cholesterol‑rich membranes in cryosections of cultured human lymphoblastoid cells, using high‑pressure freezing and fixation during thawing to preserve ultrastructure. Cholesterol labeling was strongest at the plasma membrane and filopodium‑like processes, also present on multivesicular body vesicles and exosomes, with occasional positivity of tubulovesicular elements near endosomes and Golgi, while methyl‑β‑cyclodextrin treatment abolished labeling, demonstrating that the modified perfringolysin O combined with improved fixation and high‑pressure freezing effectively localizes membrane cholesterol in ultrathin cryosections.

Abstract

We used a proteolytically modified and biotinylated derivative of the cholesterol-binding θ-toxin (perfringolysin O) to localize cholesterol-rich membranes in cryosections of cultured human lymphoblastoid cells (RN) by electron microscopy. We developed a fixation and immunolabeling procedure to improve the preservation of membranes and minimize the extraction and dislocalization of cholesterol on thin sections. We also labeled the surface of living cells and applied high-pressure freezing and subsequent fixation of cryosections during thawing. Cholesterol labeling was found at the plasma membrane, with strongest labeling on filopodium-like processes. Strong labeling was also associated with internal vesicles of multivesicular bodies (MVBs) and similar vesicles at the cell surface after secretion (exosomes). Tubulovesicular elements in close vicinity of endosomes and the Golgi complex were often positive as well, but the surrounding membrane of MVBs and the Golgi cisternae appeared mostly negative. Treatment of cells with methyl-β-cyclodextrin completely abolished the labeling for cholesterol. Our results show that the θ-toxin derivative, when used in combination with improved fixation and high-pressure freezing, represents a useful tool for the localization of membrane cholesterol in ultrathin cryosections.