The use of anthrolysin O and ostreolysin A to study cholesterol in cell membranes

Kristen A. Johnson, Arun Radhakrishnan

Research output: Chapter in Book/Report/Conference proceedingChapter

8 Scopus citations


Cholesterol is a major component of the plasma membranes (PMs) of animal cells, comprising 35–40 mol% of total PM lipids. Recent studies using cholesterol-binding bacterial toxins such as domain 4 of Anthrolysin O (ALOD4) and fungal toxins such as Ostreolysin A (OlyA) have revealed new insights into the organization of PM cholesterol. These studies have defined three distinct pools of PM cholesterol—a fixed pool that is essential for membrane integrity, a sphingomyelin (SM)-sequestered pool that can be detected by OlyA, and a third pool that is accessible and can be detected by ALOD4. Accessible cholesterol is available to interact with proteins and transport to the endoplasmic reticulum (ER), and controls many cellular signaling processes including cholesterol homeostasis, Hedgehog signaling, and bacterial and viral infection. Here, we provide detailed descriptions for the use of ALOD4 and OlyA, both of which are soluble and non-lytic proteins, to study cholesterol organization in the PMs of animal cells. Furthermore, we describe two new versions of ALOD4 that we have developed to increase the versatility of this probe in cellular studies. One is a dual His6 and FLAG epitope-tagged version and the other is a fluorescent version where ALOD4 is fused to Neon, a monomeric fluorescent protein. These new forms of ALOD4 together with previously described OlyA provide an expanded collection of tools to sense, visualize, and modulate levels of accessible and SM-sequestered cholesterol on PMs and study the role of these cholesterol pools in diverse membrane signaling events.

Original languageEnglish (US)
Title of host publicationPore-Forming Toxins
EditorsAlejandro P. Heuck
PublisherAcademic Press Inc.
Number of pages24
ISBN (Print)9780128238585
StatePublished - Jan 2021

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988


  • Cholesterol sensors
  • Cholesterol trafficking
  • Cholesterol-binding toxins
  • Endoplasmic reticulum
  • Fluorescence imaging of cholesterol pools
  • Plasma membrane cholesterol pools

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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