TY - CHAP
T1 - The use of anthrolysin O and ostreolysin A to study cholesterol in cell membranes
AU - Johnson, Kristen A.
AU - Radhakrishnan, Arun
N1 - Publisher Copyright:
© 2021 Elsevier Inc.
PY - 2021/1
Y1 - 2021/1
N2 - 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.
AB - 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.
KW - Cholesterol sensors
KW - Cholesterol trafficking
KW - Cholesterol-binding toxins
KW - Endoplasmic reticulum
KW - Fluorescence imaging of cholesterol pools
KW - Plasma membrane cholesterol pools
UR - http://www.scopus.com/inward/record.url?scp=85101370232&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85101370232&partnerID=8YFLogxK
U2 - 10.1016/bs.mie.2021.01.011
DO - 10.1016/bs.mie.2021.01.011
M3 - Chapter
C2 - 33712199
AN - SCOPUS:85101370232
SN - 9780128238585
T3 - Methods in Enzymology
SP - 543
EP - 566
BT - Pore-Forming Toxins
A2 - Heuck, Alejandro P.
PB - Academic Press Inc.
ER -