Abstract
Cholesterol distribution in the cell is maintained by both vesicular and non-vesicular sterol transport. Non-vesicular transport is mediated by the interaction of membrane-embedded cholesterol and water-soluble proteins. Small changes to the lipid composition of the membrane that do not change the total cholesterol content, can significantly affect how cholesterol interacts with other molecules at the surface of the membrane. The cholesterol-dependent cytolysin Perfringolysin O (PFO) constitutes a powerful tool to detect cholesterol in membranes, and the use of PFO-based probes has flourished in recent years. By using a non-lytic PFO derivative, we showed that the sensitivity of the probes for cholesterol can be tuned by modifications introduced directly in the membrane-interacting loops and/or by modifying residues away from the membrane-interacting domain. Through the use of these biosensors on live RAW 264.7 cells, we found that changes in the overall cholesterol content have a limited effect on the average cholesterol accessibility at the surface of the membrane. We showed that these exquisite biosensors report on changes in cholesterol reactivity at the membrane surface independently of the overall cholesterol content in the membrane.
Highlights
Different models have been proposed to explain the mechanism of cholesterol interaction with other lipids and the reactivity of cholesterol in model membranes, such as the condensed-complex model, the super lattice model, or the umbrella model[10]
Our pioneering work with this protein has shown that Perfringolysin O (PFO) binding relies on the exposure of cholesterol at the surface of the membrane, and that PFO is a good reporter of cholesterol accessibility on model membranes and mammalian cells[12,13,24,25]
We have previously shown that cholesterol exposure at the membrane surface is required and sufficient to trigger PFO binding and all the structural re-arrangements that lead to oligomerization[12,13,24]
Summary
Different models have been proposed to explain the mechanism of cholesterol interaction with other lipids and the reactivity of cholesterol in model membranes, such as the condensed-complex model, the super lattice model, or the umbrella model[10]. Changes in the phospholipid composition of a membrane at a constant cholesterol concentration (e.g., addition of double bonds or removal of head groups by phospholipases or sphingomyelinases) increase the cholesterol reactivity with water-soluble molecules[5,12,13,14] While these conclusions have been mostly obtained using model membrane systems, the same effects have been shown using cellular systems[7,15,16]. Variations in the phospholipid content by hydrolysis of phospholipid head groups or incorporation of double bonds in the acyl chains do not alter the fluorescent signal of filipin or cholesterol analogs (because cholesterol concentration remains unchanged) These changes in lipid composition affect cholesterol accessibility[12,13,14]. Multiple factors can affect the interaction of PFO with membranes, and detailed studies on this protein-membrane interactions are needed to properly interpret and compare the results obtained with these emerging palette of cholesterol biosensors
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