Analysis of Cholesterol Trafficking with Fluorescent Probes

Abstract

Cholesterol plays an important role in determining the biophysical properties of biological membranes, and its concentration is tightly controlled by homeostatic processes. The intracellular transport of cholesterol among organelles is a key part of the homeostatic mechanism, but sterol transport processes are not well understood. Fluorescence microscopy is a valuable tool for studying intracellular transport processes, but this method can be challenging for lipid molecules because addition of a fluorophore may alter the properties of the molecule greatly. We discuss the use of fluorescent molecules that can bind to cholesterol to reveal its distribution in cells. We also discuss the use of intrinsically fluorescent sterols that closely mimic cholesterol, as well as some minimally modified fluorophore-labeled sterols. Methods for imaging these sterols by conventional fluorescence microscopy and by multiphoton microscopy are described. Some label-free methods for imaging cholesterol itself are also discussed briefly.

A comparative study on fluorescent cholesterol analogs as versatile cellular reporters

“…However, we kept them out of the scope of this study due to their fairly poor photo-physical properties (such as UV absorption and fl uorescence, low quantum yield, high photobleaching). Such properties make UV-optimized optics in the excitation path of a conventional wide fi eld microscope or multiphoton excitation and UV-sensitive detectors (which are all not easily available in cell biological laboratories) requirements for their visualization ( 80 ). In addition, their low brightness and high bleaching propensity make DHE and CTL unsuitable for single molecule studies or STED-based experiments, which were some important criteria we assessed in our analysis.…”

A novel intrinsically fluorescent probe for study of uptake and trafficking of 25-hydroxycholesterol

“…An alternative approach takes advantage of intrinsically fl uorescent sterols, including the naturally occurring cholesterol analog, dehydroergosterol (DHE) ( 32 ), or a synthetic sterol, CTL ( 46 ). These fl uorescent sterols are similar to cholesterol in biophysical behavior and structure ( 18,39 ), but include two additional double bonds in the steroid ring system that are responsible for their fl uorescent properties.…”

“…1B ). Because CTL has previously been shown to mimic the membrane behavior of cholesterol ( 38,39 ), we reasoned that the 25-HCTL probe similarly would be a faithful mimic of 25-HC in model membrane studies.…”

Lipofuscin Accumulation into and Clearance from Retinal Pigment Epithelium Lysosomes: Physiopathology and Emerging Therapeutics

“…Furthermore, sulfo-butyl-ether-βCD (Captisol®), a βCD derivative that cannot form stacked dimers and that therefore cannot solubilize membrane cholesterol, can still reduce lysosomal buildups [120]. The model for βCD-mediated removal of lysosomal cholesterol proposes that [124,125]. In the absence of extracellular cholesterol acceptor molecules, the stoichiometric analysis of βCD clearance provides no evidence of cholesterol release to the media but rather indicates a rapid metabolic processing within the cytosolic compartment [126,127].…”

“…The model for βCD-mediated removal of lysosomal cholesterol proposes that βCD enters lysosomes by endocytosis, where it binds free cholesterol in the lumen and shutle it to the limiting lysosomal membrane [121,122]. From there, cholesterol is transferred, by a not fully characterized traicking machinery, that probably involves points of membrane contact between organelles and cholesterol binding proteins [123], to the ER, plasma membrane, peroxisomes, and mitochondria [124,125]. In the absence of extracellular cholesterol acceptor molecules, the stoichiometric analysis of βCD clearance provides no evidence of cholesterol release to the media but rather indicates a rapid metabolic processing within the cytosolic compartment [126,127].…”