Abstract
Cholesterol (Chol) is a crucial component of cellular membranes, but knowledge of its intracellular dynamics is scarce. Thus, it is of utmost interest to develop tools for visualization of Chol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently labeled Chol analogs. Unfortunately, the introduction of the label may influence the characteristics of the analog, such as its localization, interaction, and trafficking in cells; hence, it is important to get knowledge of such bias. In this report, we compared different fluorescent lipid analogs for their performance in cellular assays: 1) plasma membrane incorporation, specifically the preference for more ordered membrane environments in phase-separated giant unilamellar vesicles and giant plasma membrane vesicles; 2) cellular trafficking, specifically subcellular localization in Niemann-Pick type C disease cells; and 3) applicability in fluorescence correlation spectroscopy (FCS)-based and super-resolution stimulated emission depletion-FCS-based measurements of membrane diffusion dynamics. The analogs exhibited strong differences, with some indicating positive performance in the membrane-based experiments and others in the intracellular trafficking assay. However, none showed positive performance in all assays. Our results constitute a concise guide for the careful use of fluorescent Chol analogs in visualizing cellular Chol dynamics.
Highlights
Cholesterol (Chol) is a crucial component of cellular membranes, but knowledge of its intracellular dynamics is scarce
We used the following fluorescent Chol analogs for their performance in three different assays testing their potential to measure membrane and intracellular trafficking dynamics: the Chol binding fluorescent dye, filipin III [12, 39], and Chol analogs tagged with the organic dye, dansyl (DChol labeled at the steroid backbone) [40], NBD (22-NBD-Chol labeled at position 22 of the side alkyl chain, 25-NBD-Chol labeled at position 25 of the side alkyl chain, 3-C6-NBD-Chol and 3-C12-NBD-Chol, both labeled at position 3 via a C6- and C12linker, respectively), BODIPY-derived TopFluor cholesterol (TF-Chol), AbberiorStar512 (Star512-PEG-Chol) and KK114 [27] (KK114-PEG-Chol) labeled at position 3 via a PEG2000linker
Labeling at the alkyl chain is usually preferred over attachment of the dye to the 3-OH group, because it has been indicated that the respective –OH group may play important roles in Chol functionality and interactions [41]
Summary
Cholesterol (Chol) is a crucial component of cellular membranes, but knowledge of its intracellular dynamics is scarce. It is of utmost interest to develop tools for visualization of Chol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently labeled Chol analogs. We compared different fluorescent lipid analogs for their performance in cellular assays: 1) plasma membrane incorporation, the preference for more ordered membrane environments in phase-separated giant unilamellar vesicles and giant plasma membrane vesicles; 2) cellular trafficking, subcellular localization in Niemann-Pick type C disease cells; and 3) applicability in fluorescence correlation spectroscopy (FCS)-based and super-resolution stimulated emission depletion-FCS-based measurements of membrane diffusion dynamics. It is essential to visualize cellular Chol organization and to measure dynamics (such as uptake, diffusion, trafficking, and localization) with high specificity and sensitivity.
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