A comparative study on fluorescent cholesterol analogs as versatile cellular reporters

Erdinc Sezgin, Fatma Betul Can, Falk Schneider, Mathias P. Clausen, Silvia Galiani, Tess A. Stanly, Dominic Waithe, Alexandria Colaco, Alf Honigmann, Daniel Wüstner, Frances Platt, Christian Eggeling

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Abstrakt

Cholesterol 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 cholesterol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently-labeled cholesterol 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 (GUVs) and giant plasma membrane vesicles (GPMVs); 2) cellular trafficking, specifically subcellular localization in Niemann-Pick C (NPC) disease cells; and 3) applicability in fluorescence correlation spectroscopy (FCS) and super-resolution STED-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 cholesterol analogs in visualizing cellular cholesterol dynamics.
OriginalsprogEngelsk
TidsskriftJournal of Lipid Research
Vol/bind57
Sider (fra-til)299-309
ISSN0022-2275
DOI
StatusUdgivet - 2016

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Sezgin, E., Betul Can, F., Schneider, F., Clausen, M. P., Galiani, S., Stanly, T. A., Waithe, D., Colaco, A., Honigmann, A., Wüstner, D., Platt, F., & Eggeling, C. (2016). A comparative study on fluorescent cholesterol analogs as versatile cellular reporters. Journal of Lipid Research, 57, 299-309. https://doi.org/10.1194/jlr.M065326