Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells |
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Authors: | Daniel Wüstner Nils J Færgeman |
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Institution: | (1) Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark |
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Abstract: | Distribution and dynamics of cholesterol in the plasma membrane as well as internalization pathways for sterol from the cell
surface are of great cell biological interest. Here, UV-sensitive wide field microscopy of the intrinsically fluorescent sterols,
dehydroergosterol (DHE) and cholestatrienol (CTL) combined with advanced image analysis were used to study spatiotemporal
sterol distribution in living macrophages, adipocytes and fibroblasts. Sterol endocytosis was directly visualized by time-lapse
imaging and noise-robust tracking revealing confined motion of DHE containing vesicles in close proximity to the cell membrane.
Spatial surface intensity patterns of DHE as well as that of the lipid marker DiIC12 being assessed by statistical image analysis
persisted over several minutes in cells having a constant overall curvature. Sites of sterol endocytosis appeared indistinguishable
from other regions of the cell surface, and endocytosis contributed by 62% to total sterol uptake in J774 cells. DHE co-localized
with fluorescent transferrin (Tf) in vesicles right after onset of endocytosis and in deepened surface patches of energy depleted
cells. Surface caveolae labeled with GFP-tagged caveolin were not particularly enriched in DHE or CTL. Some sterol co-localized
with internalized caveolin suggesting that caveolar endocytosis contributes to vesicular sterol uptake. These findings demonstrate
that plasma membrane sterol is internalized by several endocytic pathways. Sterol endocytosis does not require formation of
microscopically resolvable sterol clusters or enrichment of sterol in surface caveolae.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. |
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Keywords: | Cholesterol Membrane Fluorescence Time-lapse Tracking Wavelet Fourier Caveolin |
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