Showing papers by "Daniel Wüstner published in 2008"

Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells

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

Chromatic aberration correction and deconvolution for UV sensitive imaging of fluorescent sterols in cytoplasmic lipid droplets

Abstract: Intrinsically fluorescent sterols, like dehydroergosterol (DHE), mimic cholesterol closely and are therefore suitable to determine cholesterol transport by fluorescence microscopy. Disadvantages of DHE are its low quantum yield, rapid bleaching, and the fact that its excitation and emission is in the UV region of the spectrum. Thus, one has to deal with chromatic aberration and low signal-to-noise ratio. We developed a method to correct for chromatic aberration between the UV channel and the red/green channel in multicolor imaging of DHE compared with the lipid droplet marker Nile Red in living macrophage foam cells and in adipocytes. We used deconvolution microscopy and developed image segmentation techniques to assess the DHE content of lipid droplets in both cell types in an automated manner. Pulse-chase studies and colocalization analysis were performed to monitor the redistribution of DHE upon adipocyte differentiation. DHE is targeted to transferrin-positive recycling endosomes in preadipocytes but associates with droplets in mature adipocytes. Only in adipocytes but not in foam cells fluorescent sterol was confined to the droplet-limiting membrane. We developed an approach to visualize and quantify sterol content of lipid droplets in living cells with potential for automated high content screening of cellular sterol transport.