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.

Potential of ultraviolet wide‐field imaging and multiphoton microscopy for analysis of dehydroergosterol in cellular membranes

“…This value can be calculated from s 5 ln 2/k, with k being the fitted decay rate constant in the monoexponential model. This is very close to the value we determined previously with the same type of analysis from WF timelapse sequences of DHE-stained cells (Wü stner and Faergeman, 2008b). In those experiments, we also observed a large immobile fraction of sterol in the plasma membrane [i.e., a monoexponential fit of the form given earlier shows that temporal correlation leveled off to a value of R(t ?…”

“…In those experiments, we also observed a large immobile fraction of sterol in the plasma membrane [i.e., a monoexponential fit of the form given earlier shows that temporal correlation leveled off to a value of R(t ? 1) 5 b 5 0.77 (Wü stner and Faergeman, 2008b)]. Note that the time resolution of this experiment is !1 min, whereas that of the sequence used in the N&B analysis (stated earlier) was much higher (around 2 s between frames).…”

“…For analysis of plasma membrane staining patterns of DHE as measured by MP microscopy, intensity line scans were extracted from the cell perimeter as described (Wü stner and Faergeman, 2008b). From these intensity profiles, spatial and temporal correlations were calculated separately.…”

“…Note that we focused at the perimeter of the cell for this experiment to distinguish between sterol membrane dynamics and movement of DHE-containing vesicles. For data analysis, we used our previously developed protocol (Wü stner, 2008;Wü stner and Faergeman, 2008b) and extracted intensity profiles along the plasma membrane over the complete time-lapse sequence (Figs. 5A and 5B).…”

“…Temporal correlation of the time-dependent fluorescence intensity profiles of DHE-stained plasma membrane was calculated by a bivariate correlation analysis implemented in SigmaPlot 9.0 (SPSS, Chicago, IL) defining the correlation coefficient R as a function of time, t, relative to the first intensity pattern at t 5 t 0 , as described (Wü stner and Faergeman, 2008b …”