Use of dansyl-cholestanol as a probe of cholesterol behavior in membranes of living cells

Abstract

While plasma membrane cholesterol-rich microdomains play a role in cholesterol trafficking, little is known about the appearance and dynamics of cholesterol through these domains in living cells. The fluorescent cholesterol analog 6-dansyl-cholestanol (DChol), its biochemical fractionation, and confocal imaging of L-cell fibroblasts contributed the following new insights: i) fluorescence properties of DChol were sensitive to microenvironment polarity and mobility; (ii) DChol taken up by L-cell fibroblasts was distributed similarly as cholesterol and preferentially into cholesterol-rich vs. -poor microdomains resolved by affinity chromatography of purified plasma membranes; iii) DChol reported similar polarity (dielectric constant near 18) but higher mobility near phospholipid polar head group region for cholesterol in purified cholesterol-rich versus -poor microdomains; and iv) real-time confocal imaging, quantitative colocalization analysis, and fluorescence resonance energy transfer with cholesterol-rich and -poor microdomain markers confirmed that DChol preferentially localized in plasma membrane cholesterol-rich microdomains of living cells. Thus, DChol sensed a unique, relatively more mobile microenvironment for cholesterol in plasma membrane cholesterol-rich microdomains, consistent with the known, more rapid exchange dynamics of cholesterol from cholesterol-rich than -poor microdomains.

Fig. 1.

Structure and fluorescence properties of DChol. A: Structure of DChol. The properties of DChol in solvents are shown in B–H as follows. B: Excitation and emission spectra of DChol in ethanol (5 μg/ml). Effect of solvent polarity (100% to 0% dioxane in water) on DChol (5 μg/ml) excitation and emission maximum (C), stokes shift (D), fluorescence excitation intensity at maximum (E), fluorescence emission intensity at maximum (F), polarization changes (G), and light scattering (H). The properties of DChol in LUV are shown in I and J. LUV (POPC:total sterol = 65:35) were prepared with increasing DChol (0.1–10% of total sterol). I: Maximal fluorescence emission intensity (excitation at 336 nm) of DChol in LUV with increasing amount of DChol. J: Fluorescence polarization of DChol (excitation at 336 nm; emission at 522 nm) of DChol in LUV with increasing of DChol.

Fig. 2.

Colocalization of DChol with different microdomain markers in living cells. L-cells were double labeled with DChol and another membrane domain marker, which were then simultaneously imaged through separate photomultipliers as described in Methods. Images of DChol are shown in green in B, F, J, and N. The images of membrane domain markers are shown in red: Alexa Fluor 594 CT-B (A), DiD (E), BCθ (I), and N-Rh-DOPE (M). The superposition of red and green images are shown in C, G, K, and O, and the yellow colocalized pixels are shown in D, H, L, and P. Changes in DChol uptake into cholesterol-rich and -poor microdomains with time were followed by measuring the ratio dansyl fluorescence (cholesterol-rich/-poor microdomains) with time based on DChol colocalization with Alexa Flour 594 CT-B as shown in Q. ***, Significantly different from 6 min (P < 0.001); #, significantly different from 10 min (P < 0.05). R: DChol fluorescence distribution (cholesterol-rich/-poor microdomains) with time based on colocalization with DiD. *, Significantly different from 10 min (*, P < 0.05, ***, P < 0.001); #, significantly different from 15 min (P < 0.05).

Fig. 3.

FRET between DChol and cholesterol-rich microdomain markers DHE, DiD, and BCθ. A and B: FRET between DChol and DHE in LUVs. A: Emission spectra (325–575 nm) of LUVs containing 5% DHE and increasing amount of DChol. The spectra were recorded at 37°C with 300 nm excitation. B: The energy transfer efficiency E% as a function of increasing DChol (mol%). Open circles, E% was calculated from experimental data by E = 1 − I_DA/I_D, where I_DA and I_D were donor DHE fluorescence intensity at 373 nm in the presence and absence of acceptor DChol, respectively; lines, E% was calculated using a mathematical model of random distribution of DChol relative to DHE in LUV bilayer as described in Methods. Solid line, R = 15.5; short dashed line, R = 19.1; long dashed line, R = 13.1. C–H: FRET between DChol and DHE in living cells by multi-photon imaging. Images were taken with multi-photon excitation using a 900 nm laser and a D375/50 (350–400 nm) emission filter for DHE (C, E), BGG22 (410–490 nm) emission filter for DChol (D, F). C: DHE emission when L-cells were incubated with DHE only. D: DChol emission when L-cells were incubated with DChol only. E: DHE emission when L-cells were incubated with both DHE and DChol. F: DChol emission when L-cells were incubated with both DHE and DChol. G: Average fluorescence intensity of whole cells (mean ± SE, n = 16–24). H: Average fluorescence intensity of PM (mean ± SE, n = 16–24). **, P < 0.01 t-test, significantly different from DHE only for DHE emission and significantly different from DChol only for DChol emission. FRET between DChol and DiD (I–K) and BCθ (L–N). The excitation laser 408 nm was chosen to only excite the donor DChol and emission filter 680/32 was used to only detect the emission from acceptor DiD and BCθ. Upper panels show FRET between DChol (donor) and DiD (acceptor). I: Image of cells labeled with donor DChol only. J: Image of cells labeled with acceptor DiD only. K: Cells were labeled with both DChol and DiD. Lower panels show FRET between DChol (donor) and Alexa Fluor 660 BCθ (acceptor). L: DChol only. M: Alexa Fluor 660 BCθ only. N: DChol plus Alexa Fluor 660 BCθ.

Fig. 4.

Real-time confocal imaging of DChol uptake through cholesterol-rich and -poor microdomains of L-cell fibroblasts. Cells were first labeled with Alexa Fluor CT-B as described in Methods, then DChol-MβCD (DChol concentration 10 μg/ml) was added to the cells in PBS and fluorescence images were acquired continuously for the first 15 min at room temperature. A: Average DChol fluorescence in the whole cell (solid circles), in the plasma membrane (PM, solid inverted triangles), and intracellular regions (solid squares). B: Average DChol fluorescence in the PM that colocalized with the cholesterol-rich microdomain marker Alexa Fluor CT-B (solid triangles) and not colocalized with Alexa Fluor CT-B (open triangles). Images of DChol was obtained with 408 nm excitation and HQ530/40 emission filter, images of Alexa Fluor CT-B were obtained with 568 nm excitation and HQ598/40 emission filter. Data were presented as mean ± SE (n = 25).