Figure 1.
Cholesterol oxidation decreases membrane order.
A) Structures of cholesterol and cholestenone. B) HDFs were treated at 37°C with coase (10 U/ml; 1 h), MBCD (0.5 mM; 5 min or 5 mM; 1 h) or buffer only (ctrl). Cells were labeled with Laurdan, fixed, imaged with a 2-photon microscope and GP images were generated. C) Average GP values calculated from the Laurdan GP data represent three independent experiments; n = 59 fields, >300 cells per condition; ***P<0.0005. The fraction of cholesterol remaining in cells after the treatments is indicated. D) The order parameters |SCD|, from MD simulations of the palmitoyl tail of POPC molecules in various lipid bilayers. The liquid-ordered (Lo) systems were POPC/PSM/cholesterol/cholestenone 2∶1∶1∶0, POPC/PSM/cholesterol/cholestenone 2∶1∶0.75∶0.25, POPC/PSM/cholesterol/cholestenone 2∶1∶0∶1, and the liquid-disordered (Ld) systems were with 1 chol in a POPC bilayer and 1 cholestenone in a POPC bilayer. Carbon numbering starts from carbons close to the glycerol group.
Figure 2.
Transbilayer mobility of cholestenone and cholesterol.
A) Snapshots showing the spontaneous flip-flop motion of a cholestenone molecule during MD simulation in the raft-like system POPC/PSM/cholesterol/cholestenone 2∶1∶0.75∶0.25. POPC and PSM are shown as green and grey lines, respectively, with head-group phosphorus atoms as spheres. Cholesterol (chol) and cholestenone (cnone) are shown as magenta and blue ball-and-stick representation, respectively. The particular cholestenone undergoing flip-flop is highlighted as red van der Waals spheres and its 3-keto oxygen atom is shown in yellow. B) Time scale and frequency of flip-flops by cholesterol and cholestenone. Time evolution of the Z-coordinate of the head group oxygen atom of cholesterol (i) and cholestenone (ii) along the bilayer normal in the system POPC/PSM/chol/cholestenone 2∶1∶0.8∶0.2. The Y-axis zero indicates the center of the bilayer. Different colors show different cholesterol or cholestenone molecules. The figures highlight how different the flip-flop rates of cholesterol and cholestenone are: while cholesterol undergoes only a single quick flip-flop event, cholestenone undergoes a large number of flip-flops during the same period. Yet the concentration of cholesterol was four times higher than that of cholestenone. C) The potential of mean forces (kJ/mol) of cholesterol and cholestenone in a variety of lipid bilayer models (shown by dark lines), either in the liquid-disordered (Ld) or liquid-ordered (Lo) phase. The transparent area following each PMF curve represents the corresponding statistical errors.
Table 1.
Average number of hydrogen bondsa (H-bonds) and charge pairsb formed by steroids with the surrounding lipids and water.
Table 2.
Thermodynamics and kinetics associated with cholesterol/cholestenone flip-flop and desorption.
Figure 3.
Efflux of cholestenone and cholesterol from HDFs to extracellular acceptors.
A) HDFs prelabeled with [14C]cholesterol were treated with coase (10 U/ml; 1 h), MBCD (0.5 mM; 5 min), or mock treated (ctrl). Efflux of radiolabeled lipids was analyzed after a 4 h chase in serum-free buffer containing B) ApoA-I (10 µg/ml); n = 4 or C) BSA (0.2%); n = 4–6. *P<0.05; **P<0.005; ***P<0.0005.
Figure 4.
Intracellular trafficking of cholesterol and cholestenone.
A) RAW 264.7 macrophages prelabeled with [14C]cholesterol for 18 h were treated with 10 U/ml coase for 2 h. The post-nuclear supernatant was subjected to sucrose gradient fractionation. A total of 7 fractions was collected and analysed for radiolabeled [14C]cholesterol, -cholestenone, and -cholesteryl ester content. Inset: Percentage of [14C]cholesterol and -cholestenone in the lowest density fraction 1; n = 4. *P<0.05. B) Structures of Bpy-cholesterol (Bpy-chol) and Bpy-cholestenone (Bpy-cnone). C) HDFs were pulse labeled with the indicated Bpy-lipids for 8 min, and the cells were imaged by confocal microscopy during 2 h of chase. To visualize LDs, cells were pulse labeled for 1 h with Bpy568-C12 (5 µM) and Bpy-lipid (1 µM), and imaged after a 1 h chase. Scale bars 10 µm. D) HDFs were pulse labeled with Bpy-lipids and analysed for Bpy fluorescence intensity immediately after the pulse and after 4 h of chase in DMEM, 10% FBS; n = 8; ***P<0.0005.
Figure 5.
Coase treatment or cholestenone impairs fibroblast migration in a wound-healing assay.
A) Confluent HDFs were treated with coase (10 U/ml; 1 h), MBCD (0.5 mM; 5 min), or mock treated (ctrl) and collected for lipid extraction at the indicated time points. Cholesterol and cholestenone amounts were determined; n = 8–16. B) HDFs were treated as in A, and wounded with a pipette tip. Cells were fixed at the indicated time points, stained with Alexa568-phalloidin and imaged. Scale bars 20 µm. C) Cell migration area was quantified at 22 h post treatments; n = 11–15 fields. D) HDFs were incubated with MBCD-cholestenone (MB-CN) complex, collected for lipid analysis and cholesterol and cholestenone amounts were determined; n = 6–12. E) HDFs were treated with MB-CN complex as in D, wounded with a pipette tip and incubated in serum-free buffer for 22 h. Cells were stained with Alexa568-phalloidin and the wound area was imaged.
Figure 6.
Defective lamellipodial spreading of migrating coase treated cells.
A) HDFs were treated, wounded and stained with phalloidin as in Figure 5. B) The width of the lamellipodium was measured from control, MBCD and coase treated cells. Data represent 4 biological replicates per condition; n = 172–228 cells. C) Cells were fixed at 3 h post wounding and stained with fluorescent phalloidin, and anti-Arp2/3 complex component ARPC2 antibody, followed by secondary fluorescent antibody and imaging by confocal microscopy. Please note comparable image acquisition and display in Arp2/3 panels; inset with increased brightness demonstrates the largely cytoplasmic staining pattern of Arp2/3 in coase treated cells. Scale bars, 10 µm.
Table 3.
Lipid compositions for all the bilayersa used in the present work.