Fig 1.
CCPs internalisation into PC LUVs.
Time-dependent percent of total fluorescence evolution. (A) PC LUVs were incubated with NBD-labelled CPPs for 2.5 hours at 35°C. (B) Quantification of CPPs penetration into LUVs composed of PC and PC/SM/Chol. Means ± SEM of 3 experiments. * Indicates that the P value for RW9 was from 0.02 to 0.04 when comparing RW9 with penetratin and R9 (unpaired t-test). (C) PC LUVs incubated with NBD-labelled CPPs for 5 minutes at 50°C. In A and C dithionite was added (arrow head) to quench the CPPS in solution. After stabilization, melittin was added (two arrow heads) allowing the CPP quenching inside the LUVs. The difference of fluorescence before and after melittin is the percent of internalized peptide (Zoom). Penetratin green, R9 blue and RW9 red.
Fig 2.
CPPs effect on cholesterol-pyrene multimers in function of temperature on PC LUVs.
The excimers/isoemisive ratio (474/432 nm) was followed at different temperatures during heating (A, continuous lines), or cooling (B, dotted lines) of the samples. CPPs were incubated with the LUVs at a 1/25 P/L ratio. Control CPP free LUVs (black ●,○), Penetratin (green ♦,◊), R9 (blue ▲,∆) and RW9 (red ■,□). Means ± SEM of 5 to 8 independent experiments. The stars in colour correspond to their respective experimental point colours compared to the control LUVs; * P<0.05, ** P<0.01, *** P<0.001 by unpaired t-test.
Fig 3.
CPPs effects on the cholesterol-pyrene environment in function of temperature on PC LUVs.
(A) Liquid disordered contribution (cPyD9) in PC LUVs during cooling in the presence of R9 (blue ∆) and RW9 (red □). (B) Lo/Ld ratio (cPyO3/cPyD9) in PC LUVs during heating in the absence (black ●) or the presence of R9 (blue ▲) at P/L ratio 1/25. Means ± SEM of 5 to 7 independent experiments. * P<0.05 by unpaired t-test.
Fig 4.
CPPs effect on cholesterol-pyrene multimers in function of temperature on SM/Chol LUVs.
The excimers/iso-emissive ratio (474/432 nm) was followed during heating (A, continuous lines), or cooling (B, dotted lines). CPPs were incubated with the LUVs at 1/10 P/L ratio. Control CPP free LUVs (black ●,○), Penetratin (green ♦,◊), R9 (blue ▲,∆) and RW9 (red ■,□). Means ± SEM of 3 to 9 independent experiments. The stars in colour correspond to their respective experimental point colours compared to the control LUVs; * P<0.05, ** P<0.01 by unpaired t-test.
Table 1.
Peptide effects on cholesterol-pyrene fluorescence ratios in different membranes.
Fig 5.
CPPs effect on cholesterol-pyrene fluorescence anisotropy.
SM/Chol membranes were incubated at 35°C with R9 in blue or RW9 in red. Control peptide free LUVs are in Black. Means ± SEM of 3 independent experiments. Unpaired t-test was performed for 390, 400, 410 and 420 nm. The stars correspond to the comparison of RW9 and control LUVs; * P<0.05, ** P<0.01.
Fig 6.
Confocal images of GPMVs labelled with di-4-ANEPPDHQ and incubated with CPPs.
The 570-590 nm range corresponds to the ordered membrane contribution and the 620-640 nm range to the disordered membrane contribution. GP was calculated as explained in the methods section. (A) GPMVs incubated with R9 (top) and RW9 (bottom) during 190 min. (B) Peptide-free GPMV (top) and incubated with RW16 (bottom) during 60 min. Bars represent 10 μm.
Fig 7.
Time evolution of GP histograms of di-4-ANEPPDHQ-labelled PMS incubated with CPPs.
Pixel GP distribution of PMS incubated without peptide (A), with R9 (B), RW9 (C) and RW16 (D). The incubation times with or without peptides are 5-20 min in black, 20-90 min in red 90-140 min green and 140-200 min in blue. Distributions are means ± SEM of 3 to 10 GPMVs.
Fig 8.
Schematic representation of cholesterol-pyrene movements in the membrane.
A cell membrane is composed of different domains of disordered (white) and ordered (grey) character. In a starting condition (S), the cholesterol-pyrene probe (black bars) is distributed in all domains but enriched in Lo domains in which it is frequently present as dimers (multimers). If cholesterol-pyrene dimers dissociate inside the Lo domain (A), the new monomeric species would increase the Lo spectral signal contribution and consequently the Lo/Ld ratio. If the cholesterol-pyrene newly formed monomers move to an Ld domain the Ld spectral contribution increases and the Lo/Ld ratio diminishes (B). The presented experimental data suggest that R9 effect on PC membranes is well represented by the A hypothesis and that the cholesterol reorganisation induced by RW9 rather follows the hypothesis B.