13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
Membrane permeabilization and shape transition: role of asymmetry and depth of modification
Stéphanie Bonneau1, Julien Heuvingh2, Halina Mojzisova1, Daniel Brault1. 1 FRE
3207, 2 UMR 7636
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
Unsatturated lipids in mecanisms of photodynamic processes • Targets of oxidative modification • Lipid hydroperoxides = important non-radical intermediates of the peroxidative process • Oxidated lipids can mediate signaling cascades which either fortify antioxidant defenses of cells or evoke apoptotic death if oxidative pressure is too great.
PhotoDynamic Therapy (PDT) Photochemical Internalization (PCI)
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
Models of lipids membranes Lipidic unilamellar vesicles
• Water permeable, impermeable to solutes
GUV: • Volume fixed by osmolarity • Surface area fixed by the number of lipids • Optical contrast due to the difference of inner and external medium
SUV (TEM)
Used for spectroscopy
5nm
PS
5 à 20 µm
Sucrose NaCl Glucose NaCl
GUV (PCM)
Used for microscopy
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
mTHPC Foscan ®
Chlorine e6
TPCS2a
Φ∆ Three chlorin-macrocycles
Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009
Ce6
0,63
TPCS2a
0,66
m-THPC
0,68
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
Photo-induced peroxidation Methyl linoleate (ML) → conjugated diene (abs. 234 nm)
In solution Within the membrane
Proportional to Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009
m-THPC >TPCS2a>Ce6
DPPC SUV
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
τ= 3ns
τ= 35 µs D~250nm 1O
Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009
2
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
Photo-induced permeabilization
DOPC-SUV
TPCS2a>Ce6>>m-THPC Photo-oxidation and permeabilization are not proportional Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
11O O22
11 1O O O222
1 O
2
Effects induced by charges ? By the depth within the membrane ? Perturbation of the membrane symmetry ? Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009
1O
2
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
Photo-inducing membrane asymmetry Asymmetrical perturbation of the membrane: PS incubated with GUVs → External leaflet labelled
Ce6
PS added to the lipids during the preparation of the GUVs → Both leaflets labelled
DOPC-GUV
+ Rincing / centrifugation → Inner leaflet labelled
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
Chlorin within the inner leaflet
10 µm Speed 7x
Ref.: J. Heuvingh & S. Bonneau; Biophys. J.; accepted
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
Shape transitions (Inner leaflet targeted) Pear shape
Prolate vesicle
10 µm
2 seconds / picture
Outward budding Budding Pear shape
1 second / picture
Outward budding 10 µm
Ref.: J. Heuvingh & S. Bonneau; Biophys. J.; accepted
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
Chlorin within the external leaflet
10 µm Speed 7x
Ref.: J. Heuvingh & S. Bonneau; Biophys. J.; accepted
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
Shape transitions (External leaflet targeted) short time deformation
5 µm
2
11
12
13
Inward budding
14
15
permeabilization
28
45
70 time (s)
short time deformation
5 µm
2
8
9
10
deformation
11
12
Ref.: J. Heuvingh & S. Bonneau; Biophys. J.; accepted
13
14
Inward budding permeabilization
15
40
60
80
100 time (s)
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
Shape transitions and spontaneous curvature Relative contribution of various events
ADE Model 100%
Inner leaflet
Seifert 1997 Döbereiner 2000
80% 60%
Both leaflet 40% 20% 0% 20% 40% 60% 80%
100%
Outer leaflet
Outward budding Oblate to prolate transition Long-time fluctuation (>20s)
Short-time fluctuation (0
Oxidation if the inner leaflet
Negative curvature of the photo-sensitized leaflet
∆A C0
int
C120µM)
Normalized contrast
e-
(t-t0)
5µM 25 µ M 125 µ M 125 µ M-inside
180
τ
τ(s)
120
60
0 0,0
time (s) Ref.: J. Heuvingh & S. Bonneau; Biophys. J.; accepted
5,0
10,0
15,0
20,0
Diameter of the vesicule (µm)
25,0
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
Permeabilization of the membrane Ce6 outside Ce6 both (in/out) Ce6 inside
Pore diameter between 16 nm to 43 nm [Ce6]
Importance of the asymmetry in the permeabilization process Photo-oxidation and permeabilization are not quantitatively correlated Ref.: J. Heuvingh & S. Bonneau; Biophys. J.; accepted
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
Permeabilization, curvature and tension Modification of the spontaneous curvature
Area Volume
Tension of the membrane
Lysis of the GUV
Hypothesis : the budding induces a tension of 0.15 mN/m Ref.: J. Heuvingh & S. Bonneau; Biophys. J.; accepted
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
Permeabilisization, curvature and tension Modification of the spontaneous curvature
Tension of the membrane
Area Volume
Energy necessary to entropically stretch a membrane from a tension σ0 to a tension σ is:
Energy gained when budding a vesicle of radius r
∆H b = 8πk c (1 − C 0* r )
∆Hb > ∆Hs
Lysis of the GUV
∆H s =
for
A kbT ( σ − σ 0 ) = 4 πr 2 σ 8π k c
2σ C > kc * 0
Lysis tension of 0.15 mN/m if C0* > 42 µm-1 Area decrease of 2.3% in a leaflet Ref.: J. Heuvingh & S. Bonneau; Biophys. J.; accepted
Area Volume
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
Conclusions Depth and asymmetry of photo-modification within the membrane are key of its effect Importance of the localisation of the photosensitizer within the membrane Effticiency of asymmetrically charges PS in PCI depth of the interaction with the membrane flip-flop, GOVERNED BY the DEPTH (lysosomal localisation + asymmetry) Importance of the geometry parameters of the membrane-PS interaction for the induced permeabilisation (i.e. the PS PCI-effect).
Permeabilization Stenching
13th ESP 2009 - Wroclaw, Poland, September 5/10, 2009
Thanks !
Spectroscopy
ANBioPhy, Paris Biophotonics Group Halina Mojzisova Daniel Brault
Microscopy
PMMH, Paris Julien Heuvingh
