Photo-destabilization of membranes

Transcription

1 Photo-destabilization of membranes Stéphanie Bonneau 1, Daniel Brault 1, Julien Heuvingh 2, Christine Vever-Bizet 1, Frédéric Wieber 3 1 FRE 3207, 2 UMR 7636, 3 UMR 7117

2 Self assembled system Colloque «Origine des Planètes et de la Vie» 26 mai 2009 Lipidic unilamellar vesicles Model of biological membrane 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 LUV (TEM) 5nm 5 à 20 µm GUV (PCM)

3 Self assembled system under influence Photo-sensitizer Chlorin- or porphyrin-type, polycyclic aromatic chondritic compounds Amphiphilic compounds Phospho-lipidic models Coll. H. Zepick (Department of Materials ETH Zürich) Light R-OH R-O Photo-chimical reactant P 3 P P - Radicals Stability of the membrane Probability of internalization Anoxic mechanisms 1 O 2 O 2 Singlet oxygen

4 1 st part Preliminary results: Membrane permeability and membrane geometry

5 mthpc Foscan Chlorine e6 TPCS2a Φ Three chlorin-macrocycles Ce6 TPCS 2a m-thpc 0,63 0,66 0,68 Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009

6 Photo-induced peroxidation In solution Within the membrane Proportional to m-thpc >TPCS 2a >Ce6 Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009

7 τ= 3ns 1 O 2 τ= 35 µs D~250nm Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009

8 Photo-induced permeabilization TPCS 2a >Ce6>>m-THPC Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009

9 1 O 2 1 O 2 1 O 2 1 O 2 1 O 2 1 O 2 Effect induced by charges? By the depth within the membrane? Perturbation of the membrane symmetry? Ref.: H. Mojzisova & al. Photochem. Photobiol. 2009

10 2 nd part Membrane asymmetry and permeability

11 Photo-inducing membrane asymmetry OOH Chlorin e6 Lipid Peroxide Cleaved-lipid Asymmetrical perturbation of the membrane: External leaflet Inner leaflet lavage / centrifugation

12 Chlorin (12µm) within the inner leaflet 10 µm Speed 7x

13 Shape transitions (Inner leaflet targeted) Prolate vesicle Pear shape 2 seconds / picture Budding 10 µm Pear shape Budding 1 second / picture Budding 10 µm

14 Chlorin (12µm) within the external leaflet 10 µm Speed 7x

15 Shape transitions (External leaflet targeted) 5 µm short time deformation Invagination permeabilization time (s) short time deformation deformation Invagination permeabilization time (s) 5 µm

16 Shape transitions and spontaneous curvature ADE Model Seifert 1997 Döbereiner 2000

17 Shape transitions and spontaneous curvature Effective spontaneous curvature: ext C 0 *= C 0 + απ h a 0 a 0 C>0 int Oxidation if the inner leaflet Negative curvature of the photo-sensitized leaflet C<0 ext int Oxidation of the external leaflet

18 Permeabilization of the membrane External leaflet : 100% vesicles permeabilized Inner leaflet : No permeabilized vesicles (except for C>120µM) Normalized contrast e - (t-t 0 ) τ τ(s) 60 5µM 25 µ M 125 µ M 125 µ M-inside time (s) 0 0,0 5,0 10,0 15,0 20,0 25,0 Diameter of the vesicule (µm)

19 Permeabilization of the membrane [Ce6] [Ce6] Ce6 outside Ce6 both (in/out) Ce6 inside Importance of the asymmetry in the permeabilization process

20 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 0.15mN/m

21 Fusion 125µM out 10 µm Speed 7x

22 «Zoology» 5µM in 125µM out 10 µm 10 µm Speed 7x

23 Peroxidation and permeabilization DOPC DPPC DMPC (C14) Hypothesis : photo-degradation of the chlorin asymmetry

24 Photo-sensitizer Amphiphilic compound Coll. H. Zepick (Department of Materials ETH Zürich) Chlorin e6 R-OH R-O Photo-chemical reactant Light P 3 P P - Radicals

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