France - US Workshop on NanoBio Technologies, Washington, March 2-3, 2006. Lateral Heterogeneity of Membrane lipids : consequences on lipid-exoplasmic protein interactions in supported membranes Christian Le Grimellec, Marie-Cécile Giocondi, Françoise Besson*, Patrice Dosset, Pierre Emmanuel Milhiet. Centre de Biochimie Structurale, CNRS UMR 5048-Univ. Montpellier I, INSERM U554, Montpellier, France *R.T.M. CNRS UMR 5013, Univ. Claude Bernard Lyon 1, France •In eukaryotic cells, microdomains enriched in sphingomyelin (SM) and cholesterol (Chl) form functional platforms involved in key cellular processes (cell signaling, cell-cell interactions, ……). These microdomains (rafts?) are the docking sites for pathogens and toxins and are involved in a variety of pathologies (Alzheimer, Parkinson, Prions diseases, neoplasia, atherosclerosis, HIV-1, malaria,...). Mayor & Rao, 2004
Part of these functions ( hydrolases, cell adhesion molecules, transmembrane signaling) is performed by exoplasmic proteins linked to the membrane via a glycosylphosphatidylinositol (GPI) anchor. Formation of microdomains would arise primarily from lipid-lipid interactions: lipids constituting rafts are ordered (Lo, L β ’?) and phase-separate from the disordered (L α ), more fluid species Goal: •To understand the role of lipid-lipid and lipid-protein interactions in the self- assembling processes of functional platforms.
Presumed size of domains suggests a mesoscopic approach: Atomic force microscopy on supported bilayers. Atomic Force Microscope (Binnig, Quate, & Gerber. P.R.L., 1986) SUV buffer bilayer lo g f g fusion MICA MICA Bilayers under buffer Lipid-lipid interactions
DPPC SM Tc=20°-45°C Tc=41°C POPC Tc=-(2°)C Cholesterol DOPC Tc=-20°C Polymorphism of sphingomyelin microdomains DOPC/SM (1:1) bar:1 µm Existence of globular/ripple structures and gel/gel ϕ separation
Polymorphism of sphingomyelin microdomains DOPC/SM (1:1) bar:500 nm Details of globular/ripple structures Polymorphism of sphingomyelin microdomains POPC/SM (1:1) bar:500 nm Details of globular/ripple structures
Topography of cholesterol containing bilayers: DOPC/SM/Chl bilayers Salt-induced SM polymorphism: effect of hypertonic NaCl solution on POPC/SM/Chl -reproducible shape and periodicity (~215 nm) between different samples. -2-D « egg-carton » pattern, Chl- dependent -is not induced by hypertonic sucrose
What does the mesoscopic view of supported bilayer tell us about the behavior of functional platforms lipidic constituents? • Polymorphism of individual SM microdomains in plasma membrane models (different organizations energetically close). • Reversible changes in microdomains organization can be induced by limited modification of the medium. Lipid-Exoplasmic GPI-anchored protein interactions Do lipid composition and polymorphism of model microdomains affect the distribution of a GPI- anchored protein (Alkaline Phosphatase from bovine intestine, APase) within the bilayer? •Direct insertion of APase in preformed bilayers. Protein - C O - NH -(CH 2 ) 2 - P (Man) 3 GlcNH 2 Inositol P CH 2 - CH - CH 2 CO CO (CH 2 ) (CH 2 ) 14 CH 3 CH 3 16
DOPC/DPPC bilayers bar: 2 µm A,B,C 0.5 µm D z= 10 nm A & B, 5 nm D APase spontaneously inserts in the lipid ordered (L β ’) gel phase, preferentially at the periphery. Moderate increase in the ordered domains area. (Room temperature). Long range order in the APase distribution. DOPC/SM bar: 2 µm z=15 nm APase spontaneously inserts in the lipid ordered (L β ’) gel phase . APase accumulates at the gel-fluid interface but distributes homogeneously in the gel phase. Remodeling but practically no change in ordered domains area. (Room temperature)
Polymorphism of DOPC/SM samples bar: 4 µm A-C, 1.25 µm D. Z=15 nm A-C, 10 nm D. APase preferentially localizes in the thickest SM subdomains (highest Tc?) whose area is markedly increased. Heterogeneity in the APase distribution in the other gel regions ( gel phase physical state heterognenity?) POPC/SM (1:1) + APase 40 µm 15 µm 30 µm APase localizes in particular regions of the fluid/gel interface. These regions are most often induced by the interaction of the protein with SM domains.
POPC/SM (1:1) APase recruits the most ordered SM species in its environment. bar: 4 µm A-C; 1.6µm D; 0.5 µm E z=15 nm A-D; 12 nm E Distribution of APase in the ordered domains varies according to the lipid species constituting both the ordered (gel) and fluid phases. Conversely, APase induces a remodeling of gel domains most likely by recruiting the highest melting temperature lipid species. Effect of cholesterol on APase distribution?
DOPC/SM + 15 mol% Chl + APase (room temperature) Addition of 15 mol% Chl often results in the branching of lo-enriched domains. APase distributes homogeneously in the ordered phase. (room temperature) POPC/SM/Chl (1:1:0.35) + APase 25°C A A:In contrast to DOPC /SM/Chl bilayersAPase localizes into domains which do not exactly superimposed with the B ordered (Lo?) domains. B: Using 300 pN scan force sweeps away most of the APase. The domains left are thicker and more irregular than the Lo ’s. More ordered (Gel phase) domains?
POPC/SM/Chl (1:1:0.35) For low APase/lipid 18°C ratio temperature- dependent experiments support the hypothesis of a gel phase environment for the APase. 28°C 34°C Almeida et al., 2004 POPC/SM/Chl (1:1:0.35) + high amount APase At low temperature: APase localizes in SM enriched domains This localization in SM domains is maintained through the macro-ripple stage. At a higher temperature, APase localizes in both SM and POPC enriched domains.
Membrane distribution of the APase •The degree of PC unsaturation affects the distribution of APase between PC/SM domains both in the absence and in the presence of cholesterol. •APase distribution is markedly affected by the temperature (i.e. the physical state) of the sample. • APase preference for the most ordered regions. • APAse contributes to the selection of its environment which is made of the most ordered SM species. Conclusions • Unidirectional direct insertion of an exoplasmic GPI- anchored protein into a pre-formed supported bilayer under phase-separation. •Self-assembling properties upon insertion of a second exoplasmic protein ?
V.Vié, C. Domec, O. Bagdhadi, T. Plénat, S. Boichot, CBS, Montpellier D. Lévy, J-L. Rigaud, Institut Curie, Paris E. Lesniewska, J-P.Goudonnet, Laboratoire de Physique, Université de Bourgogne, Dijon. B.Roux ,F. Ronzon, F. Besson, Université Claude Bernard, Lyon N. van Mau, F. Heitz, S. Deshayes CRBM, Montpellier M. Zinke-Allmang, Dept. Physics & Astronomy, University of WesternOntario, Canada.
Membrane distribution of the APase •The degree of PC unsaturation affects the distribution of APase between PC/SM domains both in the absence and in the presence of cholesterol. •APase distribution is markedly affected by the temperature (i.e. the physical state) of the sample. • APase preference for the most ordered regions. • APAse contributes to the selection of its environment which is made of the most ordered SM species. Conclusions • Unidirectional direct insertion of an exoplasmic GPI- anchored protein into a pre-formed supported bilayer under phase-separation. •Self-assembling properties upon insertion of a second exoplasmic protein ?
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