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Supported bilayers

Friday 1 February 2013, by mcube

We describe here different works we have done in the last 10 years in order to investigate physical properties of supported bilayers. We have developed an original model system consisting in double supported bilayers: two bilayers separated by a thin (2 nm) water layer [1].

The main advantage of this system compared to classical supported bilayers consists in the weak interaction of the second bilayer, named "floating bilayer" with the solid substrate, preserving interesting physical properties. We have clearly demonstrate the stability of the floating bilayer by going from the gel to the fluid phase [2]. This is an important result which opens the doors to using floating bilayer as biomembrane models [3].

Fluctuations and Destabilization of Supported Membranes

The dynamics of lipid membranes has an intrinsic multiscale character. Equilibrium and non- equilibrium fluctuations as well as destabilization or topological changes involve complex mechanisms in a range of length scales from a nanometer to many micrometers. Thanks to optical microscopy experiments on giant vesicles, large length scales are now better understood but accessing smaller length scales is still challenging and presently only achieved by numerical simulations. A major challenge resides thus in accessing experimentally sub-optical properties and understanding the relations connecting the different scales involved in fluid membrane dynamics.

Fluctuations of Supported Membranes

Figure1: Schematic view of off-specular reflectivity experiments on floating bilayer.

A refined analysis of the bilayer fluctuations requires a full characterization of the fluctuations spectrum, that is the fluctuations amplitude at a given spatial length scale. We have developed a new x-ray off-specular scattering experiment at ESRF, in collaboration with J. Daillant (CEA-Saclay) [4]. The main difficulty of this experiment is due to the weakness of the scattering volume. Surface sensitivity was considerably enhanced by taking advantage of total external reflection at the Si–water interface, but the intensity scattered by the fluctuations bilayer remains two or three orders of magnitude lower than bulk scattering (see figure 1). We developed a rigorous calculation of specular and off-specular X-ray reflectivity of supported membranes [5]. More precisely, we computed the correlation functions of the membranes only by approximating the interaction potentials as quadratic functions. Thanks to specular reflectivity, we determine the perpendicular structure of the samples, whereas off-specular reflectivity gives access to lateral inhomogeneities of the membranes. By performing a simultaneous analysis, we characterize both structure and parameters of the fluctuation spectrum: tension, rigidity and interaction potentials. By studying single supported bilayers, we measure for the first time the effective microscopic tension related to protrusions inside the membrane (about 80 mN/m). Long-range fluctuations are determined by moving the membrane away from the substrate by the addition of an adsorbed membrane. Therefore we precisely measure the interaction potential between neutral membranes in a state of equilibrium [6].

Destabilization of Supported Membranes

We have investigated membrane fluctuations, protrusion modes and destabilization mechanisms leading to vesicle formation [7] [8].

Destabilization by temperature


Figure 2:Effect of temperature on a double supported bilayer of DSPC: inter-bilayer (water layer) thickness vs rms fluctuations (Debye-Waller factor).

We have observed a spectacular maximum in both inter-bilayer distance and bilayer roughness as a function of temperature (see figure [2]). We have investigated in details this effect for various lipid bilayer [9]. We interpret this effect as a direct consequence of the balance between energy minimization and entropic repulsion. We have then developp a theoretical model [10] allowing us to describe the thermal fluctuations of a supported bilayer in an asymetrical case. Finally we have shown that the supported bilayer could be destabilized leading to monodisperse Giant Vesicles [11].

Effect of an electric field on a floating lipid bilayer: A neutron reflectivity study


Figure 3: Effect of an electric field on a floating bilayer: (left) destabilisation of the floating by electric observed by neutron reflectivity; (b) formation of GUVs from a single supported bilayer.

We have investigated the influence of an alternative electric field on a supported phospholipid double bilayer by using neutron specular reflectivity [12]. We clearly show that the electric field can lead to a complete
unbinding of the floating bilayer in a high-conductivity solution at low frequency. In a low-conductivity solution, small reversible modifications of the reflectivity profiles are observed and described as changes of the r.m.s. roughness of the floating bilayer. These data could be interpreted with a fluctuation spectrum including a negative electrostatic surface tension term as suggested by reference.

Internal dynamics

In order to better understand the influence of the substrate on lipid bilayer dynamics, the diffusion law of DMPC and DPPC in Supported Lipid Bilayers (SLB), on different substrates, has been carefully investigated by using FRAPP (Fluorescence Recovery After Patterned Photobleaching) [13].

Over micrometer length scales, we demonstrate the validity of a Brownian diffusive law both in the gel and the fluid phases of the lipids. Measuring the diffusion coefficient as a function of temperature, we characterize the gel-to-liquid phase transition of DMPC and DPPC. It is shown that different results can be obtained, depending on the substrate and on the method used for bilayer preparation. In particular, we have shown that preparation of the bilayers from vesicle fusion leads to larger scattering of the diffusion data and of the transition temperature shifts.

Footnotes

[1T. Charitat, E. Bellet-Amalric, G. Fragneto, and F. Graner, Adsorbed and free lipid bilayers at the solid-liquid interface, European Physical Journal B, 8, 583-593 (1999)

[2G. Fragneto, T.Charitat, F. Graner, K. Mecke, L. Périno-Gallice, and E. Bellet-Amalric, A Fluid Floating Bilayers, Europhysics Letters, 53, 100-106 (2001).

[3G. Fragneto, F. Graner, T. Charitat, P. Dubos, and E. Bellet-Amalric, Interaction of the Third Helix of Antennapedia Homeodomain with a Deposited Phospholipid Bilayer : A Neutron Reflectivity Structural Study, Langmuir, 16, 4581-4588 (2000).

[4Daillant, J.; Bellet-Amalric, E.; Braslau, A.; Charitat, T.; Fragneto, G.; Graner, F.; Mora, S.; Rieutord, F.; Stidder, B., Structure and fluctuations of a single floating lipid bilayer,PNAS 102, 11639-11644 (2005)

[5Malaquin, L.; Charitat, T.; Daillant, J. Supported bilayers: Combined specular and diffuse X-ray scattering European Physical Journal E 31, 285-301 (2010)

[6Hemmerle, A.; Malaquin, L.; Charitat, T.; Lecuyer, S.; Fragneto, G.; Daillant, J. Controlling interactions in supported bilayers from weak electrostatic repulsion to high osmotic pressure, PNAS 109, 19938-19942 (2012)

[7Charitat, T.; Lecuyer, S.; Fragneto, G. Fluctuations and destabilization of single phospholipid bilayers, Biointerphases, 3 (2008)

[8Fragneto, G.; Charitat, T.; Daillant, J. Floating lipid bilayers: models for physics and biology European Biophysical Journal With Biophysics Letters 41 863-874 (2012)

[9G. Fragneto, T. Charitat, E. Bellet-Amalric, R. Cubitt, and F. Graner, Swelling of phospholipid floating bilayers: the effect of chain length, Langmuir, 19, 7695-7702 (2003).

[10K. Mecke, T.Charitat and F. Graner, Fluctuating Lipid Bilayer in an Arbitrary Potential: Theory and Experimental Determination of Bending Rigidity, Langmuir, 19, 2080-2087 (2003).

[11S. Lecuyer, T.Charitat, From supported membranes to tethered vesicles: Lipid bilayers destabilisation at the main transition, Europhysics Letters 75, 4, 652-658 (2006).

[12S. Lecuyer, G. Fragneto, T. Charitat, Effect of an electric field on a floating lipid bilayer: a neutron reflectivity study, European Physical Journal E, 21, 153-159 (2006).

[13Scomparin, C.; Lecuyer, S.; Ferreira, M.; Charitat, T.; Tinland, B. Diffusion in supported lipid bilayers: influence of substrate and preparation technique on the internal dynamics, European Physical Journal E, 28, 211 (2009)