Controlling interactions in supported bilayers from weak electrostatic repulsion to high osmotic pressure

Understanding interactions between membranes requires measurements on well-controlled systems close to natural conditions, in which fluctuations play an important role. We have determined, by grazing incidence X-ray scattering, the interaction potential between two lipid bilayers, one adsorbed on a solid surface and the other floating close by. We find that interactions in this highly hydrated model system are two orders of magnitude softer than in previously reported work on multilayer stacks. This is attributed to the weak electrostatic repulsion due to the small fraction of ionized lipids in supported bilayers with a lower number of defects. Our data are consistent with the Poisson-Boltzmann theory, in the regime where repulsion is dominated by the entropy of counter ions. We also have unique access to very weak entropic repulsion potentials, which allowed us to discriminate between the various models proposed in the literature. We further demonstrate that the interaction potential between supported bilayers can be tuned at will by applying osmotic pressure, providing a way to manipulate these model membranes, thus considerably enlarging the range of biological or physical problems that can be addressed.Comment: 14 pages, 8 figure

Fluctuations et déstabilisation d'une bicouche lipidique supportée

Nous présentons une étude expérimentale des propriétés dynamiques de bicouches lipidiques supportées. La structure et les propriétés à l'équilibre de simples et double-bicouches sont étudiées par réflectivité de rayonnement (neutrons et rayons X). La diffusion hors-spéculaire de rayons X permet d'étudier le spectre de fluctuations submicronique d'une bicouche "flottante", fluctuant quelques nanomètres au dessus d'une bicouche greffée: la tension de surface, le module de courbure et, pour la première fois par cette technique, le potentiel d'interaction intermembranaire sont déterminés.Nous montrons par microscopie de fluorescence que cette bicouche unique peut former des vésicules. Sa déstabilisation peut avoir lieu au passage de la transition gel-fluide des lipides, ou sous l'effet d'un champ électrique basse fréquence appliqué en phase fluide. Dans ce dernier cas nous étudions également l'effet du champ à l'échelle moléculaire par réflectivité de neutrons, et déterminons les conditions du décollement. Dans les deux cas, la déstabilisation conduit à des vésicules dont la taille relativement homogène pourrait permettre d'élucider le mécanisme de formation, et de mettre au point une technique de préparation plus contrôlée.Une dernière partie de ce travail concerne l'étude de la diffusion latérale des lipides dans la bicouche, par recouvrement de fluorescence. La transition gel-fluide peut être repérée grâce à la mesure du coefficient de diffusion en fonction de la température; nos résultats suggèrent son abaissement de quelques degrés pour une bicouche supportée sur une lame de verre.We study experimentally the dynamical properties of a supported lipid bilayer. The structure and equilibrium properties of single and double bilayers are studied by neutron reflectivity. The submicronic fluctuation spectrum of a "floating" bilayer is studied by off-specular X-ray scattering: its surface tension, bending modulus and, for the first time with this technique, the inter-membrane potential can be determined.Using fluorescence microscopy, we show that this single bilayer can form vesicles. Its destabilisation can occur either at the main gel-fluid transition of the lipids, and can be interpreted in terms of a drop of the bending rigidity, or under an AC low-frequency electric field applied in the fluid phase. In that last case we also study the effect of the electric field at the molecular length scale by neutron reflectivity. In both cases, the detabilisation leads to the formation of relatively monodisperse vesicles, which could give a better understading of the formation mechanism, and eventually allow better control of the preparation process.In a last part, we study the lateral diffusion of lipids in the supported bilayer thanks to fluorescence recovery after photobleaching. Measuring the diffusion coefficient as a function of temperature enables one to spot the gel-fluid transition; our results suggest that it could be lowered by a few degrees for a single bilayer supported on a glass substrate.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Continuous versus Arrested Spreading of Biofilms at Solid-Gas Interfaces: The Role of Surface Forces

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Substrate stiffness impacts early biofilm formation by modulating Pseudomonas aeruginosa twitching motility

Surface-associated lifestyles dominate in the bacterial world. Large multicellular assemblies, called biofilms, are essential to the survival of bacteria in harsh environments, and are closely linked to antibiotic resistance in pathogenic strains. Biofilms stem from the surface colonization of a wide variety of substrates encountered by bacteria, from living tissues to inert materials. Here, we demonstrate experimentally that the promiscuous opportunistic pathogen Pseudomonas aeruginosa explores substrates differently based on their rigidity, leading to striking variations in biofilm structure, exopolysaccharides (EPS) distribution, strain mixing during co-colonization and phenotypic expression. Using simple kinetic models, we show that these phenotypes arise through a mechanical interaction between the elasticity of the substrate and the type IV pilus (T4P) machinery, that mediates the surface-based motility called twitching. Together, our findings reveal a new role for substrate softness in the spatial organization of bacteria in complex microenvironments, with far-reaching consequences on efficient biofilm formation

Asymmetric adhesion of rod-shaped bacteria controls microcolony morphogenesis

It is unclear how cell adhesion and elongation coordinate during formation of bacterial microcolonies. Here, Duvernoy et al. monitor microcolony formation in rod-shaped bacteria, and show that patterns of surface colonization derive from the spatial distribution of adhesive factors on the cell envelope