Patterning and dynamics of membrane adhesion under hydraulic stress.

Hydraulic fracturing plays a major role in cavity formation during embryonic development, when pressurized fluid opens microlumens at cell-cell contacts, which evolve to form a single large lumen. However, the fundamental physical mechanisms behind these processes remain masked by the complexity and specificity of biological systems. Here, we show that adhered lipid vesicles subjected to osmotic stress form hydraulic microlumens similar to those in cells. Combining vesicle experiments with theoretical modelling and numerical simulations, we provide a physical framework for the hydraulic reconfiguration of cell-cell adhesions. We map the conditions for microlumen formation from a pristine adhesion, the emerging dynamical patterns and their subsequent maturation. We demonstrate control of the fracturing process depending on the applied pressure gradients and the type and density of membrane bonds. Our experiments further reveal an unexpected, passive transition of microlumens to closed buds that suggests a physical route to adhesion remodeling by endocytosis.

Scratching beyond the surface - minimal actin assemblies as tools to elucidate mechanical reinforcement and shape change.

The interaction between the actin cytoskeleton and the plasma membrane in eukaryotic cells is integral to a large number of functions such as shape change, mechanical reinforcement and contraction. These phenomena are driven by the architectural regulation of a thin actin network, directly beneath the membrane through interactions with a variety of binding proteins, membrane anchoring proteins and molecular motors. An increasingly common approach to understanding the mechanisms that drive these processes is to build model systems from reconstituted lipids, actin filaments and associated actin-binding proteins. Here we review recent progress in this field, with a particular emphasis on how the actin cytoskeleton provides mechanical reinforcement, drives shape change and induces contraction. Finally, we discuss potential future developments in the field, which would allow the extension of these techniques to more complex cellular processes.

Encapsulated bacteria deform lipid vesicles into flagellated swimmers.

We study a synthetic system of motile Escherichia coli bacteria encapsulated inside giant lipid vesicles. Forces exerted by the bacteria on the inner side of the membrane are sufficient to extrude membrane tubes filled with one or several bacteria. We show that a physical coupling between the membrane tube and the flagella of the enclosed cells transforms the tube into an effective helical flagellum propelling the vesicle. We develop a simple theoretical model to estimate the propulsive force from the speed of the vesicles and demonstrate the good efficiency of this coupling mechanism. Together, these results point to design principles for conferring motility to synthetic cells.

Comparative Study of Lipid- and Polymer-Supported Membranes Obtained by Vesicle Fusion.

We compare the fusion of giant lipid and block-copolymer vesicles on glass and poly(dimethylsiloxane) substrates. Both types of vesicles are similar in their ability to fuse to hydrophilic substrates and form patches with distinct heart or circular shapes. We use epifluorescence/confocal microscopy and atomic force microscopy on membrane patches to (i) characterize bilayer fluidity and patch-edge stability and (ii) follow the intermediate stages in the formation of continuous supported bilayers. Polymer membranes show much lower membrane fluidity and, unlike lipids, an inability of adjacent patches to fuse spontaneously into continuous membranes. We ascribe this effect to hydration repulsion forces acting between the patch edges, which can be diminished by increasing the sample temperature. We show that large areas of supported polymer membranes can be created by fusing giant vesicles on glass or poly(dimethylsiloxane) substrates and annealing their edges.

Dynamic mechanochemical feedback between curved membranes and BAR protein self-organization.

In many physiological situations, BAR proteins reshape membranes with pre-existing curvature (templates), contributing to essential cellular processes. However, the mechanism and the biological implications of this reshaping process remain unclear. Here we show, both experimentally and through modelling, that BAR proteins reshape low curvature membrane templates through a mechanochemical phase transition. This phenomenon depends on initial template shape and involves the co-existence and progressive transition between distinct local states in terms of molecular organization (protein arrangement and density) and membrane shape (template size and spherical versus cylindrical curvature). Further, we demonstrate in cells that this phenomenon enables a mechanotransduction mode, in which cellular stretch leads to the mechanical formation of membrane templates, which are then reshaped into tubules by BAR proteins. Our results demonstrate the interplay between membrane mechanics and BAR protein molecular organization, integrating curvature sensing and generation in a comprehensive framework with implications for cell mechanical responses.

Substrate-led cholesterol extraction from supported lipid membranes.

The lipid membrane is a principal building block in biology, technology and industry, where it often occurs supported by other hydrophilic structures. Interactions with the support can affect the physical behavior of the membrane from the local organization and diffusion of lipids and proteins, to phase transitions, and the local mechanical properties. In this study we show that supporting substrates textured with nanoscale hydrophilic and hydrophobic domains can modify the membrane's chemical composition by selectively extracting cholesterol molecules without affecting the remaining phospholipids. Using polydimethylsiloxane (PDMS) substrates with various degrees of plasma oxidation, we are able to trigger dramatic changes in the membrane morphology and biophysical properties, and relate them to the amount of extracted cholesterol. We also show that it is possible to control the cholesterol extraction through mechanical extension of the flexible PDMS support. Given the ubiquity of bio-substrates with textured surface properties and the wide use of PDMS we expect that our results will have implications not only in biological and chemical sciences but also in nanotechnologies such as organ on a chip technologies, biosensors, and stretchable bio-electronics.

Shape Transformations of Lipid Bilayers Following Rapid Cholesterol Uptake.

High cholesterol levels in the blood increase the risk of atherosclerosis. A common explanation is that the cholesterol increase in the plasma membrane perturbs the shape and functions of cells by disrupting the cell signaling pathways and the formation of membrane rafts. In this work, we show that after enhanced transient uptake of cholesterol, mono-component lipid bilayers change their shape similarly to cell membranes in vivo. The bilayers either expel lipid protrusions or spread laterally as a result of the ensuing changes in their lipid density, the mechanical constraints imposed on them, and the properties of cyclodextrin used as a cholesterol donor. In light of the increasingly recognized link between membrane tension and cell behavior, we propose that the physical adaptation of the plasma membrane to cholesterol uptake may play a substantial role in the biological response.

Biophysical dissection of schistosome septins: Insights into oligomerization and membrane binding.

Septins are GTP-binding proteins that are highly conserved among eukaryotes and which are usually membrane-associated. They have been linked to several critical cellular functions such as exocytosis and ciliogenesis, but little mechanistic detail is known. Their assembly into filaments and membrane binding properties are incompletely understood and that is specially so for non-human septins where such information would offer therapeutic potential. In this study we use Schistosoma mansoni, exhibiting just four septin genes, as a simpler model for characterizing the septin structure and organization. We show that the biochemical and biophysical proprieties of its SmSEPT5 and SmSEPT10 septins are consistent with their human counterparts of subgroups SEPT2 and SEPT6, respectively. By succeeding to isolate stable constructs comprising distinct domains of SmSEPT5 and SmSEPT10 we were able to infer the influence of terminal interfaces in the oligomerization and membrane binding properties. For example, both proteins tended to form oligomers interacting by the N- and C-terminal interfaces in a nucleotide independent fashion but form heterodimers via the G interface, which are nucleotide dependent. Furthermore, we report for the first time that it is the C-terminus of SmSETP10, rather than the N-terminal polybasic region found in other septins, that mediates its binding to liposomes. Upon binding we observe formation of discrete lipo-protein clusters and higher order septin structures, making our system an exciting model to study interactions of septins with biological membranes.

Confined bilayers passively regulate shape and stress.

Lipid membranes are commonly confined to adjacent subcellular structures or to artificial substrates and particles. We develop an experimental and theoretical framework to investigate the mechanics of confined membranes, including the influence of adhesion, strain, and osmotic pressure. We find that supported lipid bilayers respond to stress by nucleating and evolving spherical and tubular protrusions. In cells, such transformations are generally attributed to proteins. Our results offer insights into the mechanics of cell membranes and can further extend the applications of supported bilayers.

The role of the membrane confinement in the surface area regulation of cells.

We propose a new in vitro system to study the mechanics of surface area regulation in cells, which takes into an account the spatial confinement of the cell membrane. By coupling a lipid bilayer to the strain-controlled deformation of an elastic sheet, we show that upon straining the supported lipid bilayer expands its surface area by absorbing adherent lipid vesicles and upon compression decreases its area by expelling lipid tubes out of its plane. The processes are reversible and closely resemble in vivo observations on shrinking cells. Our results suggest that the mechanics of the area regulation in cells is controlled primarily by the membrane tension and the effects of the membrane confinement.

Mechanics of surface area regulation in cells examined with confined lipid membranes.

Cells are wrapped in inelastic membranes, yet they can sustain large mechanical strains by regulating their area. The area regulation in cells is achieved either by membrane folding or by membrane exo- and endocytosis. These processes involve complex morphological transformations of the cell membrane, i.e., invagination, vesicle fusion, and fission, whose precise mechanisms are still under debate. Here we provide mechanistic insights into the area regulation of cell membranes, based on the previously neglected role of membrane confinement, as well as on the strain-induced membrane tension. Commonly, the membranes of mammalian and plant cells are not isolated, but rather they are adhered to an extracellular matrix, the cytoskeleton, and to other cell membranes. Using a lipid bilayer, coupled to an elastic sheet, we are able to demonstrate that, upon straining, the confined membrane is able to regulate passively its area. In particular, by stretching the elastic support, the bilayer laterally expands without rupture by fusing adhered lipid vesicles; upon compression, lipid tubes grow out of the membrane plane, thus reducing its area. These transformations are reversible, as we show using cycles of expansion and compression, and closely reproduce membrane processes found in cells during area regulation. Moreover, we demonstrate a new mechanism for the formation of lipid tubes in cells, which is driven by the membrane lateral compression and may therefore explain the various membrane tubules observed in shrinking cells.

Membrane flow patterns in multicomponent giant vesicles induced by alternating electric fields†Electronic supplementary information (ESI) available: Vesicle preparation procedure, numerical calculations and confocal microscopy movies of domain motion. See DOI: 10.1039/b811876kClick here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.

Alternating electric (AC) fields induce circular patterns of lipid transport in membranes of giant vesicles. The flow is visualized by fluorescently labelled lipid domains.