Giant unilamellar vesicles or GUVs are systems of choice as biomimetic models of cellular membranes. Although a variety of procedures exist for making single walled vesicles of tens of microns in size, the range of lipid compositions that can be used to grow GUVs by the conventional methods is quite limited, and many of the available methods involve energy input that can damage the lipids or other molecules present in the growing solution for embedment in the membrane or in the vesicle interior. Here, we show that a wide variety of lipids or lipid mixtures can grow into GUVs by swelling lipid precursor films on top of a dried polyvinyl alcohol gel surface in a swelling buffer that can contain diverse biorelevant molecules. Moreover, we show that the encapsulation potential of this method can be enhanced by combining polyvinyl alcohol-mediated growth with inverse-phase methods, which allow (bio)molecule complexation with the lipids.

Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. We address this problem by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive. We use the cleavage of the Drosophila melanogaster embryo as a model system, where contractile actin rings drive constriction of invaginating membranes to produce an epithelium in a manner akin to cell division. In vivo functional studies show that septins are required for generating curved and tightly packed actin filament networks. In vitro reconstitution assays show that septins alone bundle actin filaments into rings, accounting for the defects in actin ring formation in septin mutants. The bundling and bending activities are conserved for human septins, and highlight unique functions of septins in the organization of contractile actomyosin rings.

We demonstrate that cytoskeletal actin-myosin networks can be encapsulated with high efficiency in giant liposomes by hydration of lipids in an agarose hydrogel. The liposomes have cell-sized diameters of 10-20 μm and a uniform actin content. We show by measurements of membrane fluorescence intensity and bending rigidity that the majority of liposomes are unilamellar. We further demonstrate that the actin network can be specifically anchored to the membrane by biotin-streptavidin linkages. These protein-filled liposomes are useful model systems for quantitative studies of the physical mechanisms by which the cytoskeleton actively controls cell shape and mechanics. In a broader context, this new preparation method should be widely applicable to encapsulation of proteins and polymers, for instance, to create polymer-reinforced liposomes for drug delivery.

Filamentous biopolymer networks in cells and tissues are routinely imaged by confocal microscopy. Image analysis methods enable quantitative study of the properties of these curvilinear networks. However, software tools to quantify the geometry and topology of these often dense 3D networks and to localize network junctions are scarce. To fill this gap, we developed a new software tool called “SOAX”, which can accurately extract the centerlines of 3D biopolymer networks and identify network junctions using Stretching Open Active Contours (SOACs). It provides an open-source, user-friendly platform for network centerline extraction, 2D/3D visualization, manual editing and quantitative analysis. We propose a method to quantify the performance of SOAX, which helps determine the optimal extraction parameter values. We quantify several different types of biopolymer networks to demonstrate SOAX's potential to help answer key questions in cell biology and biophysics from a quantitative viewpoint.

Cardiolipin is a cone-shaped lipid predominantly localized in curved membrane sites of bacteria and in the mitochondrial cristae. This specific localization has been argued to be geometry-driven, since the CL’s conical shape relaxes curvature frustration. Although previous evidence suggests a coupling between CL concentration and membrane shape in vivo, no precise experimental data are available for curvature-based CL sorting in vitro. Here, we test this hypothesis in experiments that isolate the effects of membrane curvature in lipid-bilayer nanotubes. CL sorting is observed with increasing tube curvature, reaching a maximum at optimal CL concentrations, a fact compatible with self-associative clustering. Observations are compatible with a model of membrane elasticity including van der Waals entropy, from which a negative intrinsic curvature of −1.1 nm −1 is predicted for CL. The results contribute to understanding the physicochemical interplay between membrane curvature and composition, providing key insights into mitochondrial and bacterial membrane organization and dynamics.

Significance Animal cells continuously move, divide, and transmit forces by actively reorganizing their internal scaffold or cytoskeleton. Molecular motors pull actin filaments together and generate contraction of the cytoskeleton underneath the cell membrane. We address the detailed mechanism of contraction by using a minimal in vitro assay: a liposome membrane to which we attach actin filaments and molecular motors in a controlled manner. We reproduce contraction like in cells. We show that the scaffold needs to be tightly attached to the membrane for efficient contraction, but, under some conditions, efficient contraction can lead to liposome destruction. These results suggest that cells must precisely control their contractility to remain intact during cellular events.

Septins are cytoskeletal filaments that assemble at the inner face of the plasma membrane. They are localized at constriction sites and impact membrane remodeling. We report in vitro tools to examine how yeast septins behave on curved and deformable membranes. Septins reshape the membranes of Giant Unilamellar Vesicles with the formation of periodic spikes, while flattening smaller vesicles. We show that membrane deformations are associated to preferential arrangement of septin filaments on specific curvatures. When binding to bilayers supported on custom-designed periodic wavy patterns displaying positive and negative micrometric radii of curvatures, septin filaments remain straight and perpendicular to the curvature of the convex parts, while bending negatively to follow concave geometries. Based on these results, we propose a theoretical model that describes the deformations and micrometric curvature sensitivity observed in vitro. The model captures the reorganizations of septin filaments throughout cytokinesis in vivo, providing mechanistic insights into cell division.

Biosensors based on plasmonic nanostructures are widely used in various applications and benefit from numerous operational advantages. One type of application where nanostructured sensors provide unique value in comparison with, for instance, conventional surface plasmon resonance, is investigations of the influence of nanoscale geometry on biomolecular binding events. In this study, we show that plasmonic “nanowells” conformally coated with a continuous lipid bilayer can be used to detect the preferential binding of the insulin receptor tyrosine kinase substrate protein (IRSp53) I-BAR domain to regions of negative surface curvature, i.e., the interior of the nanowells. Two different sensor architectures with and without an additional niobium oxide layer are compared for this purpose. In both cases, curvature preferential binding of IRSp53 (at around 0.025 nm−1 and higher) can be detected qualitatively. The high refractive index niobium oxide influences the near field distribution and makes the signature for bilayer formation less clear, but the contrast for accumulation at regions of negative curvature is slightly higher. This work shows the first example of analyzing preferential binding of an average-sized and biologically important protein to negative membrane curvature in a label-free manner and in real-time, illustrating a unique application for nanoplasmonic sensors.