Mechanical properties of plasma membrane vesicles correlate with lipid order, viscosity and cell density

Regulation of plasma membrane curvature and composition governs essential cellular processes. The material property of bending rigidity describes the energetic cost of membrane deformations and depends on the plasma membrane molecular composition. Because of compositional fluctuations and active processes, it is challenging to measure it in intact cells. Here, we study the plasma membrane using giant plasma membrane vesicles (GPMVs), which largely preserve the plasma membrane lipidome and proteome. We show that the bending rigidity of plasma membranes under varied conditions is correlated to readout from environment-sensitive dyes, which are indicative of membrane order and microviscosity. This correlation holds across different cell lines, upon cholesterol depletion or enrichment of the plasma membrane, and variations in cell density. Thus, polarity- and viscosity-sensitive probes represent a promising indicator of membrane mechanical properties. Additionally, our results allow for identifying synthetic membranes with a few well defined lipids as optimal plasma membrane mimetics

Building a community to engineer synthetic cells and organelles from the bottom-up

Employing concepts from physics, chemistry and bioengineering, 'learning-by-building' approaches are becoming increasingly popular in the life sciences, especially with researchers who are attempting to engineer cellular life from scratch. The SynCell2020/21 conference brought together researchers from different disciplines to highlight progress in this field, including areas where synthetic cells are having socioeconomic and technological impact. Conference participants also identified the challenges involved in designing, manipulating and creating synthetic cells with hierarchical organization and function. A key conclusion is the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives

Partitionierung von Membrankomponenten in adhärenten Vesikeln

Biological membranes are segmented into functional compartments of different length and time scales. Here we study model systems which mimic certain aspects of these multiscale phaenomena. We introduce a novel experimental setup for modulating adhesion of giant unilamellar vesicles to a planar substrate. Adhesion is induced by application of an external potential to a transparent ITO electrode (the substrate), which enables single-vesicle studies. We demonstrate tuneable and reversible adhesion of negatively charged vesicles. The adhesion energy at different potentials is calculated from the vesicle shape assessed with confocal microscopy. Two approaches for these estimates are employed: one based on the whole contour of the vesicle and a second one based on the contact curvature of the membrane in the vicinity of the substrate. Both approaches agree well with each other and show that the adhered vesicles are in the weak adhesion regime for the range of explored potentials. Using fluorescence quenching assays, we detect that, in the adhering membrane segment, only the outer bilayer leaflet of the vesicle is depleted of negatively charged fluorescent lipids, while the inner leaflet remains unaffected. We show that depletion of negatively charged lipids is consistent with solutions obtained by Poisson Boltzmann theory which accounts for lipid mobility. We also show that lipid diffusion is not significantly affected in the adhering membrane segment adhesion zone in the case of fully miscible membrane components. We then extend this method to the study of multicomponent lipid membranes, which exhibited domains due to a miscibility gap in the liquid phase, as a simple biomimetic model of a heterogeneous membrane. Upon adhesion, we find complex remodelling of membrane composition and morphology, depending on the initial phase of the membrane. Particularly we find a previously undescribed budding transition in the contact line of adhering vesicles. Initially phase separated vesicles are robust against budding transitions in the explored adhesion energies. Only under conditions where membrane components are fully miscible, buds appear. We link these buds to lipid flows between the two membrane compartments. Finally, we study specific adhesion by membrane bound proteins. By natural reconstitution of the membrane protein CD47 into giant plasma membrane vesicles, we can study adhesion complexes. This model is suitable to extract two membrane proteins, which are difficult to assess with established methods.Biologische Membranen sind in funktionale Kompartimente von verschiedenen Zeit- und Längenskalen eingeteilt. In dieser Arbeit untersuchen wir Modellsysteme, welche verschiedene Skalen von Biomembranen abbilden. Dazu haben wir eine neue Methode entwickelt, um die Adhäsionsenergie von „Giant Unilamellar Vesicles“ zu einer ebenen Oberfläche präzise einzustellen. Adhäsion wird durch ein externes elektrisches Potential induziert, welches an eine transparente ITO Elektrode angelegt wird. So wird die Untersuchung von Adhäsionsphänomenen an einzelnen Vesikeln möglich. Wir zeigen, dass so reversible und einstellbare Adhäsion von elektrisch negativ geladenen Vesikeln möglich ist. Die Abhängigkeit von Adhäsionsenergie zu angelegtem Potential haben wir auf zwei verschiedenen Wegen bestimmt. Sowohl eine Methode, die die Morphologie von adhärenten Vesikeln berücksichtigt, als auch die Bestimmung der Adhäsionsenergie durch die Membrankrümmung in der Nähe der Kontaktlinie liefern übereinstimmende Ergebnisse. Die gewonnenen Daten zeigen, dass sich die Vesikel im „weak adhesion“ Regime befinden. Durch Fluoreszenzauslöschungs-Experimente zeigen wir, dass im adhärierenden Membransegment nur die äußere Membranhälfte von negativ geladenen Lipiden verarmt wird, während die innere Membranhälfte unbeeinflusst bleibt. Die Verarmung von negativ geladenen Lipiden ist konsistent mit einer Possion-Boltzmann-Theorie, welche Lipidmobilität explizit berücksichtigt. Des Weiteren zeigen wir, dass die Lipiddiffusion in beiden Kompartimenten nicht beeinträchtigt ist, solange die Membrankomponenten ideal mischbar sind. Im Folgenden betrachten wir Multikomponenten-Vesikel, welche Domänen, hervorgerufen durch eine Mischungslücke in der flüssigen Phase, zeigen. In Abhängigkeit von der Initialen Membranphase führt die Adhäsion von solchen Vesikeln zur komplexen Remodellierung von Membranzusammensetzung und Morphologie. Insbesondere beschreiben wir bisher unbekannte Membranauswölbungen („Buds“) in der Kontaktlinie von adhärenten Vesikeln. Wenn phasenseparierte Vesikeln adhärieren, erscheinen zunächst keine Buds. Nur unter Bedingungen, in denen die Membrankomponenten vollständig verflüssigt werden, erscheinen Buds. Wir diskutieren einen möglichen Mechanismus, der auf Lipidflüsse zwischen den beiden Segmenten basiert. Abschließend untersuchen wir die spezifische Adhäsion von Vesikeln durch Membranproteine. Wir stellen fest, dass natürlich rekonstituiertes CD47 in „Giant Plasma Membrane Vesicles“ Adhäsionskomplexe bilden kann. Dieses Modell ermöglicht es, die zweidimensionale Bindungskonstante zu bestimmen, welche mit etablierten Methoden schwierig messbar ist

From beetles in nature to the laboratory: actuating underwater locomotion on hydrophobic surfaces

The controlled wetting and dewetting of surfaces is a primary mechanism used by beetles in nature, such as the ladybird and the leaf beetle for underwater locomotion.(1) Their adhesion to surfaces underwater is enabled through the attachment of bubbles trapped in their setaecovered legs. Locomotion, however, is performed by applying mechanical forces in order to move, attach, and detach the bubbles in a controlled manner. Under synthetic conditions, however, when a bubble is bound to a surface, it is nearly impossible to maneuver without the use of external stimuli. Thus, actuated wetting and dewetting of surfaces remain challenges. Here, electrowetting-on-dielectric (EWOD) is used for the manipulation of bubble particle complexes on unpatterned surfaces. Bubbles nucleate on catalytic Janus disks adjacent to a hydrophobic surface. By changing the wettability of the surface through electrowetting, the bubbles show a variety of reactions, depending on the shape and periodicity of the electrical signal. Time-resolved (mu s) imaging of bubble radial oscillations reveals possible mechanisms for the lateral mobility of bubbles on a surface under electrowetting: bubble instability is induced when electric pulses are carefully adjusted. This instability is used to control the surface-bound bubble locomotion and is described in terms of the change in surface energy. It is shown that a deterministic force applied normal can lead to a random walk of micrometer-sized bubbles by exploiting the phenomenon of contact angle hysteresis. Finally, bubble use in nature for underwater locomotion and the actuated bubble locomotion presented in this study are compared