Biology subjects

Lipowsky, R.

Publications and source records attributed to Lipowsky, R..

9 recordsLinked to original sources

bioRxiv2026

Giant Vesicle Platform for Synthetic Endoplasmic Reticulum Networks

The endoplasmic reticulum (ER) is a membrane-bound organelle found in all eukaryotic cells. This organelle has a fascinating morphology, which involves a network of membrane nanotubes and three-way junctions connecting three nanotubes. Previous reconstitution studies were based on proteo-liposomes with a size of 100 to 200 nanometers, using electron microscopy as the main method to image the resulting membrane morphologies. These studies revealed the importance of membrane GTPases, that is, membrane proteins that hydrolyse GTP. Here, we introduce an alternative platform for the formation of synthetic ER networks starting from giant unilamellar vesicles with a typical size of 10 mirometers and containing a single membrane GTPase such as atlastin from fruit flies or Sey1 from yeast. This giant vesicle approach makes the networks accessible to optical microscopy, by which one can elucidate both their structure and their dynamics. We first demonstrate that the membranes' propensity to form synthetic ER networks depends strongly on the lipid composition. More precisely, we show that this propensity varies non-monotonically with the mole fraction of cholesterol and that the frequency of three-way junctions has a maximum for an optimal mole fraction between 10 and 15 mol%. This optimal value is comparable to the mole fraction of cholesterol as estimated for ER membranes in vivo. We also demonstrate the formation of synthetic ER networks when the membranes contain only the membrane GTPase Sey1 but no other membrane protein, which implies that Sey1 is able on its own to generate a high membrane curvature. Based on the reconstitution approach introduced here, the synthetic ER networks can now be further investigated using the versatile toolbox that has been developed for giant vesicles.

synthetic biology↗

bioRxiv2024

Lipid packing and cholesterol content regulate membrane wetting by biomolecular condensates.

Biomolecular condensates play a pivotal role in cellular processes by interacting with membranes and leading to wetting transitions and to mutual remodeling. Using a combination of hyperspectral imaging, phasor analysis, and fluid-elastic parameter measurements, we investigated how membrane lipid packing affects condensate wetting. Our results show that it is not only the membrane phase state, but rather the degree of lipid packing that determines the condensate affinity for membranes. Increasing lipid chain length or cholesterol content enhances lipid packing, thereby decreasing condensate affinity. This regulatory mechanism is consistent across various condensate-membrane systems, underscoring the critical role of the membrane interface. Additionally, protein adsorption promotes extensive membrane remodeling, including tube and double-membrane sheet formation. This work provides a novel mechanism by which membrane composition fine-tunes condensate wetting, highlighting its potential impact on cellular functions and organelle interactions.

biophysics↗

bioRxiv2024

Photoswitchable endocytosis of biomolecular condensates in giant vesicles

Interactions between membranes and biomolecular condensates can give rise to complex phenomena such as wetting transitions, mutual remodeling, and endocytosis. In this study, we demonstrate a light-triggered manipulation of condensate engulfment using giant vesicles containing photoswitchable lipids. UV irradiation increases the membrane area, facilitating a rapid condensate endocytosis, which can be reverted by blue light. The affinity of the protein-rich condensates to the membrane and the reversibility of the engulfment processes is quantified from confocal microscopy images. The degree of engulfment, whether partial or complete, depends on the initial membrane excess area and the relative sizes of vesicles and condensates. Theoretical estimates suggest that utilizing the light-induced excess area to increase the vesicles-condensate adhesion interface is energetically more favorable than the energy gain from folding the membrane into invaginations and tubes. Our overall findings demonstrate that membrane-condensate interactions can be easily and quickly modulated via light, providing a versatile system for building platforms to control cellular events and design intelligent drug delivery systems for cell repair.

biophysics↗

bioRxiv2022

Membrane wetting, molding and reticulation by protein condensates

Cells compartmentalize their components in liquid-like condensates, which can be reconstituted in vitro. Although these condensates interact with membrane-bound organelles, the potential of membrane remodeling and the underlying mechanisms are not well understood. Here, we demonstrate that interactions between protein condensates (including hollow ones) and membranes can lead to remarkable morphological transformations and describe these with theory. Modulation of solution salinity or membrane composition drives the condensate-membrane system through two wetting transitions, from dewetting, through a broad regime of partial wetting, to complete wetting. A new phenomenon, namely fingering or ruffling of the condensate-membrane interface is observed when sufficient membrane area is available, producing intricately curved structures. The observed morphologies are governed by the interplay of adhesion, membrane elasticity, and interfacial tension. Our results highlight the relevance of wetting in cell biology, and pave the way for the design of synthetic membrane-droplet based biomaterials and compartments with tunable properties.

biophysics↗

bioRxiv2022

Stepwise remodelling and subcompartment formation in individual vesicles by three ESCRT-III proteins

The endosomal sorting complex required for transport (ESCRT) is a multi-protein complex involved in several membrane remodelling processes. Different approaches have been used to dissect the mechanism by which ESCRT proteins produce scission in the membranes. However, the underlying mechanisms generating the membrane deformations remain poorly understood. In this study, giant unilamellar vesicles (GUVs), microfluidic technology and micropipette aspiration are combined to continuously follow the ESCRT-III-mediated membrane remodelling on the single-vesicle level for the first time. With this approach, we identify different mechanisms by which a minimal set of three ESCRT-III proteins from the phagocytic parasite Entamoeba histolytica reshape the membrane. These proteins modulate the membrane stiffness and spontaneous curvature to regulate the bud size and generate intraluminal vesicles in GUVs even in the absence of ATP. We show that the bud stability depends on the protein concentration and membrane tension. The approach introduced here should open the road to diverse applications in synthetic biology for establishing artificial cells with several membrane compartments.

biophysics↗

bioRxiv2022

Binding of His-tagged fluorophores to lipid bilayers and giant vesicles

His-tagged molecules can be attached to lipid bilayers via certain anchor lipids, a method that has been widely used for the bio-functionalization of membranes and vesicles. To observe the membrane-bound molecules, it is useful to consider His-tagged molecules that are fluorescent as well. Here, we study two such molecules, green fluorescence protein (GFP) and green-fluorescent fluorescein isothiocyanate (FITC), both of which are tagged with a chain of six histidines (6H) that bind to the anchor lipids within the bilayers. The His-tag 6H is much smaller than the GFP molecule but somewhat larger than the FITC dye. The lipid bilayers form giant unilamellar vesicles (GUVs), the behavior of which can be directly observed in the optical microscope. We apply and compare three well-established preparation methods for GUVs: electroformation on platinum wire, polyvinyl alcohol (PVA) hydrogel swelling, and electroformation on indium tin oxide (ITO) glass. Microfluidics is used to expose the GUVs to a constant fluorophore concentration in the exterior solution. The brightness of membrane-bound 6H-GFP exceeds the brightness of membrane-bound 6H-FITC, in contrast to the quantum yields of the two fluorophores in solution. In fact, 6H-FITC is observed to be strongly quenched by the anchor lipids which bind the fluorophores via Ni2+ ions. For both 6H-GFP and 6H-FITC, the membrane fluorescence is measured as a function of the fluorophores molar concentration. The theoretical analysis of these data leads to the equilibrium dissociation constants Kd = 37.5 nM for 6H-GFP and Kd = 18.5 nM for 6H-FITC. We also observe a strong pH-dependence of the membrane fluorescence.

biophysics↗

bioRxiv2021

Super-resolution imaging of highly curved membrane structures in giant vesicles encapsulating molecular condensates

Molecular crowding is an inherent feature of the cell interior. Synthetic cells as provided by giant unilamellar vesicles (GUVs) encapsulating macromolecules (polyethylene-glycol and dextran) represent an excellent mimetic system to study membrane transformations associated with molecular crowding and protein condensation. Similarly to cells, such GUVs loaded with macromolecules exhibit highly curved structures such as internal nanotubes. In addition, upon liquid-liquid phase separation as inside living cells, the membrane of GUVs encapsulating an aqueous two-phase system deforms to form apparent kinks at the contact line of the interface between the two aqueous phases. These structures, nanotubes and kinks, have dimensions below optical resolution and if resolved, can provide information about material properties such as membrane spontaneous curvature and intrinsic contact angle describing the wettability contrast of the encapsulated phases to the membrane. Previous experimental studies were based on conventional optical microscopy which cannot resolve these membrane and wetting properties. Here, we studied these structures with super-resolution microscopy, namely stimulated emission depletion (STED) microscopy, together with microfluidic manipulation. We demonstrate the cylindrical nature of the nanotubes with unprecedented detail based on the superior resolution of STED and automated data analysis. The spontaneous curvature deduced from the nanotube diameters is in excellent agreement with theoretical predictions. Furthermore, we were able to resolve the membrane "kink" structure as a smoothly curved membrane demonstrating the existence of the intrinsic contact angle. We find very good agreement between the directly measured values and the theoretically predicted ones based on the apparent contact angles on the micrometer scale. During different stages of cellular events, biomembranes undergo a variety of shape transformations such as the formation of buds and nanotubes regulated by membrane necks. We demonstrate that these highly curved membrane structures are amenable to STED imaging and show that such studies provide important insights in the membrane properties and interactions underlying cellular activities.

biophysics↗

bioRxiv2021

Structural variability and concerted motions of the T cell receptor - CD3 complex

We investigate the structural and orientational variability of the membrane-embedded T cell receptor (TCR) - CD3 complex in extensive atomistic molecular dynamics simulations based on the recent cryo-EM structure determined by Dong et al. (2019). We find that the TCR extracellular (EC) domain is highly variable in its orientation by attaining tilt angles relative to the membrane normal that range from 15{degrees} to 55{degrees}. The tilt angle of the TCR EC domain is both coupled to a rotation of the domain and to characteristic changes throughout the TCR - CD3 complex, in particular in the EC interactions of the C{beta} FG loop of the TCR, as well as in the orientation of transmembrane helices. The concerted motions of the membrane-embedded TCR - CD3 complex revealed in our simulations provide atomistic insights for force-based models of TCR activation, which involve such structural changes in response to tilt-inducing forces on antigen-bound TCRs.

immunology↗

bioRxiv2020

Super-elasticity of plasma- and synthetic membranes by coupling of membrane asymmetry and liquid-liquid phase separation

Biological cells are contained by a fluid lipid bilayer (plasma membrane, PM) that allows for large deformations, often exceeding 50% of the initial (or projected) PM area. Biochemically isolated lipids self-organize into membranes, but the extraordinary deformability of the plasma membrane is lost. Pure lipid bilayers are prone to rupture at small (<2-4%) area strains and this limits progress for synthetic reconstitution of cellular features such as migration, phagocytosis and division. Here, we show that by preserving PM structure and composition during isolation from cells, vesicles with cell-like elasticity are obtained. We found that these plasma membrane vesicles store significant area in the form of nanotubes in their lumen. These are recruited by mechanical tension applied to the outer vesicle membrane showing an apparent elastic response. This "super-elastic" response emerges from the interplay of lipid liquid-liquid phase separation and membrane asymmetry. This finding allows for bottom-up engineering of synthetic vesicles that appear over one magnitude softer and with three fold larger deformability than conventional lipid vesicles.

biophysics↗