Biology subjects

Dimova, R.

Publications and source records attributed to Dimova, R..

36 records · Page 2Linked to original sources

bioRxiv2023

Bilayer charge asymmetry destabilizes membranes upon poration

Transmembrane asymmetry is ubiquitous in cells, particularly with respect to lipids, where charged lipids are mainly restricted to one monolayer. We investigate the influence of anionic lipid asymmetry on the stability of giant unilamellar vesicles (GUVs), minimal plasma membrane models. To quantify asymmetry, we apply the fluorescence quenching assay, which is often difficult to reproduce and caution in handling the quencher is generally underestimated. Thus, we first optimize this assay and then apply it to GUVs prepared with the inverted emulsion transfer protocol using increasing fractions of anionic lipids restricted to one leaflet. This protocol is found to produce highly asymmetric bilayers, but with [~]20% interleaflet mixing. To probe the stability of asymmetric vs symmetric membranes, we expose the GUVs to porating electric pulses and monitor the fraction of destabilized vesicles. The pulses open macropores, and the GUVs either completely recover or exhibit leakage or bursting/collapse. Residual oil destabilizes porated membranes and destabilization is even more pronounced in asymmetrically charged membranes. This is corroborated by the measured pore edge tension decrease with increasing charge asymmetry. Using GUVs with membranes with imposed transmembrane pH asymmetry, we confirm that poration-triggered destabilization does not depend on the approach used to generate membrane asymmetry.

biophysics↗

bioRxiv2023

Wetting by biomolecular condensates increases membrane lipid packing and dehydration

Membrane wetting by biomolecular condensates recently emerged as a key phenomenon in cell biology, playing an important role in a diverse range of processes across different organisms. However, an understanding of the molecular mechanisms behind condensate formation and interaction with lipid membranes is still missing. To study this, we exploited the properties of the dyes ACDAN and LAURDAN as nano-environmental sensors in combination with phasor analysis of hyperspectral and lifetime imaging microscopy. Using glycinin as a model condensate-forming protein and giant vesicles as model membranes, we obtained vital information on the process of condensate formation and membrane wetting. Our results reveal that glycinin condensates display differences in water dynamics when changing the salinity of the medium as a consequence of rearrangements in the secondary structure of the protein. Remarkably, analysis of membrane-condensates interaction with protein as well as polymer condensates indicated a correlation between increased wetting affinity and enhanced lipid packing. This is demonstrated by a decrease in the dipolar relaxation of water across all membrane-condensate systems, suggesting a general mechanism to tune membrane packing by condensate wetting.

biophysics↗

bioRxiv2023

Modulating membrane shape and mechanics of minimal cells by light: area increase, softening and interleaflet coupling of membrane models doped with azobenzene-lipid photoswitches

Light can effectively interrogate biological systems in a reversible and physiologically compatible manner with high spatiotemporal precision. Understanding the biophysics of photo-induced processes in bio-systems is crucial for achieving relevant clinical applications. Employing membranes doped with the photolipid azobenzene-phosphatidylcholine (azo-PC), we provide a holistic picture of light-triggered changes in membrane kinetics, morphology and material properties obtained from correlative studies on cell-sized vesicles, Langmuir monolayers, supported lipid bilayers and molecular dynamics simulations. Light-induced membrane area increase as high as [~]25% and a 10-fold decrease in the membrane bending rigidity is observed upon trans-to-cis azo-PC isomerization associated with membrane leaflet coupling and molecular curvature changes. Vesicle electrodeformation measurements and atomic force microscopy reveal that trans azo-PC bilayers are thicker than POPC bilayer but have higher specific membrane capacitance and dielectric constant suggesting an increased ability to store electric charges across the membrane. Lastly, incubating POPC vesicles with azo-PC solutions resulted in the insertion of azo-PC in the membrane enabling them to become photoresponsive. All these results demonstrate that light can be used to finely manipulate the shape, mechanical and electric properties of photolipid-doped minimal cell models and liposomal drug carriers, thus, presenting a promising therapeutic alternative for the repair of cellular disorders.

biophysics↗

bioRxiv2022

Effect of leaflet asymmetry on mechanical properties of lipid bilayers with phosphatidic acid

The asymmetry of membranes has a significant impact on their biophysical characteristics and behavior. This study investigates the composition and mechanical properties of symmetric and asymmetric membranes in giant unilamellar vesicles (GUVs) made of phosphatidylcholine (POPC) and phosphatidic acid (POPA). A combination of fluorescence quantification, zeta potential measurements, micropipette aspiration and bilayer molecular dynamics simulations are used to characterize these membranes. The outer leaflet composition in vesicles is found consistent across the two preparation methods we employed, namely electroformation and inverted emulsion transfer. However, characterizing the inner leaflet poses challenges. Micropipette aspiration of GUVs show that oil residues do not substantially alter membrane elasticity, but simulations reveal increased membrane thickness and decreased interleaflet coupling in the presence of oil. Asymmetric membranes with a POPC:POPA mixture in the outer leaflet and POPC in the inner leaflet display similar stretching elasticity values to symmetric POPC:POPA membranes, suggesting potential POPA insertion into the inner leaflet during vesicle formation and suppressed asymmetry. The inverse compositional asymmetry, with POPC in the outer leaflet and POPC:POPA in the inner yield less stretchable membranes with higher compressibility modulus compared to their symmetric counterparts. Challenges in achieving and predicting compositional correspondence highlight the limitations of phase-transfer-based methods. Additionally, caution is advised when using fluorescently labeled lipids (even at low fractions of 0.5 mol%), as unexpected gel-like domains in symmetric POPC:POPA membranes were observed only with a specific type of labeled DOPE (dioleoylphosphatidylethanolamine) and the same fraction of unlabeled DOPE. The latter suggest that such phase separation may result from interactions between lipids and membrane fluorescent probes. Overall, this study underscores the complexity of factors influencing GUV membrane asymmetry, emphasizing the need for further research and improvement of characterization techniques. SIGNIFICANCEAsymmetrically charged lipid bilayer models are superior to commonly used symmetrical ones, exhibiting naturally present asymmetry, thereby exhibiting a more adequate range of biophysical membrane characteristics better reflecting biological membranes. This study focuses on the mechanical properties of phosphatidic acid (PA)-enriched membranes, a crucial lipid for cellular lipid metabolism, e.g. glycerophospholipid synthesis, and for signal transduction. Micropipette aspiration, fluorescent PA-sensor, and zeta potential studies demonstrate that asymmetric membranes are less stretchable than symmetric ones. Accompanying in silico studies on the symmetric membranes confirm that oil impurities do not influence the membrane stretching elasticity but increase its thickness and decrease the coupling of the two leaflets, which sheds light on the elastic behavior of experimental models of asymmetric lipid bilayers.

biophysics↗

bioRxiv2022

Kinetic relaxation of giant vesicles validates diffusional softening in a binary lipid mixture

The stiffness of biological membranes determines the work required by cellular machinery to form and dismantle vesicles and other lipidic shapes. Model membrane stiffness can be determined from the equilibrium distribution of giant unilamellar vesicle surface undulations observable by phase contrast microscopy. With two or more components, lateral fluctuations of composition will couple to surface undulations depending on the curvature sensitivity of the constituent lipids. The result is a broader distribution of undulations whose complete relaxation is partially determined by lipid diffusion. In this work, kinetic anaysis of the undulations of giant unilamellar vesicles made of phospatidylcholine-phosphatidylethanolamine mixtures validates the molecular mechanism by which the membrane is made 25% softer than a single-component one. The mechanism is relevant to biological membranes, which have diverse and curvature-sensitive lipids.

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↗

bioRxiv2022

GM1 asymmetry in the membrane stabilizes pores

Cell membranes are highly asymmetric and their stability against poration is crucial for survival. We investigated the influence of membrane asymmetry on electroporation of giant unilamellar vesicles with membranes doped with GM1, a ganglioside asymmetrically enriched in the outer leaflet of neuronal cell membranes. Compared to symmetric membranes, the lifetimes of micronsized pores are about an order of magnitude longer suggesting that pores are stabilized by GM1. Internal membrane nanotubes caused by the GM1 asymmetry, obstruct and additionally slow down pore closure, effectively reducing pore edge tension and leading to leaky membranes. Our results point to the drastic effects this ganglioside can have on pore resealing in biotechnology applications based on poration as well as on membrane repair processes. SIGNIFICANCEMembrane pore closure is crucial for cell survival and is important for biotechnological and medicine applications based on transfer of material, e.g. drugs, genes, through pores. Electroporation is widely used as means to perforate the membrane but factors governing membrane resealing are still a matter of debate, in particular the large variations of pore lifetimes. Here, we probed the effect of bilayer asymmetry on pore dynamics employing cell-sized giant unilamellar vesicles doped with the ganglioside GM1 (asymmetrically enriched in neurons). We find that the presence of GM1 and its asymmetric distribution in the bilayer dramatically slows down pore resealing, not only by mere molecular stabilization of the pore rim, but also by generating membrane nanotubes.

biophysics↗

bioRxiv2022

Controlled adhesion, membrane pinning and vesicle transport by Janus particles

The interactions between biomembranes and particles are key to many applications, but the lack of controllable model systems to study them limits the progress in their research. Here, we describe how Janus polystyrene microparticles, half coated with iron, can be partially engulfed by artificial cells, namely giant vesicles, with the goals to control and investigate their adhesion and degree of encapsulation. The interaction between the Janus particles and these model cell membrane systems is mediated by electrostatic charge, offering a further mode of modulation in addition to the iron patches. The ferromagnetic particle coatings also enable the ability to manipulate and transport the vesicles by magnetic fields.

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

Viscosity of fluid membranes measured from vesicle deformation

Viscosity is a key property of cell membranes that controls mobility of embedded proteins and membrane remodeling. Measuring it is challenging because existing approaches involve complex experimental designs and/or models, and the applicability of some is limited to specific systems and membrane compositions. As a result there is scarcity of data and the reported values for membrane viscosity vary by orders of magnitude for the same system. Here, we show how viscosity of bilayer membranes can be obtained from the transient deformation of giant unilamellar vesicles. The approach enables a non-invasive, probe-independent and high-throughput measurement of the viscosity of bilayers made of lipids or polymers with a wide range of compositions and phase state. Pure lipid and single-phase mixed bilayers are found to behave as Newtonian fluids with strain-rate independent viscosity, while phase-separated and diblock-copolymers systems exhibit shear-thinning in the explored range of strain rates 1-2000 s-1. The results also reveal that electrically polarized bilayers can be significantly more viscous than charge-neutral bilayers. These findings suggest that biomembrane viscosity is a dynamic property that can be actively modulated not only by composition but also by membrane polarization, e.g., as in action potentials.

biophysics↗

bioRxiv2020

The ESCRT-III machinery participates in the production of extracellular vesicles and protein export during Plasmodium falciparum infection

Infection with Plasmodium falciparum enhances extracellular vesicles (EVs) production in parasitized red blood cells (pRBC), an important mechanism for parasite-to-parasite communication during the asexual intraerythrocytic life cycle. The endosomal sorting complex required for transport (ESCRT), and in particular the ESCRT-III sub-complex, participates in the formation of EVs in higher eukaryotes. However, RBCs have lost the majority of their organelles through the maturation process, including an important reduction in their vesicular network. Therefore, the mechanism of EV production in P. falciparum-infected RBCs remains to be elucidated. Here we demonstrate that P. falciparum possesses a functional ESCRT-III machinery that is activated by an alternative recruitment pathway involving the action of PfBro1 and PfVps32/PfVps60 proteins. Additionally, multivesicular bodies formation and membrane shedding, both reported mechanisms of EVs production, were reconstituted in the membrane model of giant unilamellar vesicles using the purified recombinant proteins. Moreover, the presence of PfVps32, PfVps60 and PfBro1 in EVs purified from a pRBC culture was confirmed by super-resolution microscopy. In accordance, disruption of the Pfvps60 gene led to a reduction in the number of the produced EVs in the KO strain when compared with the parental 3D7 strain. Overall, our results increase the knowledge on the underlying molecular mechanisms during malaria pathogenesis and demonstrate that ESCRT-III P. falciparum proteins participate in EVs production.

microbiology↗

bioRxiv2020

To close or to collapse: the role of charges on membrane stability upon pore formation

Resealing of membrane pores is crucial for cell survival. We study membrane surface charge and medium composition as defining regulators of membrane stability. Pores are generated by electric field or detergents. Giant vesicles composed of zwitterionic and negatively charged lipids mixed at varying ratios are subjected to a single strong electric pulse. Charged vesicles are prone to catastrophic collapse transforming them into tubular structures. The spectrum of destabilization responses includes the generation of long-living submicroscopic pores and partial vesicle bursting. The origin of these phenomena is related to the membrane edge tension, which governs pore closure. This edge tension significantly decreases as a function of the fraction of charged lipids. Destabilization of charged vesicles upon pore formation is universal - it is also observed with other poration stimuli. Disruption propensity is enhanced for membranes made of lipids with higher degree of unsaturation. It can be reversed by screening membrane charge in the presence of calcium ions. We interpret the observed findings in light of theories of stability and curvature generation and discuss mechanisms acting in cells to prevent total membrane collapse upon poration. Enhanced membrane stability is crucial for the success of electroporation-based technologies for cancer treatment and gene transfer.

biophysics↗

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↗

bioRxiv2020

Interferon-Induced Transmembrane Protein 3 Blocks Fusion of Diverse Enveloped Viruses by Locally Altering Mechanical Properties of Cell Membranes

Interferon-induced transmembrane protein 3 (IFITM3) potently inhibits entry of diverse enveloped viruses by trapping the viral fusion at a hemifusion stage, but the underlying mechanism remains unclear. Here, we show that recombinant IFITM3 reconstituted into lipid vesicles induces negative membrane curvature and that this effect maps to its small amphipathic helix (AH). We demonstrate that AH: (i) partitions into lipid-disordered domains where IAV fusion occurs, (ii) induces negative membrane curvature, and (iii) increases lipid order and membrane stiffness. Additionally, replacing the IFITM3 AH with AH from an unrelated protein did not compromise its antiviral activity. These effects on membrane properties correlate with the fusion-inhibitory activity, as exogenous addition of AH to insulin-producing cells reduces glucose-stimulated insulin secretion. Our results thus imply that IFITM3 inhibits the transition from hemifusion to full fusion by imposing an unfavorable membrane curvature and increasing the order and stiffness of the cytoplasmic leaflet of endosomal membranes.

biophysics↗

bioRxiv2020

Fluctuation spectroscopy of giant unilamellar vesicles using confocal and phase contrast microscopy

A widely used method to measure the bending rigidity of bilayer membranes is fluctuation spectroscopy, which analyses the thermally-driven membrane undulations of giant unilamellar vesicles recorded with either phase-contrast or confocal microscopy. Here, we analyze the fluctuations of the same vesicle using both techniques and obtain consistent values for the bending modulus. We discuss the factors that may lead to discrepancies.

biophysics↗

bioRxiv2019

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

Plasma membranes dynamically respond to external cues and changing environment. Quantitative measurements of these adaptations can elucidate the mechanism that cells exploit to survive, adapt and function. However, cell-based assays are affected by active processes while measurements on synthetic models suffer from compositional limitations. Here, as a model system we employ giant plasma membrane vesicles (GPMVs), which largely preserve the plasma membrane lipidome and proteome. From analysis of fluorescence emission and lifetime of environment-sensitive dyes, and membrane shape fluctuations, we investigate how plasma membrane order, viscosity and bending rigidity are affected by different stimuli such as cell seeding density in three different cell models. Our studies reveal that bending rigidity of plasma membranes vary with lipid order and microviscosity in a highly correlated fashion. Thus, readouts from polarity- and viscosity-sensitive probes represent a promising indicator of membrane mechanical properties. Quantitative analysis of the data allows for comparison to synthetic lipid membranes as plasma membrane mimetics.

biophysics↗