Biology subjects

Dimova, R.

Publications and source records attributed to Dimova, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Single-protein diffusion in micrometer-size vesicles is governed by viscosity, surface charge, and membrane interactions

Giant unilamellar vesicles (GUVs) are a powerful model system for studying protein dynamics in cell-sized compartments with a precisely tunable physicochemical environment. Here, we apply Single-Molecule Displacement Mapping (SMdM) to quantify how luminal viscosity, ionic strength, membrane lipid composition, and protein surface charge of soluble proteins govern intraluminal single-molecule diffusion. Increasing viscosity led to a predictable decrease in the diffusion coefficient. However, spatially resolved diffusion measurements reveal reduced protein mobility near the membrane, suggesting additional membrane-proximal interactions not captured by particle-based simulations. We observe long-range (~1 um) effects of membrane charge on protein mobility that extend well beyond classical Debye screening, which may reflect shape fluctuations of the GUV membrane. For the membrane-associated protein LactC2, we resolve two distinct diffusing populations within a single vesicle, corresponding to free and membrane-bound states. Our workflow provides a quantitative framework for probing single-protein mobility in vesicle-based synthetic cells. These results advance our understanding of how molecular crowding and transient membrane interactions shape protein diffusion within cell-sized compartments, with possible implications for metabolic organization.

biophysics↗

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↗

bioRxiv2026

Light-controlled membrane remodeling in gel-fluid phase-separated giant vesicles using photoswitchable lipids

Photoswitchable lipids enable optical control of membrane area, mechanics and phase behavior, offering a platform to study stimuli-responsive biomimetic systems. However, it remains unclear how the spatial organization of coexisting membrane phases governs photoinduced mechanical responses. Here, we incorporate the photoswitch azobenzene-phosphatidylcholine (azoPC) and dipalmitoyphosphatidylcholine (DPPC) into gel-fluid phase-separated giant unilamellar vesicles (GUVs) to probe how domain architecture controls light-driven deformation. We combine differential scanning calorimetry, temperature-controlled confocal microscopy, and a calibrated heating stage to quantify phase transitions and visualize domain dynamics. Dispersed domains produce global GUV crumpling upon UV-light-induced trans-to-cis isomerization of azoPC, whereas coarsened fluid domains locally confine deformation to budding regions of the GUVs; both responses are reversed by blue light. Temperature-controlled imaging reveals that the gel-fluid transition in GUVs is considerably broader than the calorimetric profile suggests, with coexisting phases detectable well above the calorimetry peak transition temperature. Well above the transition temperature, i.e. in the fully melted membrane, UV irradiation unexpectedly induces reversible nucleation of gel-like flower domains, consistent with an increased transition temperature in the cis azoPC state due to lipid packing incompatibility with DPPC. These results demonstrate that membrane domain architecture dictates the spatial distribution of photoinduced remodeling and that photoswitchable lipids can tune both membrane morphology and phase equilibria, enabling new strategies for stimuli-responsive synthetic cells and soft actuators.

biophysics↗

bioRxiv2025

Protein-free membrane fusion: a refined view of the delicate fusogenic properties of calcium

In the late 20th century, calcium took on the identity of an independent fusogen, when it was found to induce fusion of anionic large unilamellar vesicles (LUVs), yet its ability to drive fusion in cell-sized membranes remains poorly understood. Here, we directly quantify calcium-mediated fusion of giant unilamellar vesicles (GUVs) using a microfluidic trapping platform combined with confocal microscopy, enabling simultaneous measurement of lipid mixing, content mixing, and fusion outcomes across hundreds of single vesicles. We systematically map fusion efficiency as a function of calcium concentration, membrane composition, and mechanically imposed tension. We find that calcium-induced fusion of GUVs in the absence of proteins is remarkably fickle and composition-sensitive, as the vesicles need to be sufficiently instable to allow the opening of the fusion pore, yet stable enough to prevent bursting and collapse. Negatively charged GUVs containing high fractions of DOPE exhibit the highest fusogenic responsiveness, whereas other compositions undergo extensive lipid mixing without pore formation. Increasing membrane tension can shift this balance and promote full fusion, revealing a narrow parameter space in which calcium acts as an effective protein-free fusogen for cell-sized membranes. These findings clarify longstanding discrepancies between LUV- and GUV-based calcium fusion assays and provide quantitative design rules for employing calcium as a fusogen in synthetic biology and membrane-reconstitution studies, where controlled membrane growth, vesicle-vesicle fusion, and module integration are central to building and sustaining artificial cells. SIGNIFICANCECalcium is widely regarded as a potent, protein-free fusogen in nanometer-scale lipid vesicles, yet its relevance for fusion between cell-sized membranes remains unresolved. This gap limits our ability to translate decades of LUV-based fusion studies to more physiologically realistic systems and restricts the use of calcium in synthetic cell engineering. By directly quantifying calcium-mediated fusion in giant unilamellar vesicles (GUVs), we identify the membrane compositions and mechanical conditions under which calcium can reliably drive full fusion rather than mere lipid mixing. Our findings provide the first systematic map of calciums fusogenic parameter space in cell-sized membranes, enabling more informed design of fusion-based assays, reconstitution experiments, and strategies for membrane growth in artificial cells.

biophysics↗

bioRxiv2025

Label-free in situ approach for characterizing the macromolecular composition and water content in biomolecular condensates

Biomolecular condensates are membraneless cellular organelles that form via liquid-liquid phase separation of proteins and nucleic acids. Their functional roles are tightly coupled to material properties like viscosity and hydrophobicity, which serve as key markers of cellular state. However, conventional determination of condensate composition and water content relies on invasive procedures that can damage samples. Here, we introduce Raman spectroscopy coupled with spectral phasor analysis as an in situ, label-free approach to resolve the chemical profiles and molecular concentrations within both the dense and dilute phases of biomolecular condensates. This method outperforms traditional regression and deconvolution approaches, yielding a precise readout of client molecule partitioning. By accounting for contributions of the protein backbone to the Raman spectra of condensates, we assess the signature of "solid-like" hydrogen-bonded water from protein hydration, revealing that most water molecules within condensates retain bulk, "liquid-like" properties. Finally, using environment-sensitive fluorescent probes, we demonstrate that macromolecular structure and water content--rather than hydrogen bonding alone--drive condensate hydrophobicity; notably, the dense phase remains predominantly water-rich even at low apparent dielectric constants.

biophysics↗

bioRxiv2025

The Hidden Cost of Fusion: Intrinsic Asymmetry in Vesicle Fusion Restricts Synthetic Cell Growth

Membrane fusion is essential for signaling, cargo delivery, and synthetic cell growth, yet its mechanical consequences remain poorly defined. How fusion-driven membrane growth can be sustained without compromising compartment stability remains an unresolved challenge. Here, we established a minimal reconstituted system where content-loaded small liposomes fuse with single cell-sized giant unilamellar vesicles (GUVs), combining micropipette delivery, electrodeformation, and live imaging. Fusion outcomes were quantified through lipid and content mixing assays, GUV electrodeformation to track area and tension, and phase contrast imaging to monitor leakage. GUVs incorporated lipids and cargo from hundreds of thousands of vesicles at unprecedented efficiency rates, enabling substantial growth. However, accumulation of leaflet asymmetries induced curvature and tension, driving budding, rupture and leakage. Hemifusion amplified these destabilizing effects. Lipid number asymmetries emerge as a dominant mechanical cost of fusion, highlighting how cells may regulate these processes and guiding the design of therapeutic delivery systems and synthetic cells capable of robust and stable growth. Significance StatementMembrane fusion enables essential biological processes from secretion to cell growth. Using a synthetic system, we show that rapid fusion of small vesicles with model cell-like compartments drives dramatic growth but also introduces leaflet asymmetries that build curvature and tension, limit expansion and compromise membrane stability. By uncovering lipid number asymmetry as a key mechanical cost of fusion, our study explains why cells tightly regulate fusion events and provides principles for designing delivery vehicles and synthetic cells capable of robust, sustainable growth.

biophysics↗

bioRxiv2025

Integrating experiments and simulations to unravel coacervate-membrane interactions: Insights into de-mixing and morphology modulation

Intrinsically disordered proteins and polypeptides can undergo liquid-liquid phase separation (LLPS) to form condensates/coacervates, which play numerous regulatory roles in the cell. Recently, the relevance of such LLPS occurring in the vicinity of membranes has been brought to light by several experimental studies. Membrane-adsorbed condensates are crucial for biomolecular localization, and in some cases, phase separation of proteins at the membrane surface induces significant changes in membrane morphology. A detailed microscopic understanding of the mechanisms behind these observations remains incomplete. Here we combine experiments and molecular simulations to unravel structural and dynamic features of the coacervate/membrane interface across scales. We study poly-Lysine/poly-Aspartate (K10/D10) coacervates as a prototype of phase-separated condensates with different unilamellar liposomes. Using a multiscale characterization approach that combines confocal microscopy, hyperspectral imaging, fluorescence recovery after photobleaching, and two complementary coarse-grained approaches, we show that the membrane-condensate affinity can be tuned by the anionic lipid content and quantified through the intrinsic contact angle - a material property derived from system geometry - both in vitro and in silico. We find that the membrane region in contact with the condensate displays a nearly two-fold reduced fluidity compared to the bare membrane. This is attributed to orientational ordering of lipid tails, resulting in decreased area per lipid. Moreover, we observed local lipid de-mixing induced by the coacervate adsorption. This study provides an effective framework for integrating experiment and computation to characterize the properties of coacervate/membrane interfaces that are critical to the functional impacts of these interactions.

biophysics↗

bioRxiv2025

Fluorescence-based mapping of dielectric permittivity of condensates and their environment

Biomolecular condensates, essential for cellular organization, possess mesoscale properties largely governed by hydrophobicity, influencing molecule partitioning and material characteristics like viscosity, surface tension, and hydration. While hydrophobicitys role is increasingly recognized, its impact on membrane-condensate interactions remains unexplored. Here, we introduce a novel approach, combining hyperspectral imaging of an environment-sensitive dye and phasor analysis, to quantitatively map the local dielectric permittivity of both condensates and their environment with pixel resolution as sensed by the dye. This robust method reveals a surprisingly broad range of condensate permittivities, from oil-like to water-like. Importantly, we uncover that membrane affinity is not dictated by condensate permittivity itself, but by the permittivity contrast with their surroundings. Indeed, membrane wetting affinity is found to scale linearly with this contrast, unveiling a unifying dielectric principle governing condensate-membrane interactions. Compatible with live-cell and in vitro imaging, this technique provides unprecedented insights into condensate biophysics and function and opens new avenues for studying biomolecular condensate biology.

biophysics↗

bioRxiv2025

Insights into CPSFL1 Induced Membrane Dynamics: A Multifaceted Regulator Linking Vesicle Formation to Thylakoid Biogenesis

Light drives plant life through photosynthesis, a process that takes place in the thylakoid membrane of the chloroplast, an organelle of cyanobacterial origin. The formation of thylakoid membranes within the chloroplast involves the eukaryote-specific factor CHLOROPLAST SEC14 LIKE PROTEIN 1 (CPSFL1), which shares strong sequence homology with the vesicle trafficking regulator SEC14. CSPFL1 is essential for vesicle formation, yet its specific molecular function in this process has remained unclear. In this study, we characterized CSPFL1 functions both in vitro and in vivo. Using a minimal membrane system of giant unilamellar vesicles (GUVs), we show that CPSFL1 alone can induce vesiculation. This process is mediated by lipid binding and membrane deformation, driven by curvature sensing and lipid-protein electrostatics. When expressed in the prokaryote E. coli, the eukaryote-specific CSPFL1 induces membrane curvature and vesicle formation. Plastid CPSFL1 co-purifies with vesicular structures. Lipid compositional analysis of CPSFL1-induced vesicles from bacteria reveals the presence of quinone precursors as cargo, linking CSPFL-mediated vesicle formation to prenylquinone transport. Together, our data suggest that during plant evolution, the eukaryotic vesicle formation system was co-opted for the transport of membrane integral metabolites from the inner envelope to the thylakoid membrane.

plant biology↗

bioRxiv2025

Peptide-induced hydration of lipid bilayers modulates packing pattern and conformations of hydrocarbon chains - a potential pathway for peptide translocation?

Cell-penetrating peptides (CPPs) with a cationic-hydrophobic character are recognized as carriers for delivering various therapeutics and diagnostic agents across cell membranes and into the cells. Among the most studied CPPs, nona-arginine (R9) exhibits superior penetration compared to nona-lysine (K9), suggesting that the penetration ability depends not only on charge, distribution and concentration of peptides but also on the lipid membrane composition. However, for heptapeptides composed of arginine (R), lysine (K) and phenylalanine (F) residues, which show some CPPs properties, these interactions remain unexplored. This study sheds light on the adsorption of R5F2/K5F2 on model prokaryotic (PRO) and eukaryotic (EU) lipid membranes containing a zwitterionic lipid (phosphatidylcholine; PC) and an anionic lipid (either phosphatidylglycerol, PG, in the PRO model, or phosphatidylserine, PS in EU) at the 90:10 molar ratio. Using differential scanning calorimetry (DSC) and temperature-dependent UV-Vis spectroscopy, we observed peptide-induced changes in the interfacial water layer that affect the fluidity and rigidity of lipid bilayers. The distinct adsorption behavior of R5F2/K5F2 on PRO and EU lipid bilayers revealed the changes in lipid packing and hydrocarbon chain conformations as exclusively peptide-dependent features. The peptide-induced formation of vacancies in the non-polar bilayer part is consistent with partial leakage observed in giant unilamellar vesicles. The synchronized arrangement could represent a mechanism for the concerted translocation of CPPs, along with their potential cargo across the lipid membrane. This study provides new insights into the peptide-lipid interactions underlying CPPs functionality.

biophysics↗

bioRxiv2024

Bacteriophages inject DNA into giant unilamellar vesicles mimicking Gram-negative lipopolysaccharide outer membranes

Giant unilamellar vesicles (GUVs) are a versatile platform to study cell membrane functions. We have constructed phospholipid GUVs presenting lipopolysaccharide on their external surface (LPS-GUVs) to mimic the outer membrane (OM) of Gram-negative bacteria. GUVs allow for adjusting a defined OM composition, unlike the dynamic changes of LPS structures typically observed in vivo. The OM is a major control point for the viral genome transfer from bacteriophages into bacterial hosts. We found that siphovirus 9NA specifically binds to the surface of GUVs when presenting its Salmonella Typhimurium LPS phage receptor. Using LPS-GUVs filled with DNA-sensitive dyes we show that after surface fixation, the bacteriophage particle opens and injects its DNA into the GUV lumen. No OM proteins were included in the LPS-GUV membrane, emphasizing that the presence of the LPS membrane glycolipid assembly alone is sufficient to trigger the start of bacteriophage genome transfer. LPS-GUVs thus open a sustainable route to systematic studies of viral infection mechanisms at the host envelope and provide a cell-free platform to study surfaces of pathogenic bacteria. This is an important prerequisite for developing effective antimicrobial therapies based on bacteriophages that target Gram-negative pathogens.

biochemistry↗

bioRxiv2024

Breast cancer cell-derived extracellular vesicles accelerate collagen fibrillogenesis and integrate into the matrix

Extracellular vesicles (EVs) within the extracellular matrix (ECM) are often studied as passive elements whose diffusion and behaviour are subject to the composition and structure of the ECM. While EV diffusion and distribution in tissues are indeed governed by matrix interactions, accumulating evidence suggests that EVs contain much of the cellular machinery required for actively remodeling ECM. Using rheology and confocal reflectance microscopy, we investigate the gelation of collagen I hydrogels formed in the presence of EVs, and show that EVs can play an active role in ECM formation. EVs appear to nucleate new fibrils, recruiting collagen molecules from solution and accelerating their polymerization. Trypsinization of EVs shows that collagen-EV interactions are primarily mediated by surface proteins. The use of extruded plasma membrane vesicles shows that membrane composition determines final fibril length and matrix structure. EVs also become integrated into the fibril structures that they help form, reminiscent of matrix vesicles found in situ within tissues. This represents a plausible way by which EVs are deposited into the ECM, becoming signaling cues for resident cells. Our data show that EV-matrix interactions are dynamic and can contribute to the remodeling of tissue microenvironments. SignificanceExtracellular vesicles (EVs) are nanoscale membrane structures known for their role in facilitating cellto-cell trafficking of proteins, lipids, RNA, and other signaling molecules. In this report, we show that EVs derived from breast cancer cells are not merely passive messengers, but also direct active effectors of extracellular matrix (ECM) remodeling processes. Bulk rheology and confocal microscopy show that these EVs have the ability to nucleate new collagen fibrils and accelerate the formation of dense fibrillar collagenous networks. This has important implications in cancer pathology, where matrix density is often associated with worse disease outcomes, but could also potentially be exploitable in future tissue engineering applications.

biophysics↗

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

Extracellular vesicle mobility in collagen I hydrogels is modulated by RGD-binding integrins

Extracellular vesicles (EVs) are a diverse population of membrane structures produced and released by cells into the extracellular space for the intercellular trafficking of cargo molecules. They are implicated in various biological processes, including angiogenesis, immunomodulation, and cancer cell signaling. While much research has focused on their biogenesis or their effects on recipient cells, less is understood about how EVs are capable of traversing diverse tissue environments and crossing biological barriers. Their interactions with extracellular matrix components are of particular interest, as such interactions govern diffusivity and mobility, providing a potential basis for organotropism. To start to untangle how EV-matrix interactions affect diffusivity, we use highspeed epifluorescence microscopy, single particle tracking, and confocal reflectance microscopy to analyze particle mobility and localization in extracellular matrix-mimicking hydrogels composed of collagen I. EVs are compared with synthetic liposomes and extruded plasma membrane vesicles to better understand the importance of membrane composition on these interactions. By treating EVs with trypsin to digest surface proteins, we determine that proteins are primarily responsible for EV immobilization in collagen I hydrogels. We next use a synthetic peptide competitive inhibitor to narrow down the identity of the proteins involved to argynylglycylaspartic acid (RGD) motif-binding integrins, which interact with unincorporated or denatured non-fibrillar collagen. Moreover, the effect of integrin inhibition with RGD peptides has strong implications for the use of RGD-peptide-based drugs to treat certain cancers, as integrin inhibition appears to increase EV mobility, improving their ability to infiltrate tissue-like environments.

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↗

bioRxiv2023

Adhesion energy controls lipid binding-mediated endocytosis

Several bacterial toxins and viruses can deform membranes through multivalent binding to lipids for clathrin-independent endocytosis. However, it remains unclear, how membrane deformation and endocytic internalization are mechanistically linked. Here we show that many lipid-binding virions induce membrane deformation and clathrin-independent endocytosis, suggesting a common mechanism based on multivalent lipid binding by globular particles. We create a synthetic cellular system consisting of a lipid-anchored receptor in the form of GPI-anchored anti-GFP nanobodies and a multivalent globular binder exposing 180 regularly-spaced GFP molecules on its surface. We show that these globular, 40 nm diameter, particles bind to cells expressing the receptor, deform the plasma membrane upon adhesion and become endocytosed in a clathrin-independent manner. We explore the role of the membrane adhesion energy in endocytosis by using receptors with affinities varying over 7 orders of magnitude. Using this system, we find that once a threshold in adhesion energy is overcome to allow for membrane deformation, endocytosis occurs reliably. Multivalent, binding-induced membrane deformation by globular binders is thus sufficient for internalization to occur and we suggest it is the common, purely biophysical mechanism for lipid-binding mediated endocytosis of toxins and pathogens.

biophysics↗

bioRxiv2023

Non-specific membrane-matrix interactions influence diffusivity of lipid vesicles in hydrogel environments

The diffusion of extracellular vesicles and liposomes in vivo is affected by different tissue environmental conditions and is of great interest in the development of liposome-based therapeutics and drug-delivery systems. Here, we use a bottom-up biomi-metic approach to better isolate and study steric and electrostatic interactions and their influence on the diffusivity of synthetic large unilamellar vesicles in hydrogel environments. Single-particle tracking of these extracellular vesicle-like particles in agarose hydrogels as an extracellular matrix model shows that membrane deformability and surface charge affect the hydrogel pore spaces that vesicles have access to, which determines overall diffusivity. Moreover, we show that passivation of vesicles with PEGylated lipids, as often used in drug delivery systems enhances diffusivity, but that this effect cannot be fully explained with electrostatic interactions alone. Finally, we compare our experimental findings with existing computational and theoretical work in the field to help explain the non-specific interactions between diffusing particles and gel matrix environments. Statement of SignificanceThe diffusion of nanoparticles in human tissues is dependent on interactions with the surrounding environment. This has wide implications for the development of nanoparticle-based therapeutics and drug delivery systems. Studying these interactions in human tissues and even in model hydrogels composed of reconstituted tissue components can be hampered by the many different complex interactions that can occur. By using a bio-inert hydrogel material like agarose, we remove the influence of specific biochemical interactions, allowing the study of how particle diffusion can be tuned with simple material properties like charge and rigidity. Taking advantage of these non-specific interactions, nanoparticles could one day be engineered to target specific organs by optimizing diffusion in certain tissue environments or retention and immobilization in others.

biophysics↗

bioRxiv2023

Tunable biomimetic bacterial membranes from binary and ternary lipid mixtures and their application in antimicrobial testing

Reconstruction of accurate yet simplified mimetic models of cell membranes is a very challenging goal of synthetic biology. To date, most of the research focuses on the development of eukaryotic cell membranes, while reconstitution of their prokaryotic counterparts has not been fully addressed, and the proposed models do not reflect well the complexity of bacterial cell envelopes. Here, we describe the reconstitution of biomimetic bacterial membranes with an increasing level of complexity, developed from binary and ternary lipid mixtures. Giant unilamellar vesicles composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE); PC and phosphatidylglycerol (PG); PE and PG; PE, PG and cardiolipin (CA) at varying molar ratios were successfully prepared by the electroformation method. Each of the proposed mimetic models focuses on reproducing specific membrane features such as membrane charge, curvature, leaflets asymmetry, or the presence of phase separation. GUVs were characterized in terms of size distribution, surface charge, and lateral organization. Finally, the developed models were tested against the lipopeptide antibiotic daptomycin. The obtained results showed a clear dependency of daptomycin binding efficiency on the amount of negatively charged lipid species present in the membrane. We anticipate that the models proposed here can be applied not only in antimicrobial testing but also serve as platforms for studying fundamental biological processes in bacteria as well as their interaction with physiologically relevant biomolecules.

biophysics↗