Emilia Krok

Model membrane systems, including Langmuir monolayers, supported lipid bilayers (SLBs), and vesicles, are widely used to investigate the structure and dynamics of biological membranes and membrane-associated processes. It is widely assumed that lipid monolayers compressed to approximately 30 mN/m provide a physicochemical environment comparable to that of lipid bilayers. However, the validity of this correspondence remains debated. Here, we systematically test monolayer-bilayer equivalence using the fluorescence spectral responses of three molecular probes, DOPE-Atto633, Laurdan, and NR12S. Their spectra were recorded in DOPC and DPPC monolayers over a broad range of surface pressures, with and without cholesterol, and compared with corresponding bilayer systems formed as SLBs and small unilamellar vesicles (SUVs). Our results reveal pronounced differences between monolayer and bilayer environments depending on the lipid composition, cholesterol content, and the molecular probe used. For pure DOPC and DPPC, Laurdan revealed close monolayer–bilayer correspondence at approximately 30 mN/m, whereas NR12S required substantially higher surface pressures to reproduce bilayer-like spectral responses. Cholesterol further altered this correspondence, and for some compositions full spectral correspondence was not reached even at the highest surface pressures investigated. These findings challenge the general applicability of the established monolayer–bilayer correspondence and demonstrate that a single surface-pressure criterion cannot reliably reproduce the physicochemical properties of bilayer membranes across different lipid systems. They also underpin the importance of second leaflet and interleaflet coupling in shaping membrane structure and packing. More broadly, our findings emphasize the need to critically consider appropriate model systems when investigating biological membranes and their functions.

The remarkable complexity of bacterial cell membranes, combined with their diverse chemical composition and constantly changing internal physicochemical parameters, makes them exceptionally challenging to study in vivo. With many novel antibiotics targeting the bacterial cell envelope, considerable effort is now focused on the development of model membrane analogues that allow rapid initial screening of potential antimicrobial candidates. In this study, we successfully developed models based on lipid bilayers that mimic the lipid membrane of Gram-positive bacteria and the inner cell membrane of Gram-negative bacteria. These models incorporate varying levels of complexity in lipid composition and structural organization. Using models composed of DMoPC, DOPG, or DOPE and POPE:POPG binary lipid mixtures, we determined the impact of lipid composition on membrane dynamical properties and structural organization. To this end, we employed fluorescence microscopy and fluorescence recovery after photobleaching techniques. Building on these insights, we then replicated the cellular membranes of the most common bacterial strains. Finally, our investigation focused on replicating the lateral heterogeneities observed in vivo in the membranes of E. coli and B. subtilis. We anticipate that the proposed platforms, with their fully tunable lipid compositions, will be highly effective in elucidating the fundamental biological processes associated with bacterial cell membranes.

For over 40 years, Laurdan has been widely used as a universal fluorescent probe for the study of lipid membranes. However, recent molecular dynamics simulations have uncovered previously unknown properties of Laurdan, revealing that it can adopt distinct conformations within the lipid bilayer, thereby influencing its molecular orientation. Despite these findings, experimental and quantitative validation has been lacking. Here, we present the first experimental study of the orientation of Laurdan in a phase-separated supported lipid bilayer, directly linking its spatial orientation to its emission spectra in liquid-ordered (L_o) and liquid-disordered (L_d) phases. Using azimuthally and radially polarized excitation beams, we show that in the L_o phase, Laurdan molecules align more parallel to the membrane normal, whereas in the L_d phase, they adopt a more planar orientation within the membrane. Interestingly, the emission spectra for both excitation modes converge at shorter wavelengths, but show deviations at longer wavelengths, particularly in the L_d phase. By refining our understanding of the behavior of Laurdan in lipid membranes, this study underlines the critical role of the molecular orientation of the dye in fluorescence-based membrane studies and highlights the need for orientation-sensitive analysis in biophysical investigations.

Hydroxyapatite (HAP) is a well-established material in biomedical applications, especially for bone tissue regeneration, dental implants, and drug delivery systems. Recent research emphasizes enhancing the biocompatibility and osteoconductivity of orthopedic implants using HAP. This study explores the potential of combining HAP with a lipid matrix to improve the surface properties and biocompatibility of poly(lactic acid) (PLA)-based, 3D-printed, resorbable bone implants. We utilized the Langmuir–Blodgett method to deposit HAP within a dihexadecyl phosphate (DHP) matrix onto PLA substrates. This study demonstrates that DHP and HAP form stable monolayers at the air/water interface with HAP particles distributed within a homogeneous lipid matrix. The presence of HAP and the resulting changes in surface free energy (SFE) are hypothesized to enhance the biocompatibility of PLA implants. Our findings indicate that films composed of DHP + HAP 5:1 are particularly effective in altering PLA surface characteristics, potentially improving osteointegration, and reducing microbial adherence. Overall, this work highlights that surface modification of PLA with HAP and lipid matrices is the first step towards new, promising, and cost-effective strategies for developing advanced biomaterials for bone regeneration.

An imbalance between cells’ intrinsic excitability and synaptic excitation levels is the basis of spinal motoneuron (MN) pathophysiology in Amyotrophic Lateral Sclerosis. Recently, a restoration of the deficient Ia synaptic excitation of spinal MNs was achieved by applying acute trans-spinal direct current stimulation (tsDCS) to presymptomatic SOD1 G93A mice. Here we investigate whether two-week repeated tsDCS applied to presymptomatic SOD1 animals can provoke spinal MN neuroplasticity and reduce the disease burden. Anodal, cathodal or sham polarisation of 100 µA was applied to P30-P35 SOD1 G93A mice; passive membrane properties and Ia excitatory post-synaptic potential (EPSP) characteristics were investigated by intracellular recordings of spinal MNs in vivo. A second cohort of polarized animals was used to test the impact of our intervention on Ia synapse morphology, MN intracellular metabolic pathways activity, and disease markers. Anodal tsDCS evoked a strong increase in maximal Ia EPSPs, coupled with a significant upregulation of vesicular glutamate transporter levels and GlurR4 subunits of AMPA receptors at the Ia synapse. On the other hand, cathodal polarisation failed to induce any significant alteration to Ia synapse morphology but did increase both peak and plateau input resistance and recovered the abnormal paired-pulse ratio. Unexpectedly, the changes in MN electrophysiological profile and Ia synapse morphology did not translate into alterations of intracellular pathways ctivity and did not decrease the disease burden. Altogether our results indicate a strong polarity-dependent plasticity of spinal MNs in SOD1 G93A mice in response to tsDCS, which nevertheless appears insufficient to alter disease dynamics. Highlights: 14-days of trans-spinal direct current stimulation (tsDCS) alters the electrophysiological properties and morphology of Ia proprioceptive synapses on spinal MNs in SOD1 G93A mouse model of ALS Anodal (depolarising) tsDCS increases MN synaptic excitation and restores the postsynaptic elements of the Ia synapse Cathodal (hyperpolarising) tsDCS increases MN input resistance but does not impact Ia synapse morphology Both anodal and cathodal tsDCS fail to significantly modify the cellular burden of the disease

Cholesterol-rich lipid rafts are found to facilitate membrane fusion, central to processes like viral entry, fertilization, and neurotransmitter release. While the fusion process involves local, transient membrane dehydration, the impact of reduced hydration on cholesterol's structural organization in biological membranes remains unclear. Here, we employ confocal fluorescence microscopy and atomistic molecular dynamics simulations to investigate cholesterol behavior in phase-separated lipid bilayers under controlled hydration. We unveiled that dehydration prompts cholesterol release from raft-like domains into the surrounding fluid phase. Unsaturated phospholipids undergo more significant dehydration-induced structural changes and lose more hydrogen bonds with water than sphingomyelin. The results suggest that cholesterol redistribution is driven by the equalization of biophysical properties between phases and the need to satisfy lipid hydrogen bonds. This underscores the role of cholesterol-phospholipid-water interplay in governing cholesterol affinity for a specific lipid type, providing a new perspective on the regulatory role of cell membrane heterogeneity during membrane fusion.

Although cell membranes exist in excess of water under physiological conditions, there are a number of biochemical processes, such as adsorption of biomacromolecules or membrane fusion events, that require partial or even complete transient dehydration of lipid membranes. Even though the dehydration process is crucial for understanding all fusion events, still little is known about the structural adaptation of lipid membranes when their interfacial hydration layer is perturbed. Here, we present the study of the nanoscale structural reorganization of phase-separated, supported lipid bilayers (SLBs) under a wide range of hydration conditions. Model lipid membranes were characterised using a combination of fluorescence microscopy and atomic force microscopy and, crucially, without applying any chemical or physical modifications that have previously been considered essential for maintaining the membrane integrity upon dehydration. We revealed that decreasing the hydration state of the membrane leads to an enhanced mixing of lipids characteristic of the liquid-disordered (L_d) phase with those forming the liquid-ordered (L_o) phase. This is associated with a 2-fold decrease in the hydrophobic mismatch between the L_d and L_o lipid phases and a 3-fold decrease in the line tension for the fully desiccated membrane. Importantly, the observed changes in the hydrophobic mismatch, line tension, and lipid miscibility are fully reversible upon subsequent rehydration of the membrane. These findings provide a deeper insight into the fundamental processes, such as cell–cell fusion, that require partial dehydration at the interface of two membranes.

Although cell membranes in physiological conditions exist in excess of water, there is a number of biochemical processes, such as adsorption of biomacromolecules or membrane fusion events, that require partial or even complete, transient dehydration of lipid membranes. Even though the dehydration process is crucial for understanding all fusion events, still little is known about the structural adaptation of the lipid membranes when their interfacial hydration layer is perturbed. Here, we introduce the study on the nanoscale structural reorganization of the phase-separated, supported lipid bilayers (SLBs) under a wide range of hydration conditions. Model lipid membranes were characterized with the combination of fluorescence microscopy and atomic force microscopy, and crucially, without applying any chemical or physical modifications, that so far have been considered to be indispensable for maintaining the membrane integrity upon dehydration. We revealed that decreasing hydration state of the membrane leads to an enhanced mixing of lipids characteristic for the liquid-disordered (L _d ) phase with those forming liquid-ordered (L _o ) phase. This is associated with a 2-fold decrease in the hydrophobic mismatch between the L _d and L _o lipid phases and a 3-fold decrease of line tension for the completely desiccated membrane. Importantly, the observed changes in the hydrophobic mismatch, line tension, and miscibility of lipids are fully reversible upon subsequent rehydration of the membrane. These findings provide deeper insights into the fundamental processes such as cell-cell fusion that require partial dehydration at the interface of two membranes.