Drabik, D. | Search bioRxiv

bioRxiv2024

The interplay between proton diffusion across biological membranes and their biophysical properties highlights the role of defects in mixed lipid membranes

Proton circuits within biological membranes are at the heart of natural bioenergetic systems, whereas different biological membranes are characterized by different lipid compositions. In this study, we investigate how the composition of mixed lipid membranes influences the proton transfer (PT) properties of the membrane by following the excited-state PT (ESPT) process from a tethered probe to the membrane with time-scales and length-scales of PT that are relevant to bioenergetic systems. Two processes can happen during ESPT: the initial PT from the probe to the membrane at short timescales, followed by diffusion of dissociated protons around the probe on the membrane, and the possible geminate recombination with the probe at longer timescales. Here, we use membranes that are composed of mixtures of phosphatidylcholine (PC) and phosphatidic acid (PA). We show that the changes in the ESPT properties are not monotonous with the concentration of the lipid mixture; at low concentration of PA in PC, we find that the membrane is a poor proton acceptor. Molecular dynamics simulations indicate that at this certain lipid mixture, the membrane has the least defects (more structured and unflawed). Accordingly, we suggest that defects can be an important factor in facilitating PT. We further show that the composition of the membrane affects the geminate proton diffusion around the probe, whereas, on a time-scale of tens of nanoseconds, the dissociated proton is mostly lateral restricted to the membrane plane in PA membranes, while in PC, the diffusion is less restricted by the membrane.

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

Investigation of nano- and micro-domains formed by ceramide-1-phosphate in freestanding lipid bilayers

Biological membranes are known for their complex nature and formation of domains is crucial for proper execution of multiple cellular processes. Such domains, mostly due to their nanoscale character, are rarely studied since implementation of techniques for their quantitative investigation is difficult. In this article we introduce spot-variation z-scan FCS implemented for the first time for artificial lipid vesicles of defined lipid composition. We used this approach to investigate behaviour of different species of ceramide-1-phosphate within membranes. We were able to provide quantitative description of lipid domains generated by ceramide-1-phosphate in nano and microscale. Aligning these results with in silico studies helped us to validate the approach and draw conclusions on complex behaviour of signalling lipids within biological membranes.

biophysics↗