Large Unilamellar Vesicles Research Articles

Palladium responsive liposomes for triggered release of aqueous contents

Palladium (Pd) is a promising metal catalyst for novel bioorthogonal chemistry and prodrug activation. This report describes the first example of palladium responsive liposomes. The key molecule is a new caged phospholipid called Alloc-PE that forms stable liposomes (large unilamellar vesicles, ∼220 nm diameter). Liposome treatment with PdCl2 removes the chemical cage, liberates membrane destabilizing dioleoylphosphoethanolamine (DOPE), and triggers liposome leakage of encapsulated aqueous contents. The results indicate a path towards liposomal drug delivery technologies that exploit transition metal triggered leakage.

Synthesis and Spectral Characterisation of Fabricated Cerium-Doped Magnesium Oxide Nanoparticles: Evaluation of the Antimicrobial Potential and Its Membranolytic Activity through Large Unilamellar Vesicles

Considerable attention has been given to Magnesium oxide nanoparticles lately due to their antimicrobial potential, low toxicity to humans, high thermal stability, biocompatibility, and low cost of production. However, their successful transformation into sustainable drugs is limited due to their low membrane permeability, which reduces their bioavailability in target cells. Herein we propose Cerium-doped magnesium oxide nanoparticles (MgOCeNPs) as a powerful solution to above mentioned limitations and are compared with MgO NPs for their membrane permeability and antimicrobial activity. Both pure and Ce-doped were characterized by various spectroscopic and microscopic techniques, in which an X-ray diffraction (XRD) examination reveals the lattice patterns for doped nanoparticles. Furthermore, Atomic Force Microscopy (AFM) revealed the three-dimensional (3D) structure and height of the nanoparticle. The crystal structure (FCC) of MgO did not change with Ce doping. However, microstructural properties like lattice parameter, crystallite size and biological activity of MgO significantly changed with Ce doping. In order to evaluate the antimicrobial potential of MgOCeNPs in comparison to MgO NPs and to understand the underlying mechanisms, the antibacterial activity was investigated against human pathogenic bacteria E. coli and P. aeruginosa, and antifungal activity against THY-1, a fungal strain. MgOCeNPs were studied by several methods, which resulted in a strong antibacterial and antifungal activity in the form of an elevated zone of inhibition, reduced growth curve, lower minimum inhibitory concentration (MIC80) and enhanced cytotoxicity in both bacterial and fungal strain as compared to MgO nanoparticles. The study of the growth curve showed early and prolonged stationary phase and early decline log phase. Both bacterial and fungal strains showed dose-dependent cytotoxicity with enhancement in intracellular reactive oxygen species (ROS) generation and formation of pores in the membrane when interacting with egg-phosphatidylcholine model Large Unilamellar Vesicles (LUVs). The proposed mechanism of MgOCeNPs toxicity evidently is membranolytic activity and induction of ROS production, which may cause oxidative stress-mediated cytotoxicity. These results confirmed that MgOCeNPs are a novel and very potent antimicrobial agent with a great promise of controlling and treating other microbes.

Phospholipid lateral diffusion in the presence of cationic peptides as measured via 31P CODEX NMR

The effects of two cationic peptides on phospholipid lateral diffusion in binary mixtures of POPC with various anionic phospholipids were measured via 31P CODEX NMR. Large unilamellar vesicles composed of POPC/POPG (70/30 mol/mol), or POPC/DOPS (70/30 mol/mol), or POPC/TOCL (85/15 mol/mol), or POPC/DOPA (50/50 mol/mol) were exposed to either polylysine (pLYS, N = 134 monomers) or KL-14 (KKLL KKAKK LLKKL), a model amphipathic helical peptide, in an amount corresponding to 80% neutralization of the anionic phospholipid charge by the cationic lysine residues. In the absence of added peptide, phospholipid lateral diffusion coefficients (all measured at 10 °C) increased with increasing reduced temperature (T-Tm). The POPC/DOPA mixture was an exception to this generalization, in that lateral diffusion for both components was far slower than any other mixture investigated, an effect attributed to intermolecular hydrogen bonding. The addition of pLYS or KL-14 decreased lateral diffusion in the POPC/DOPS LUV, but had minimal effects in the POPC/POPG LUV, indicating that ease of access of the cationic peptide residues to the anionic phospholipid groups was important. Both cationic peptides produced the opposite effect in the POPC/DOPA case, in that lateral diffusion increased significantly in their presence, with KL-14 being most effective. This latter observation was interpreted in terms of the electrostatic / H-bond model proposed by Kooijman et al. [Journal of Biological Chemistry, 282:11356–11,364, 2007] to describe the mechanism of interaction between the phosphomonoester head group of PA and the tertiary amine of lysine.

Ion channels activity of SARS-CoV-2 envelope protein through calcium influx assay using large unilamellar vesicles.

The envelope (E) protein of the SARS-CoV-2 virus is a membrane-bound viroporin that conducts cations across the endoplasmic reticulum Golgi intermediate compartment (ERGIC) membrane of the host cell to cause virus pathogenicity. Channel opening by acidic pH and Ca2+ increases the population of the dynamic lipid-facing conformation. Here, we developed an assay for the calcium channel activity of SARS-CoV-2 envelope (ETM) protein. The calcium-sensitive fluorescence indicator, frua-2, was incorporated into large unilamellar vesicles (LUVs). After establishing a calcium gradient across the liposomal membranes, native or mutated ETM protein was added. The resulting calcium influx into the LUV detected through the fluorescence changes of fura-2 was used as a qualitative test for the electrophysiological properties of ETM protein.This discovery of ETM protein calcium influx activity may be a target for E inhibitors against SARS-CoV-2 and related coronaviruses.

Leaflet dependent effect of anionic phospholipids on the permeation of cell penetrating peptides into model membranes.

Cell-penetrating peptides (CPPs) are often short, highly positively charged molecules that cross the cell membrane without requiring any protein transport. In eukaryotic and bacterial membranes, negatively charged lipids such as phosphatidylglycerol (PG) and phosphatidylserine (PS) are often distributed asymmetrically between the two bilayer leaflets. Notably, changes in anionic lipid asymmetry are implicated in antimicrobial resistance and disease states. Thus, a physical understanding of how CPPs interact with and permeate asymmetric membranes could reveal design principles for future CPPs as drugs or drug conjugates. Here, we systematically investigate the effect of anionic lipid asymmetric leaflet distribution on interactions of two CPPs, NAF-144-67 and RW9, using large unilamellar vesicles (LUVs) as a model system. LUVs with varied inner and outer leaflet distributions of PS, phosphatidylglycerol (PG), and phosphatidic acid (PA) were generated using methyl-β-cyclodextrin exchange and incubated with RW9 or Dansyl-NAF-144-67 at a 1/100 peptide/lipid ratio. Fluorescence spectroscopy was used to probe the chemical environment of the peptides at equilibrium using the solvatochromic properties of tryptophan and Dansyl. As anionic lipids were doped into the outer leaflet, more CPP permeated into the membrane interior for both peptides while inner leaflet anionic leaflets had minimal effect on peptide-membrane permeation. The effect of anionic lipid gradient between leaflets was also explored and the partition coefficient of each peptide into the hydrophobic interior of the membrane was quantified, with PA in the outer leaflet resulting in the most membrane-partitioning by both peptides. These results suggest that positively charged CPPs may permeate to a greater extent into cells with anionic outer leaflet lipids, such as cancer cells, while cells with inner leaflet anionic lipids may not produce the same effect, thus informing the design of CPPs for selective drug delivery.

Phase state of lipid bilayers modulates ammonia and water permeability through AtTip2;1.

Lipid-protein interactions determine the structure, stability and function of transmembrane proteins. Protein conformation can be influenced by local interactions at the lipid-protein interface or global interactions such as the lateral pressure profile of the membrane, which in turn is influenced by the lipid composition of the membrane. However, despite its methodological importance for in vitro protein characterization and its biological implications the influence of lipid bilayer fluidity on membrane protein activity is largely unknown. Reconstituting the aqua-ammonia channel AtTip2;1, from Arabidopsis thaliana, into OSPC containing large unilamellar vesicles we modulate its unitary water and ammonia permeability switching between the liquid-disordered state and the gel phase state of the bilayer. Quantitative activity characterization utilizing fluorescence correlation spectroscopy, stopped-flow methodology, and dynamic light scattering is complemented by molecular dynamics simulations of AtTIP2;1 in OSPC bilayers. Our in vitro experiments revealed a unitary water permeability in the liquid-disordered phase of 2.5·10−13 cm³s−1. On the contrary, selective transport of ammonia is an order of magnitude lower questioning if ammonia transport is the main function of AtTIP2;1. Moreover, results obtained from our MD simulations show, similar to our in vitro efforts, that AtTIP2;1 exhibits lower activity in the gel state as compared to its activity in the liquid-disordered state. This finding highlights the crucial importance of the lipid matrix in in vitro and in silico protein characterization and is expected to trigger a wealth of future investigations on permeability and regulation of AQPs and other membrane proteins.

Vesicle budding caused by lysolipid-induced asymmetry stress.

The lipid membrane undergoes continuous and dynamic changes in a multitude of cellular processes. Exo- and endovesiculation, asexual reproduction or the forming of organelles require a change in local curvature. Although many protein-centered mechanisms have been identified to induce these changes, the properties of the lipid membrane itself have been neglected in most cases. Changes in lipid composition and asymmetry can also contribute to membrane curvature generation. Lysolipids dissolved or dispersed in the outer medium insert into the accessible membrane leaflet and expand its area but show only very slow translocation to the trans leaflet. This resulting asymmetry stress is partially relaxed by vesicle budding even in the absence of proteins. Budding induced by lysolipids have been studied with giant unilamellar vesicles (GUVs), but they lack a quantitative perspective, since bulk measurements are difficult to implement using GUVs. Using Asymmetric Flow Field-Flow Fractionation (AF4), we were able to establish an assay quantifying the fraction of lipid budding off from large unilamellar vesicles (LUVs) depending on specific conditions. We discovered that the extent of budding depends, above a threshold, not on the amount of lysolipid added. Instead, it is limited by the excess surface area stored in the initial LUV. As the vesicle becomes ideally spherical, a hypotonic vesicle interior opposes further budding and further lysolipid is prevented from entering the membrane. Our studies provide a detailed picture how lysolipids and other membrane-impermeant amphiphiles contribute to membrane curvature generation. Furthermore, it provides insight about alternative mechanisms of a membrane to relax asymmetry stress, including transient membrane leakage as a mode of action of antibiotic lipopeptides.

Disease-associated mutations in Drp1 have fundamentally different effects on the mitochondrial fission machinery.

Patient mutations have been identified throughout dynamin-related protein 1 (Drp1), the key mediator of mitochondrial fission. These mutations generally impact children and often result in severe neurological defects and, in some cases, death. Previously, the underlying functional defect leading to patient phenotypes has been speculated based on comparisons with synthetic mutations at similar sites. Drp1 is comprised by four domains, including a GTPase domain (G-domain) that regulates GTP binding and hydrolysis and a middle domain (MD) that mediates Drp1 self-assembly. We analyzed six mutations, four in the MD and two in the G-domain. All the MD mutants studied were found to be dimer-limited in solution, and three of these MD mutants were found to be impaired in self-assembly. However, one of the MD mutants retains oligomerization capability despite being located within this self-assembly region. Further, this mutant retains its ability to interact with a pre-formed lipid template but is unable to re-shape large, unilamellar vesicles (LUVs). This indicates that Drp1 dimers alone are sufficient to form a helical polymer on lipid template. Differences were also observed between the two G-domain mutations. One of the mutants is in a nucleotide-binding loop that is critical for functional hydrolysis. This mutant exhibited impaired GTP hydrolysis but can still oligomerize in solution and with a lipid template. In contrast, the other G-domain mutation is located relatively distant from the nucleotide binding site. This mutant poorly tubulates LUVs, highlighting the role of G-domain interactions in driving membrane curvature. Overall, the functional defects caused by mutations in Drp1 are highly variable even when the mutations occur within the same functional domain. This study provides a framework for characterizing additional Drp1 mutations to provide a comprehensive understanding of functional sites within this essential protein.

Photodynamic activity rather than drilling causes membrane damage by a light-powered molecular nanomotor.

The chase toward endowing chemical compounds with machine-like functions mimicking those of biological molecular machineries has yielded a variety of artificial molecular motors (AMMs). Pharmaceutical applications of photoexcited monomolecular unidirectionally-rotating AMMs have been envisioned in view of their ability to permeabilize biological membranes. Nonetheless, the mechanical properties of lipid membranes render the proposed drilling activity of AMMs doubtful. Here, we show that singlet oxygen released by a photoexcited "molecular drill" oxidized unsaturated lipids composing giant unilamellar vesicles. In contrast, giant liposomes built of saturated lipids were inert to AMM photoactuation. The AMM did not mechanically destroy gramicidin A ion channels in planar bilayer lipid membranes but instead photoinactivated them. Sodium azide, a singlet oxygen quencher, reduced both AMM-mediated light-induced dye release from unsaturated large unilamellar vesicles and protected gramicidin A from photoinactivation. Upon additional consideration of the underlying bilayer mechanics, we conclude that AMMs' envisioned therapeutic and pharmaceutical applications rely on their photodynamic activity rather than their nanomechanical drilling abilities.

Quantification of biophysical properties of candidalysin, a fungal peptide toxin secreted by C. albicans during invasion.

Candida albicans fungus can cause invasive candidiasis. To become pathogenic and translocate across the intestinal epithelium C.abicans fungus needs to change its morphology from yeast to invasive filamentous. Virulence factor candidalysin secreted during fungal invasion, is found to be crucial in direct cell membrane damage, the stimulation of a danger response signalling pathway, and the activation of epithelial immunity. Due to its amphiphilic alpha-helical structure peptide has a high binding affinity to plasma membranes. With increasing accumulation on the membrane surfaces, the alpha-helix peptide starts penetration of the lipid bilayer until the plasma membrane is damaged. However, whether there is specific affinity to different membrane compositions is still unknown. Here we determined the concentrations of candidalysin required for membrane permeabilization of large unilamellar vesicles with various lipid compositions. Then, we used spectral imaging to assess the lipid packing changes in giant unilamellar vesicles of different lipid composition resembling human epithelial, bacterial and fungal membranes under the treatment of candidalysin. Solvatochromic polarity-sensitive dyes were used to label model membranes. Spectral imaging data allowed to calculate generalized polarization map of the labelled membrane and further investigate lipid packing during peptide binding.

The Unusual Aggregation and Fusion Activity of the Antimicrobial Peptide W-BP100 in Anionic Vesicles

Cationic antimicrobial peptides (CAMPs) offer a promising strategy to counteract bacterial resistance, mostly due to their membrane-targeting activity. W-BP100 is a potent broad-spectrum cecropin-melittin CAMP bearing a single N-terminal Trp, which was previously found to improve its antibacterial activity. W-BP100 has high affinity toward anionic membranes, inducing membrane saturation at low peptide-to-lipid (P/L) ratios and membrane permeabilization, with the unique property of promoting the aggregation of anionic vesicles only at specific P/L ratios. Herein, we aimed to investigate this unusual behavior of W-BP100 by studying its aggregation and fusion properties with negatively-charged large (LUVs) or giant (GUVs) unilamellar vesicles using biophysical tools. Circular dichroism (CD) showed that W-BP100 adopted an α-helical conformation in anionic LUVs, neutralizing its surface charge at the aggregation P/L ratio. Its fusion activity, assessed by Förster resonance energy transfer (FRET) using steady-state fluorescence spectroscopy, occurred mainly at the membrane saturation/aggregation P/L ratio. Confocal microscopy studies confirmed that W-BP100 displays aggregation and detergent-like effects at a critical P/L ratio, above which it induces the formation of new lipid aggregates. Our data suggest that W-BP100 promotes the aggregation and fusion of anionic vesicles at specific P/L ratios, being able to reshape the morphology of GUVs into new lipid structures.

Lipid packing is disrupted in copolymeric nanodiscs compared with intact membranes

Discoidal lipid-protein nanoparticles known as nanodiscs are widely used tools in structural and membrane biology. Amphipathic, synthetic copolymers have recently become an attractive alternative to membrane scaffold proteins for the formation of nanodiscs. Such copolymers can directly intercalate into, and form nanodiscs from, intact membranes without detergents. Although these copolymer nanodiscs can extract native membrane lipids, it remains unclear whether native membrane properties are also retained. To determine the extent to which bilayer lipid packing is retained in nanodiscs, we measured the behavior of packing-sensitive fluorescent dyes in various nanodisc preparations compared with intact lipid bilayers. We analyzed styrene-maleic acid (SMA), diisobutylene-maleic acid (DIBMA), and polymethacrylate (PMA) as nanodisc scaffolds at various copolymer-to-lipid ratios and temperatures. Measurements of Laurdan spectral shifts revealed that dimyristoyl-phosphatidylcholine (DMPC) nanodiscs had increased lipid headgroup packing compared with large unilamellar vesicles (LUVs) above the lipid melting temperature for all three copolymers. Similar effects were observed for DMPC nanodiscs stabilized by membrane scaffolding protein MSP1E1. Increased lipid headgroup packing was also observed when comparing nanodiscs with intact membranes composed of binary mixtures of 1-palmitoyl-2-oleoyl-phosphocholine (POPC) and di-palmitoyl-phosphocholine (DPPC), which show fluid-gel-phase coexistence. Similarly, Laurdan reported increased headgroup packing in nanodiscs for biomimetic mixtures containing cholesterol, most notable for relatively disordered membranes. The magnitudes of these ordering effects were not identical for the various copolymers, with SMA being the most and DIBMA being the least perturbing. Finally, nanodiscs derived from mammalian cell membranes showed similarly increased lipid headgroup packing. We conclude that nanodiscs generally do not completely retain the physical properties of intact membranes.

Designing a green-emitting viscosity-sensitive 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) probe for plasma membrane viscosity imaging.

Viscosity is a key characteristic of lipid membranes - it governs the passive diffusion of solutes and affects the lipid raft formation and membrane fluidity. Precise determination of viscosity values in biological systems is of great interest and viscosity-sensitive fluorescent probes offer a convenient solution for this task. In this work we present a novel membrane-targeting and water-soluble viscosity probe BODIPY-PM, which is based on one of the most frequently used probes BODIPY-C10. Despite its regular use, BODIPY-C10 suffers from poor integration into liquid-ordered lipid phases and lack of water solubility. Here, we investigate the photophysical characteristics of BODIPY-PM and demonstrate that solvent polarity only slightly affects the viscosity-sensing qualities of BODIPY-PM. In addition, with fluorescence lifetime imaging microscopy (FLIM), we imaged microviscosity in complex biological systems - large unilamellar vesicles (LUVs), tethered bilayer membranes (tBLMs) and live lung cancer cells. Our study showcases that BODIPY-PM preferentially stains the plasma membranes of live cells, equally well partitions into both liquid-ordered and liquid-disordered phases and reliably distinguishes lipid phase separation in tBLMs and LUVs.

Anion binding and transport with meso-alkyl substituted two-armed calix[4]pyrroles bearing urea and hydroxyl groups.

Calix[4]pyrroles bearing hydroxyl (1) or urea (3) groups attached to the meso-positions with propyl linkers were synthesized as cis- and trans-isomers. The anion binding properties of cis-1 and cis-3 were screened with ion-mobility mass spectrometry, where cis-1 formed complexes with Cl-, Br- and H2PO4-, whereas cis-3 formed complexes with most of the investigated anions, including Cl-, Br-, I-, NO3-, ClO4-, OTf-, SCN- and PF6-. The structures of the chloride complexes were further elucidated with density functional theory calculations and a crystal structure obtained for cis-1. In solution, chloride and dihydrogenphosphate anion binding with cis-1 and cis-3 were compared using 1H NMR titrations. To assess the suitability of two-armed calix[4]pyrroles as anion transporters, chloride transport studies of cis-1, cis-3 and trans-3 were performed using large unilamellar vesicles. The results revealed that cis-3 had the highest activity among the investigated calix[4]pyrroles, which was related to the improved affinity and isolation of chloride inside the binding cavity of cis-3 in comparison to cis-1. The results indicate that appending calix[4]pyrroles with two hydrogen bonding arms is a feasible strategy to obtain anion transporters and receptors with high anion affinity.

Model Membranes and Antimicrobial Activities of pH-Sensitive Copolymers

Polymers are options as antimicrobials for skin protection, antifouling surfaces, and fabrics. Here we analyzed the interaction of polymers based on poly(methacrylate) (PMMA) and poly((dimethylamino ethyl) methacrylate) (PDMAEMA) with model membranes and bacteria. We used the homopolymers PMMA, PDMAEMA, and the diblock copolymer(s) prepared with different PMMAm:PDMAEMAn ratios (m/n). The interactions of PDMAEMA and PMMAm-b-PDMAEMAn with large unilamellar vesicles (LUVs) prepared with phosphatidylcholine and phosphatidylglycerol at different pHs, were analyzed by nuclear magnetic resonance (NMR), dynamic light scattering, and zeta potential. These polymers promoted LUVs leakage of a fluorescent probe (5,6-carboxyfluorescein) localized exclusively in the internal aqueous compartment. Interestingly, all copolymers exhibit a bell-shaped pH dependence for the polymer-induced LUVs leakage. The interaction of the positively charged polymers and the pH effect was also demonstrated using giant unilamellar vesicles. These copolymers inhibited bacterial growth in the micromolar range and can be used to prevent bacterial growth on surfaces.

Nano-infrared analysis of amyloid β1-42 fibrils formed in the presence of lipids with unsaturated fatty acids.

Alzheimer's disease (AD) is characterized by progressive memory loss and serious impairment of cognitive abilities. AD is the most common cause of dementia, affecting more than 44 million people around the world. The hallmark of AD is amyloid plaques, extracellular deposits primarily found in the frontal lobe, that are composed of amyloid β (Aβ) aggregates. In this study, we utilized nano-infrared spectroscopy, also known as Atomic Force Microscopy Infrared (AFM-IR) spectroscopy to investigate the effect of unsaturated phospholipids on the rate of Aβ1-42 aggregation. We found that unsaturated phosphatidylcholine, phosphatidylserine, and cardiolipin strongly suppressed aggregation of Aβ1-42. Furthermore, Aβ1-42 fibrils formed in the presence of such lipids exerted significantly lower cell toxicity compared to the protein aggregates formed in the lipid-free environment. These findings suggest that dietary changes linked to the increased consumption of unsaturated phospholipids could be considered as a potential therapeutic approach that can decelerate the progression of AD. These results also suggest that large unilamellar vesicles with unsaturated lipids can be used as potential therapeutics to delay the onset and decelerate the progression of AD.

Analytical probing of membranotropic effects of antimicrobial copper nanoparticles on lipid vesicles as membrane models.

Copper nanoparticles (CuNPs) are antimicrobial agents that are increasingly being used in several real-life goods. However, concerns are arising about their potential toxicity and thus, appropriate legislation is being issued in various countries. In vitro exploration of the permeability and the distribution of nanoparticles in cell membranes should be explored as the first step towards the investigation of the toxicity mechanisms of metal nanoantimicrobials. In this work, phosphatidylcholine-based large unilamellar vesicles have been explored as mimics of cellular membranes to investigate the effect of ultra-small CuNPs on the physicochemical features of phospholipid membranes. 4 nm-sized CuNPs were synthesized by a wet-chemical route that involves glutathione as a stabilizer, with further characterization by UV-vis absorption spectroscopy, fluorescence spectroscopy, transmission electron microscopy, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FTIR) spectroscopy. Two fluorescent membrane probes bearing naphthalene moieties (laurdan and prodan) were used to monitor the bilayer structure and dynamics, as well as to demonstrate the strong membranotropic effects of CuNPs. The fluorescence spectroscopic studies were supported by dynamic light scattering (DLS) measurements and the calcein leakage assay. Additionally, the degree of perturbation of the phospholipid bilayer by CuNPs was compared against that of Cu2+ ions, the latter resulting in negligible effects. The findings suggested that CuNPs are able to damage the phospholipid membranes, leading to their agglomeration or disruption.

A Kinetic Map of the Influence of Biomimetic Lipid Model Membranes on Aβ42 Aggregation.

The aggregation of the amyloid β (Aβ) peptide is one of the molecular hallmarks of Alzheimer's disease (AD). Although Aβ deposits have mostly been observed extracellularly, various studies have also reported the presence of intracellular Aβ assemblies. Because these intracellular Aβ aggregates might play a role in the onset and progression of AD, it is important to investigate their possible origins at different locations of the cell along the secretory pathway of the amyloid precursor protein, from which Aβ is derived by proteolytic cleavage. Senile plaques found in AD are largely composed of the 42-residue form of Aβ (Aβ42). Intracellularly, Aβ42 is produced in the endoplasmatic reticulum (ER) and Golgi apparatus. Since lipid bilayers have been shown to promote the aggregation of Aβ, in this study, we measure the effects of the lipid membrane composition on the in vitro aggregation kinetics of Aβ42. By using large unilamellar vesicles to model cellular membranes at different locations, including the inner and outer leaflets of the plasma membrane, late endosomes, the ER, and the Golgi apparatus, we show that Aβ42 aggregation is inhibited by the ER and Golgi model membranes. These results provide a preliminary map of the possible effects of the membrane composition in different cellular locations on Aβ aggregation and suggest the presence of an evolutionary optimization of the lipid composition to prevent the intracellular aggregation of Aβ.

Efficient liposome fusion to phase-separated giant vesicles

Lateral phase heterogeneity in biomembranes can govern cellular functions and may serve as a platform for enrichment or depletion of membrane-anchored molecules. In this work, we address the question of how the process of membrane fusion is affected by the membrane phase state (fluid or gel) and by phase coexistence, as well as the effects of fusion-mediated incorporation of exogeneous lipids on phase separation. Our system is based on the fusion of cationic fluid large unilamellar vesicles (LUVs) composed of dioleoyl trimethylammonium propane (DOTAP) and dioleoyl phosphoethanolamine (DOPE) with neutral and anionic giant unilamellar vesicles (GUVs) composed of phosphatidylcholine and phosphatidylglycerol. By changing the lipid composition of the GUVs, we modulated the phase state and charge of the different phases (charged or neutral, fluid or gel) and identified systems in which we can target fusion to specific domains on phase-separated membranes. Fusion efficiency was quantified using fluorescence microscopy-based lipid and content mixing assays, and flow chamber devices were used to assess the real-time sequence of events of the fusion process. To investigate the bilayer thermal behavior, differential scanning calorimetry (DSC) experiments were performed on LUVs. The results show that fusion is extensive in single-component GUVs only for fluid and negatively charged acceptor membranes. On the other hand, in phase-separated GUVs, high fusion efficiency was observed even when the gel phase was anionic and phase separation somewhat increased the fusion efficiency. Extensive fusion led to dissolution of the gel domains as a result of extensive incorporation of lipids in the fluid state from the fusogenic liposomes. Altogether, these findings have the potential to unravel the important role of membrane phase state, phase separation, charge, and the effects of extensive fusion on membrane organization and may give insights in the regulation of the interactions between cells and liposomes that are used in drug delivery systems.

Biochemical and Biophysical Characterization of the Caveolin-2 Interaction with Membranes and Analysis of the Protein Structural Alteration by the Presence of Cholesterol

Caveolin-2 is a protein suitable for the study of interactions of caveolins with other proteins and lipids present in caveolar lipid rafts. Caveolin-2 has a lower tendency to associate with high molecular weight oligomers than caveolin-1, facilitating the study of its structural modulation upon association with other proteins or lipids. In this paper, we have successfully expressed and purified recombinant human caveolin-2 using E. coli. The structural changes of caveolin-2 upon interaction with a lipid bilayer of liposomes were characterized using bioinformatic prediction models, circular dichroism, differential scanning calorimetry, and fluorescence techniques. Our data support that caveolin-2 binds and alters cholesterol-rich domains in the membranes through a CARC domain, a type of cholesterol-interacting domain in its sequence. The far UV-CD spectra support that the purified protein keeps its folding properties but undergoes a change in its secondary structure in the presence of lipids that correlates with the acquisition of a more stable conformation, as shown by differential scanning calorimetry experiments. Fluorescence experiments using egg yolk lecithin large unilamellar vesicles loaded with 1,6-diphenylhexatriene confirmed that caveolin-2 adsorbs to the membrane but only penetrates the core of the phospholipid bilayer if vesicles are supplemented with 30% of cholesterol. Our study sheds light on the caveolin-2 interaction with lipids. In addition, we propose that purified recombinant caveolin-2 can provide a new tool to study protein-lipid interactions within caveolae.