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Transmembrane protein channels enable fast and highly sensitive electrical detection of single molecules. Nanopore sequencing of DNA was achieved using an engineered Mycobacterium smegmatis porin A (MspA) in combination with a motor enzyme. Due to its favorable channel geometry, the octameric MspA pore exhibits the highest current level as compared to other pore proteins. To date, MspA is the only protein nanopore with a published record of DNA sequencing. While widely used in commercial devices, nanopore sequencing of DNA suffers from significant base-calling errors due to stochastic events of the complex DNA-motor-pore combination and the contribution of up to five nucleotides to the signal at each position. Asymmetric mutations within subunits of the channel protein offer an enormous potential to improve nucleotide resolution and sequencing accuracy. However, random subunit assembly does not allow control of the channel composition of MspA and other oligomeric protein pores. In this study, we showed that it is feasible to convert octameric MspA into a single-chain pore by connecting eight subunits using peptide linkers. We constructed single-chain MspA trimers, pentamers, hexamers and heptamers to demonstrate that it is feasible to alter the subunit stoichiometry and the MspA pore diameter. All single-chain MspA proteins formed functional channels in lipid bilayer experiments. Importantly, we demonstrated that single-chain MspA discriminated all four nucleotides identical to MspA produced from monomers. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications.STATEMENT OF SIGNFICANCENanopore sequencing of DNA is a fast and cheap technology that uniquely delivers multi-kilobase reads. It is currently used world-wide in many applications such as genome sequencing, epigenetics, and surveillance of viral and bacterial pathogens and has started to revolutionize human lives in medicine, agriculture and environmental studies. However, the high base-calling error rates prevent nanopore DNA sequencing from reaching its full potential. In this study, we converted octameric MspA into a single-chain pore enabling asymmetric mutations to fine-tune the pore geometry and chemistry and address the shortcomings of nanopores. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications.

Lipid cubic phase (LCP) formulations enhance the intestinal solubility and bioavailability of hydrophobic drugs by reducing precipitation and facilitating their mass transport to the intestinal surface for absorption. LCPs with an ester linkage connecting the acyl chain to the glycerol backbone (monoacylglycerols), are susceptible to chemical digestion by several lipolytic enzymes including lipases, accelerating the release of hydrophobic agents from the lipid bilayers of the matrix. Unlike regular enzymes that transform soluble substrates, lipolytic enzymes act at the interface of water and insoluble lipid. Therefore, compounds that bind to this interface can enhance or inhibit the activity of enzymes to varying extent. Here, we explore how the lipolysis rate can be tuned by the interfacial interaction of porcine pancreatic lipase with monoolein LCPs containing a known lipase inhibitor, tetrahydrolipstatin. Release of the Biopharmaceutical Classification System (BCS) class IV drug, paclitaxel, from the inhibitor-modified LCP was examined in the presence of lipase and its effectors colipase and calcium. By combining experimental dynamic digestion studies, thermodynamic measurements and molecular dynamics simulations of the competitive inhibition of lipase by tetrahydrolipstatin, we reveal the role and mode of action of lipase effectors in creating a precisely-balanced degradation-controlled LCP release system for the poorly soluble paclitaxel drug.

Summary Photo-generation of a proton gradient over a lipid bilayer is of interest due to its essential role in photosynthetic bacteria. Membrane asymmetry is key to the proton gradient generation via directional proton transport. Here, we report a light-driven proton pump based on two-dimensional, porphyrin-based Janus metal-organic layers (Janus-MOLs). The Janus-MOL, functionalized with carboxyquinone on one side and Acitretin on the other via a microemulsion-based method, was attached to liposome surface. Upon photoexcitation, the porphyrins initiate electron and hole transfers to carboxyquinone and Acitretin, respectively, which undergo redox reactions with freely diffusing quinone (Q)/hydrosemiquinone (HQ·) in the lipid bilayer to produce a concentration gradient of quinone-based species. Owing to different pKa values of HQ+ and HQ·, these redox reactions trigger proton transport across the membrane to create a pH gradient, which drives ATP production by CFoF1-ATP synthase in a similar fashion as photosynthetic bacteria.

Bacterial infections are considered as a critical healthcare concern worldwide. Timely infection detection is crucial to effective antibiotic administration which can reduce the severity of infection and the occurrence of antibiotic resistance. We have developed label–free polydiacetylene (PDA) liposome-based colorimetric biosensor to detect and identify bacterial cultures at the genus and species level with naked eyes by simple color change. We found that among the various liposomal systems, moderate concentration of PDA, phospholipids and cholesterol in liposome assemblies can greatly influence the sensitivity to different bacteria, exhibiting unique chromatic properties of each bacterial strain. The strikingly different chromatic color change was due to the various mechanisms of interactions between bacterial toxins and biomimetic lipid bilayers. Furthermore, increase of cholesterol in liposome assemblies greatly enhanced the sensitivity of bacterial strains related to membrane destruction mediated by pore-formation mechanism such as S. aureus and E.coli, whereas the detection of the two bacterial strains was believed to rely on the specific recognition elements coupled with PDA moiety. As a proof of concept, a colorimetric finger-print array for distinguishing 6 bacterial species was studied. Particularly, the proposed bacterial detection platform is achieved through the interaction between bacterially secreted toxins and liposome bilayers instead of specific recognition of receptors-ligands. The results of both response time and sensitivity of label-free-liposome-based system show superior to previous reports on chromatic bacterial detection assays. By combing these results, the label-free-liposome-based colorimetric sensing platform shows great importance as a bacterial-sensing and discrimination platform.

The cellular membrane has been identified to play a critical role in various biological processes including the assembly of biological systems. Membranes are complex, primarily two-dimensional assemblies with varied lipid compositions depending on the particular region of the cell. Supported lipid bilayers are considered as appropriate models for physio-chemical studies of membranes including numerous single molecule techniques. Atomic force microscopy (AFM) as a topographic technique is a fully appropriate single molecule technique capable of direct observation of molecular processes on membranes. However, reliable experimental AFM studies require the preparation of the bilayer with a sub-nanometer smooth morphology, which remains stable over long-time observation. Here we present the methodology, which allows one to prepare a smooth, stable, structurally homogeneous lipid bilayer without the presence of any trapped vesicles. We described the application of such lipid bilayers to probe time-dependent early stages of aggregation of monomeric amyloid proteins. Importantly, the proposed methodology can be extended to bilayers with various compositions, by incorporating different lipids for on-membrane aggregation study including cholesterol. Furthermore, this methodology development allowed us to monitor the aggregation of amyloid protein at its physiologically relevant low protein concentration. The flexibility of altering the membrane composition allows to identify the specific role of a particular lipid towards the aggregation kinetics, revealing the plausible mechanism of disease development.

NMDAR (N-methyl-d-aspartate receptor) consisted of GluN1 and GluN2, and/or GluN3 subunits. As the obligatory subunit of NMDAR, GluN1 contains variant N-terminal domain (NTD) and C-terminal domain (CTD). The CTD contains allosteric signal and mediates the metabotropic function of NMDAR, which has been confirmed by previous studies. However, the allosteric signaling mechanism of GluN1 CTD has not been studied. In our study, we found that GluN1 CTD could bind to the lipid bilayers and affect the antigen epitope of GluN1 C-terminal antibody, suggesting that membrane binding may determine the allosteric signal of GluN1 CTD. In addition, we discovered that the membrane binding of GluN1 CTD could be regulated by the phosphorylation of GluN1 CTD C1 region.

Gramicidin A (gA) is a hydrophobic pentadecapeptide readily incorporating into a planar bilayer lipid membrane (BLM), thereby inducing a large macroscopic current across the BLM. This current results from ion-channel formation due to head-to-head transbilayer dimerization of gA monomers with rapidly established monomer-dimer equilibrium. Any disturbance of the equilibrium, e.g., by sensitized photoinactivation of a portion of gA monomers, causes relaxation toward a new equilibrium state. According to previous studies, the characteristic relaxation time of the gA-mediated electric current decreases as the current increases upon elevating the gA concentration in the membrane. Here, we report data on the current relaxation kinetics for gA analogs with N-terminal valine replaced by glycine or tyrosine. Surprisingly, the relaxation time increased rather than decreased upon elevation of the total membrane conductance induced by these gA analogs, thus contradicting the classical kinetic scheme. We developed a general theoretical model that accounts for lateral interaction of monomers and dimers mediated by membrane elastic deformations. The modified kinetic scheme of the gramicidin dimerization predicts the reverse dependence of the relaxation time on membrane conductance for gA analogs, with a decreased dimerization constant that is in a good agreement with our experimental data. The equilibration process may be also modulated by incorporation of other peptides (“impurities”) into the lipid membrane.

The response rate of anti-PD therapy in most cancer patients remains low. Therapeutic drug and tumor-infiltrating lymphocytes (TILs) are usually obstructed by the stromal region within tumor microenvironment (TME) rather than distributed around tumor cells, thus unable to induce the immune response of cytotoxic T cells. Here, we constructed the cationic thermosensitive lipid nanoparticles IR780/DPPC/BMS by introducing cationic NIR photosensitizer IR-780 iodide (IR780) modified lipid components, thermosensitive lipid DPPC and PD-1/PD-L1 inhibitor BMS202 (BMS). Upon laser irradiation, IR780/DPPC/BMS penetrated into deep tumor, and reduced cancer-associated fibroblasts (CAFs) around tumor cells to remodel the spatial distribution of TILs in TME. Interestingly, the cationic IR780/DPPC/BMS could capture released tumor-associated antigens (TAAs), thereby enhancing the antigen-presenting ability of DCs to activate cytotoxic T lymphocytes. Moreover, IR780/DPPC/BMS initiated gel-liquid crystal phase transition under laser irradiation, accelerating the disintegration of lipid bilayer structure and leading to the responsive release of BMS, which would reverse the tumor immunosuppression state by blocking PD-1/PD-L1 pathway for a long term. This combination treatment can synergistically exert the antitumor immune response and inhibit the tumor growth and metastasis.

The outer membrane of Gram-negative bacteria presents a robust physicochemical barrier protecting the cell from both the natural environment and acting as the first line of defense against antimicrobial materials. The proteins situated within the outer membrane are responsible for a range of biological functions including controlling influx and efflux. These outer membrane proteins (OMPs) are ultimately inserted and folded within the membrane by the β-barrel assembly machine (Bam) complex. The precise mechanism by which the Bam complex folds and inserts OMPs remains unclear. Here, we have developed a platform for investigating Bam-mediated OMP insertion. By derivatizing a gold surface with a copper-chelating self-assembled monolayer, we were able to assemble a planar system containing the complete Bam complex reconstituted within a phospholipid bilayer. Structural characterization of this interfacial protein-tethered bilayer by polarized neutron reflectometry (PNR) revealed distinct regions consistent with known high-resolution models of the Bam complex. Additionally, by monitoring changes of mass associated with OMP insertion by quartz crystal microbalance with dissipation monitoring (QCM-D), we were able to demonstrate the functionality of this system by inserting two diverse OMPs within the membrane, pertactin and OmpT. This platform has promising application in investigating the mechanism of Bam-mediated OMP insertion, in addition to OMP function and activity within a phospholipid bilayer environment.

The two-dimensional (2D) affinity between protein molecules across contacting cells is a key parameter regulating and initiating several cellular processes. However, measuring 2D affinity can be challenging and experimental data are limited. In addition, the obtained 2D affinities are typically averaged over the cell population. We here present a method to measure 2D affinity on single cells binding to polyhistidine-tagged fluorescent ligands anchored to a supported lipid bilayer (SLB). By decreasing the density of ligands in the SLB using imidazole a new steady-state accumulation in the contact is obtained, and from this change, both the 2D affinity and the number of receptors on the cell can be determined. The method was validated on an SLB containing rat CD2 binding to the rat CD48 mutant T92A expressed on Jurkat T cells. The addition of imidazole did not influence the average 2D affinity (1/Kd), and the spread in affinities within the cell population was low, Kd = 4.9 ± 0.9 molecules/μm2 (mean ± SD), despite an order of magnitude spread in ligand accumulation due to differences in receptor density. It was also found that cell contact size increased both with ligand density and with the number of receptors per cell, but that the contact size stayed approximately constant when lowering the ligand density, above a density of around 10 rCD2 molecules/μm2, after the contact first had formed, indicative of a heterogeneous process. In summary, this method not only allows for single-cell affinities to be measured, but it can also reduce measurement and analysis time and improve measurement accuracy. Due to the low spread in 2D Kd within the cell population, the analysis can further be restricted to the cells showing the strongest binding, paving the way for using this method to study weak binding events.

Antimicrobial peptides (AMPs), which typically disrupt the bacterial wall prompting leakage or lysis of the cell, form a growing contingent in the arsenal against antibiotic resistant bacteria. The effectiveness of AMPs is, however, hampered by their low solubility, general chemical and physical instability, and short half-life in vivo. Lipid nanocarriers such as cubosomes are effective at encapsulating and protecting proteins while simultaneously showing promise in delivery applications. Here, the efficacy of cubosome mediated delivery of AMPs is evaluated by the in-situ surface characterization of model membranes with varying composition. The cubosomes were observed to initially fuse with the membranes, with subsequent membrane disruption observed after approximately 20 - 60 min. The time for the disruption was sensitive to the charge of the cubosome as well as the composition of the bilayer. More physiologically relevant bilayers including lipids with phospho-(1'-rac-glycerol) (PG) or phosphoethanolamine (PE) headgroups were more vulnerable than those of neat phosphocholine (PC). Notably, disruption to the bilayer occurred an order of magnitude faster for encapsulated AMP compared to free AMP.

It is known that giant vesicles undergo dynamic morphological changes when exposed to a detergent. The solubilization process may take multiple pathways. In this work, we identify lipid vesicle shape dynamics before the solubilization of 1,2-dioleoyl-sn-glycero-3-phosphocholine giant vesicles with Triton X-100 (TR) detergent. The violent lipid vesicle dynamics was observed with laser confocal scanning microscopy and was qualitatively explained via a numerical simulation. A three-dimensional Monte Carlo scheme was constructed that emulated the nonequilibrium conditions at the beginning stages of solubilization, accounting for a gradual addition of TR detergent molecules into the lipid bilayers. We suggest that the main driving factor for morphology change in lipid vesicles is the associative tendency of the TR molecules, which induces spontaneous curvature of the detergent inclusions, an intrinsic consequence of their molecular shape. The majority of the observed lipid vesicle shapes in the experiments were found to correspond very well to the numerically calculated shapes in the phase space of possible solutions. The results give an insight into the early stages of lipid vesicle solubilization by amphiphilic molecules, which is nonequilibrium in nature and very difficult to study.

Highlights: Extracellular vesicles (EVs) are small lipid bilayer particles containing biologically important cargo and impart regulatory changes in target cells. Despite the importance of EVs in cellular communication, there remains a gap in our understanding of how EVs influence HSC fate and, in turn, how aging and longevity are affected. This review summarizes the current literature dealing with how age-altered intercellular communication mediated by EVs influences HSC biology.

Time-resolved fluorescence and differential scanning calorimetry (DSC) were used to examine how two amino acids, L-phenylalanine (L-PA) and N-acetyl-DL-tryptophan (NAT), affect the temperature-dependent membrane affinity of two structurally similar coumarin solutes for 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles. The 7-aminocoumarin solutes, coumarin 151 (C151) and coumarin 152 (C152), differ in their substitution at amine position—C151 is a primary amine, and C152 is a tertiary amine—and both solutes show different tendencies to associate with lipid bilayers consistent with differences in their respective log-P-values. Adding L-PA to the DPPC vesicle solution did not change C151’s propensity to remain freely solvated in aqueous solution, but C152 showed a greater tendency to partition into the hydrophobic bilayer interior at temperatures below DPPC’s gel-liquid crystalline transition temperature (Tgel-lc). This finding is consistent with L-PA’s ability to enhance membrane permeability by disrupting chain-chain interactions. Adding NAT to DPPC-vesicle-containing solutions changed C151 and C152 affinity for the DPPC membranes in unexpected ways. DSC data show that NAT interacts strongly with the lipid bilayer, lowering Tgel-lc by up to 2°C at concentrations of 10 mM. These effects disappear when either C151 or C152 is added to solution at concentrations below 10 μM, and Tgel-lc returns to a value consistent with unperturbed DPPC bilayers. Together with DSC results, fluorescence data imply that NAT promotes coumarin adsorption to the vesicle bilayer surface. NAT’s effects diminish above Tgel-lc and imply that unlike L-PA, NAT does not penetrate into the bilayer but instead remains adsorbed to the bilayer’s exterior. Taken in their entirety, these discoveries suggest that amino acids—and by inference, polypeptides and proteins—change solute affinity for lipid bilayers with specific effects that depend on individualized amino-acid-lipid-bilayer interactions.

KRAS4B is a membrane-anchored signaling protein and a primary target in cancer research. Predictions from molecular dynamics simulations that have previously shaped our mechanistic understanding of KRAS signaling disagree with recent experimental results from neutron reflectometry, NMR, and thermodynamic binding studies. To gain insight into these discrepancies, we compare this body of biophysical data to back-calculated experimental results from a series of molecular simulations that implement different subsets of molecular interactions. Our results show that KRAS4B approximates an entropic ensemble of configurations at model membranes containing 30% phosphatidylserine lipids, which is not significantly shaped by interactions between the globular G-domain of KRAS4B and the lipid membrane. These findings revise our understanding of KRAS signaling and promote a model in which the protein samples the accessible conformational space in a near-uniform manner while being available to bind to effector proteins.

The ability to detect and characterize drug binding to a target protein is of high priority in drug discovery research. However, there are inherent challenges when the target of interest is an integral membrane protein (IMP). Assuming successful purification of the IMP, traditional approaches for measuring binding such as surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) have been proven valuable. However, the mass dependence of SPR signals may preclude the detection of binding events when the ligand has a significantly smaller mass than the target protein. In FRET-based experiments, protein labeling through modification may inadvertently alter protein dynamics. Graphene Bio-Electronic Sensing Technology (GBEST) aims to overcome these challenges. Label-free characterization takes place in a microfluidic chamber wherein a fluid lipid membrane is reconstituted directly above the GBEST sensor surface. By leveraging the high conductivity, sensitivity, and electrical properties of monolayer graphene, minute changes in electrostatic charges arising from the binding and unbinding of a ligand to a native IMP target can be detected in real time and in a mass-independent manner. Using crude membrane fractions prepared from cells overexpressing monocarboxylate transporter 1 (MCT1), we demonstrate the ability to (1) form a fluid lipid bilayer enriched with MCT1 directly on top of the GBEST sensor and (2) obtain kinetic binding data for an anti-MCT1 antibody. Further development of this novel technology will enable characterization of target engagement by both low- and high-molecular-weight drug candidates to native IMP targets in a physiologically relevant membrane environment.

Magnetotactic bacterium, Magnetospirillum magneticum, produces biogenic magnetic nanoparticles termed magnetosomes, which are primarily composed of a magnetite core and a surrounding lipid bilayer membrane. We have fabricated human transmembrane protein-magnetosome complexes by genetic engineering with embedding the transmembrane proteins of interest, in particular G protein-coupled receptors (GPCRs), in the magnetosome membrane. The magnetosomes provide a promising platform for high throughput ligand screening towards drug discovery, and this is a critical advantage of the magnetosome display system beyond conventional membrane platforms such as liposomes and lipid nano-discs. However, the human GPCRs expressed on the magnetosomes were not fully functionalized in bacterial membranes the most probably due to the lack of essential phospholipids such as phosphatidylcholine (PC) for GPCR functionalization. To overcome this issue, we expressed two types of PC-producing enzymes, phosphatidylcholine synthase (PCS) and phosphatidylethanolamine N-methyltransferase (PMT) in M. magneticum. As a result, generation and incorporation of PC in cell- and magnetosome-membranes were demonstrated. To the best of our knowledge, M. magneticum is the second bacterial species which had the PC-incorporated lipid membrane by genetic engineering. Subsequently, a GPCR, thyroid-stimulating hormone receptor (TSHR) and PCS were simultaneously expressed. We found that PC in the magnetosome membrane assisted the binding of TSHR and its ligand, indicating that the genetic approach demonstrated in this study is useful to enhance the function of the GPCRs displayed on the magnetosomes.

The advent of amphiphilic copolymers enables integral membrane proteins to be solubilized into stable 10–30 nm native nanodiscs to resolve their multisubunit structures, post-translational modifications, endogenous lipid bilayers, and small molecule ligands. This breakthrough has positioned biological membrane:protein assemblies (memteins) as fundamental functional units of cellular membranes. Herein, we review copolymer design strategies and methods for the characterization of transmembrane proteins within native nanodiscs by cryo-electron microscopy (cryo-EM), transmission electron microscopy, nuclear magnetic resonance spectroscopy, electron paramagnetic resonance, X-ray diffraction, surface plasmon resonance, and mass spectrometry.

The endoplasmic reticulum (ER) is the main harbor for newly synthesized proteins in eukaryotic cells. Through a continuous membrane network of sheets and tubules, the ER hosts secretory proteins, integral membrane proteins, and luminal proteins of the endomembrane system. These proteins are translated by ribosomes outside the ER and require subsequent integration into or translocation across the lipid bilayer of the ER. They are then modified post-translationally and folded in the ER. Some of these proteins are packaged into coat protein complex II-coated vesicles for export. Here, we review recent advances in understanding the mechanism of protein translocation and transmembrane domain insertion in the ER, summarize new insights into selective cargo packaging, and discuss the roles of ER morphological dynamics in these processes.

A controlled reaction of a mixture of structural isomers of diacylglycerol (DAG) with N,N-disuccinimidyl carbonate and then with b-alanine provided intermediates of 1,2-diacylglycerol (1,2-DAG) which were further modified with carboxy- and amino-terminated oligoethylene glycol spacer arms of different length. As an example of bioactive molecules intended for the incorporation in lipid bilayer of artificial or cell membranes, 1,2-DAGs bearing tetrasaccharide Sialyl Lewis X or triglycine at the terminus of polar spacer were synthesized. The synthetic scheme can be readily scaled-up by the use of DAGs from food industry.

Experimental observations in lipid monolayers at the air-water interface have demonstrated that solitary sound pulses can be excited. These pulses propagate electrical, chemical, and thermal variations in addition to the mechanical changes in lateral pressure and lipid density, and can interact with nearby ions, polymers, and water. In addition, it was demonstrated that sound pulses that reversibly traverse the melting transition between the so-called liquid-expanded and liquid-condensed phases display unusual nonlinear properties that are strikingly similar to those of action potentials in living cells. This review describes recent experimental and theoretical investigations of sound in lipid membranes and their potential function in biology.

Supramolecular chemistry has garnered important interest in recent years toward improving therapeutic efficacy via drug delivery approaches. Although self-assemblies have been deeply investigated, the design of novel drugs leveraging supramolecular chemistry is less known. In this contribution, we show that a Low Molecular Weight Gel (LMWG) can elicit cancer cell apoptosis. This biological effect results from the unique supramolecular properties of a bolaamphiphile-based gelator, which allow for strong interaction with the lipid membrane. This novel supramolecular-drug paradigm opens up new possibilities for therapeutic applications targeting membrane lipids.

The geometry of the channel formed by nontoxic derivative of diphtheria toxin CRM197 in lipid bilayer was determined using the dependence of single-channel conductance upon the hydrodynamic radii of different nonelectrolytes. It was found that the cis entrance of CRM197 channel on the side of membrane to which the toxoid was added at pH 4.8 and the trans entrance on the opposite side at pH 6.0 had effective radii of 3.90 and 3.48 A, respectively. The 3-alkyloxycarbonylmethyl-5-(2-hydroxyethyl)-4-methyl-1,3-thiazolium salts reversibly reduced current via CRM197 channels. The potency of the blockers increased with increasing length of alkyl chain at symmetric pH 6.0 and remained high and stable at pH 4.8 on the cis side. Comparative analysis of CRM197 and amphotericin B pore size with the inhibitory action of thiazolium salts revealed a significant increase in CRM197 pore dimension at pH 6.0. Addition of thiazolium salt with nine carbons alkyl tail increased by ∼30% the viability of human carcinoma cells A431 treated with diphtheria toxin.

Membranes surrounding cells or organelles represent barriers to proteins and other molecules. However, specific proteins can cross membranes by different translocation systems, the best studied being the Sec61/SecY channel. This channel forms a hydrophilic, hourglass-shaped membrane channel, with a lateral gate towards the surrounding lipid. However, recent studies show that an aqueous pore is not required in other cases of protein translocation. The Hrd1 complex, mediating the retro-translocation of misfolded proteins from the endoplasmic reticulum (ER) lumen into the cytosol, contains multi-spanning proteins with aqueous luminal and cytosolic cavities and lateral gates juxtaposed in a thinned membrane region. A locally thinned, distorted lipid bilayer also allows protein translocation in other systems, suggesting a new paradigm to overcome the membrane barrier.

Desmin, an intermediate filament protein expressed in muscle cells, plays a key role in the integrity and regulation of the contractile system. Furthermore, the distribution of desmin in cells and its interplay with plasma and organelle membranes are crucial for cell functions; however, the fundamental properties of lipid-desmin interactions remain unknown. Using a water-in-oil method for a limited space system in vitro, we examined the distribution of desmin in three types of phospholipid droplets: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS). When fluorescent-labeled desmin was observed for 60 min after desmin assembly was initiated by adding 25 mM KCl, desmin accumulated on both the DOPE and DOPS layers; however, it did not accumulate on the DOPC layer of droplets. An increase in salt concentration did not moderate the accumulation. The initial form of either oligomer or mature filament affected the accumulation on each lipid layer. When liposomes were included in the droplets, desmin was associated with DOPE but not on DOPC liposomes. These results suggest that desmin has the potential for association with phospholipids concerning desmin form and lipid shape. The behavior and composition of living membranes may affect the distribution of desmin networks.

Piezo1 forms a mechanically activated calcium-permeable nonselective cation channel that is functionally important in many cell types. Structural data exist for C-terminal regions, but we lack information about N-terminal regions and how the entire channel interacts with the lipid bilayer. Here, we use computational approaches to predict the three-dimensional structure of the full-length Piezo1 and simulate it in an asymmetric membrane. A number of novel insights are suggested by the model: 1) Piezo1 creates a trilobed dome in the membrane that extends beyond the radius of the protein, 2) Piezo1 changes the lipid environment in its vicinity via preferential interactions with cholesterol and phosphatidylinositol 4,5-bisphosphate (PIP2) molecules, and 3) cholesterol changes the depth of the dome and PIP2 binding preference. In vitro alteration of cholesterol concentration inhibits Piezo1 activity in a manner complementing some of our computational findings. The data suggest the importance of N-terminal regions of Piezo1 for dome structure and membrane cholesterol and PIP2 interactions.

SNARE-mediated membrane fusion proceeds via the formation of a fusion pore. This intermediate structure is highly dynamic and can flicker between open and closed states. In cells, cholesterol has been reported to affect SNARE-mediated exocytosis and fusion pore dynamics. Here, we address the question of whether cholesterol directly affects the flickering rate of reconstituted fusion pores in vitro. These experiments were enabled by the recent development of a nanodisc⋅black lipid membrane recording system that monitors dynamic transitions between the open and closed states of nascent recombinant pores with submillisecond time resolution. The fusion pores formed between nanodiscs that bore the vesicular SNARE synaptobrevin 2 and black lipid membranes that harbored the target membrane SNAREs syntaxin 1A and SNAP-25B were markedly affected by cholesterol. These effects include strong reductions in flickering out of the open state, resulting in a significant increase in the open dwell-time. We attributed these effects to the known role of cholesterol in altering the elastic properties of lipid bilayers because manipulation of phospholipids to increase membrane stiffness mirrored the effects of cholesterol. In contrast to the observed effects on pore kinetics, cholesterol had no effect on the current that passed through individual pores and, hence, did not affect pore size. In conclusion, our results show that cholesterol dramatically stabilizes fusion pores in the open state by increasing membrane bending rigidity.

In plants, defense-associated signal transduction involves key membrane-related processes, such as phospholipid-based signaling and membrane trafficking. Coordination of these processes occurs in the lipid bilayer of plasma membrane (PM) and luminal/extracellular membranes. Deciphering the spatiotemporal organization of phospholipids and lipid-protein interactions provides crucial information on the mechanisms that link phospholipid-based signaling and membrane trafficking in plant immunity. In this review, we summarize recent advances in our understanding of these connections, including deployment of key enzymes and molecules in phospholipid pathways, and roles of lipid diversity in membrane trafficking. We highlight the mechanisms that mediate feedback between phospholipid-based signaling and membrane trafficking to regulate plant immunity, including their novel roles in balancing endocytosis and exocytosis.

The actinoporins are cytolytic toxins produced by sea anemones. Upon encountering a membrane, preferably containing sphingomyelin, they oligomerize and insert their N-terminal helix into the membrane, forming a pore. Whether sphingomyelin is specifically recognized by the protein or simply induces phase coexistence in the membrane has been debated. Here, we perform multimicrosecond molecular dynamics simulations of an octamer of fragaceatoxin C, a member of the actinoporin family, in lipid bilayers containing either pure 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC) or a 1:1 mixture of DOPC and palmitoyl sphingomyelin (PSM). The complex is highly stable in both environments, with only slight fraying of the inserted helices near their N-termini. Analyzing the structural parameters of the mixed membrane in the course of the simulation we see signs of a phase transition for PSM in the inner leaflet of the bilayer. In both leaflets, cross-interactions between lipids of different type decrease over time. Surprisingly, the aromatic loop thought to be responsible for sphingomyelin recognition interacts more with DOPC than PSM by the end of the simulation. These results support the notion that the key membrane property that actinoporins recognize is lipid phase coexistence.SIGNIFICANCE STATEMENTThe mechanism of selectivity of naturally produced toxins for their target membranes is not well understood. For example, actinoporins, toxins produced by sea anemones, have been reported to selectively target sphingomyelin-containing membranes. Whether they bind this lipid preferentially or recognize the phase coexistence that sphingomyelin induces is not clear. This work examines this issue by long computer simulations of an actinoporin oligomer embedded in lipid bilayers and finds no preferential interactions of the protein with sphingomyelin. Instead, the simulations show signs of phase separation, suggesting that phase coexistence is the key property that actinoporins recognize.

Cell penetration after recognition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus by the ACE2 receptor and the fusion of its viral envelope membrane with cellular membranes are the early steps of infectivity. A region of the Spike protein of the virus, identified as the "fusion peptide" (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor-binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca2+ ions to the FPs of coronaviruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca2+ binding to the SARS-CoV-2-FP, the role of Ca2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca2+-bound SARS-CoV-2-FP, and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca2+-bound peptide models with lipid membranes, SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif, which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca2+ ions. These findings provide novel mechanistic insights regarding the role of Ca2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.

Boron neutron capture therapy (BNCT) remains an important treatment arm for cancer patients with locally invasive malignant tumors. This therapy needs a significant amount of boron to deposit in cancer tissues selectively, sparing other healthy organs. Most of the liposomes contain water-soluble polyhedral boron salts stay in the core of the liposomes and have low encapsulation efficiency. Thus, modifying the polyhedral boron core to make it hydrophobic and incorporating those into the lipid layer could be one of the ways to increase drug loading and encapsulation efficiency. Additionally, a systematic study about the linker-dependent effect on drug encapsulation and drug-release is lacking, particularly for the liposomal formulation of hydrophobic-drugs. To achieve these goals, liposomal formulations of a series of lipid functionalized cobalt bis(dicarbollide) compounds have been prepared, with the linkers of different hydrophobicity. Hydrophobicity of the linkers have been evaluated through logP calculation and its effect on drug encapsulation and release have been investigated. The liposomes have shown high drug loading, excellent encapsulation efficiency, stability, and non-toxic behavior. Release experiment showed minimal release of drug from liposomes in phosphate buffer, ensuring some amount of drug, associated with liposomes, can be available to tumor tissues for Boron Neutron Capture Therapy.

Release of neuronal transmitters from nerve terminals is triggered by the molecular Ca2+ sensor synaptotagmin 1 (Syt1). Syt1 is a transmembrane protein attached to the synaptic vesicle (SV), and its cytosolic region comprises two domains, C2A and C2B, which are thought to penetrate into lipid bilayers upon Ca2+ binding. Before fusion, SVs become attached to the presynaptic membrane (PM) by the four-helical SNARE complex, which is thought to bind the C2B domain in vivo. To understand how the interactions of Syt1 with lipid bilayers and the SNARE complex trigger fusion, we performed molecular dynamics (MD) simulations at a microsecond scale. We investigated how the isolated C2 modules and the C2AB tandem of Syt1 interact with membranes mimicking either SV or PM. The simulations showed that the C2AB tandem can either bridge SV and PM or insert into PM with its Ca2+-bound tips and that the latter configuration is more favorable. Surprisingly, C2 domains did not cooperate in penetrating into PM but instead mutually hindered their insertion into the bilayer. To test whether the interaction of Syt1 with lipid bilayers could be affected by the C2B-SNARE attachment, we performed systematic conformational analysis of the C2AB-SNARE complex. Notably, we found that the C2B-SNARE interface precludes the coupling of C2 domains and promotes their insertion into PM. We performed the MD simulations of the prefusion protein complex positioned between the lipid bilayers mimicking PM and SV, and our results demonstrated in silico that the presence of the Ca2+ bound C2AB tandem promotes lipid merging. Altogether, our MD simulations elucidated the role of the Syt1-SNARE interactions in the fusion process and produced the dynamic all-atom model of the prefusion protein-lipid complex.

Rhamnolipids (RLs) comprise a class of glycolipids produced by Pseudomonas aeruginosa under appropriate culture medium. They act as biosurfactants being composed by a hydrophilic head of either one (mono-RL) or two (di-RL) rhamnose moieties coupled to hydroxyaliphatic chains. It is well accepted that RLs present low biolitic activity as compared to other synthetic surfactants. However, their mechanisms of action in biological systems are not well defined yet. The interaction of RLs with lipid bilayers are here investigated to address how they impact on plasma membrane at molecular level. Our experimental approach was based on a deep analysis of optical microscopy data from giant unilamellar vesicles (GUVs) dispersed in aqueous solutions containing up to 0.5 mM of commercially available RLs (a mixture of mono-RL, 33–37 mol%, and di-RL, 63–67 mol%, cmc of 0.068 ± 0.005 mM). GUVs were made up of a single lipid POPC and a ternary system containing DOPC, sphingomyelin and cholesterol, which mimic lipid raft platforms. Our results demonstrate that RLs have a low partition in the lipid bilayer in respect to the total molecules in solution. We suppose that RLs insert in the outer leaflet with low propensity to flip-flop. In the case of POPC GUVs, the insertion of RL molecules in the outer leaflet impairs changes in spontaneous membrane curvature with incubation time. Then, small buds are formed that remain linked to the original membrane. No changes in membrane permeability have been detected. A remarkable result refers to the insertion of RLs in membranes containing liquid ordered ( L o ) - liquid disordered ( L d ) phase coexistence. The rate of interaction has been observed to be higher for L d phase than for L o phase ( 0.12 · 10 - 6 s−1 and 0.023 · 10 - 6 s−1 for L d and L o , respectively, at RL concentration of 0.5 mM). As a consequence, the preferential RL insertion in L d phase may also alter the membrane spontaneous curvature which, coupled to the change in the line tension associated to the domains boundary, conducted to L o domain protrusion. Even if it has been observed on a model system, such membrane remodelling might correlate to endocytic processes activated in cell membranes, regardless of the participation of specific proteins. Further, changes imposed by RLs in lipid rafts may affect the association of key proteins enrolled in cell signaling, which may perturb cell homeostasis.

To understand various interactions with cell membranes for in-vivo studies array of different lipid compositions becomes important. Work presented here shows how to prepare different lipid compositions for two different lipids with the help of micro-contact printing and photolithography. Firstly, barriers were created on hydrophilic surface by chrome patterning and/or by protein patterning. Secondly, lipid membrane of desired fluid lipid composition containing one or more lipids in it can be printed by micro-contact printing on to the surface containing chrome or protein patterns. It results in formation of 5 nm thick lipid membrane pattern partitioned with protein or chrome patterns. Thirdly, secondary intercalation can be done by adding small unilamellar vesicles of fluid lipid composition containing one or more lipids to the earlier patterned surface. Upon backfilling with the secondary vesicles it results in formation of compositional array lipid membrane patterns upon mixing with the existing patterned bilayer due to lateral fluidity.

Liposomes, a bio nanomaterial are known as drug delivery agents as they have their unique properties. A liposome encapsulates contained by a hydrophobic membrane a region of aqueous solution; this liquefies hydrophilic solutes which is not able to go through the lipids. Hydrophobic chemicals are also dissolvable in the membrane. Likewise Liposome carries both hydrophilic molecules and hydrophobic molecules. In order to bring the 10 sites action of molecules, the lipid bilayer may be combined along with different bilayers as the cell membrane carries the elements of liposome. Liposomes are used as artificial cell models. The objective of this paper is to present a picture of how liposomes (a bio nanomaterial) came into being as drug delivery agents, its advantages and disadvantages and discuss its present status, the recent developments and the future prospectus.

To develop an efficient vector for mitochondria-targeted drug delivery, we synthesized triphenylphosphonium (TPP)-modified glycol chitosan polymeric microspheres that had a unique chemical structure with both lipophilic phenyl groups and cationic phosphonium. Notably, TPP can easily pass through the phospholipid bilayer of mitochondria, thereby resulting in specific accumulation of a combined drug molecule in the mitochondria due to the membrane potential between TPP and its membrane. Therefore, TPP has been widely used as a mitochondria-targeting moiety. Triphenylphosphonium-glycol chitosan derivatives (GC-TPP and GME-TPP) with two different degrees of substitution (11% and 36%) were prepared by amidation and Michael addition. The chemical structures of GC-TPP and GME-TPP were characterized by 1H nuclear magnetic resonance and Fourier-transform infrared spectroscopy, and their sizes were measured via field emission scanning electron microscopy and dynamic light scattering. Cellular uptake through flow cytometric analysis and confocal microscopy confirmed that both GC-TPP and GME-TPP were well introduced into cells, targeting the mitochondria. In addition, cytotoxicity testing of the most common cell lines, such as HEK293, HeLa, NIH3T3, and HepG2, indicated the absence of polymer toxicity. To evaluate the carrier effectiveness of TPP for drug delivery, doxorubicin (Dox) was used as an anticancer drug. Confocal microscopy images showed that Dox-loaded GME-TPP accumulated inside cells more than Dox-loaded GC-TPP. The anticancer effects of Dox were also determined by MTT assay, apoptosis/necrosis assay, and three-dimensional spheroids. In summary, the results indicate that GC-TPP and GME-TPP microspheres possess great potential as effective drug delivery carriers.

Nanotubes are introduced into lipid bilayer resulting from the self-assembly of cyclic peptides formed by six a-amino acids. The insertion of peptide nanotubes into lipid bilayers modulates the cell wall's permeability qualities and thus confers potential bacteriocidal capacity. Relation of a peptide nanotube developed by cyclic hexapeptides, cyclo (KKLWLW) subunits, organized in an anti parallel; β-sheet like network joined in a hydrated dipalmitoylphosphatidylcholine bilayer surface was investigated using Schrodinger software in a molecular dynamics trajectory of 4-ns. Peptide nanotubes that results from the self-assembly of cyclic peptides having alternated D-L-a-amino acids, via an intermolecular hydrogen bond network, give a wide variety of biologically important structural and functional capabilities.

We studied the curvature induced phase separation of unsaturated and saturated lipids using fluorescence microscopy. In our work, 5 nm thick supported lipid membranes of various compositions unsaturated and saturated lipids were prepared by vesicle fusion method. For introducing curvature we choose poly(dimethyl)siloxane (PDMS) substrates of depressed square and elevated grids patterns of sizes varying from 5 µm to 150 µm and height of features is ∼ 1.5 µm. When we introduced the mixed lipid vesicles above the transition temperature of both lipids resulted in forming continuous lipid bilayer membrane proved by fluorescence recovery after photobleaching studies (FRAP) on the PDMS substrate. Upon slow cooling of lipid membrane on patterned surfaces, which presents an array of curvatures gave rise to a spontaneous self sorting phenomenon. We observed with the help of DiIC18(3) dye which prefers the saturated lipids, that saturated lipids (DPPC) migrated to the depressed square patterns and the unsaturated lipids (POPC) in the reverse direction i.e., to elevated grid patterns resulting in phase separation of lipids on curvatures. This migration of lipids is due to difference in bending rigidity, mechanical bending and preferential organization of the lipid molecules.

Liposomes are widely investigated as vaccine delivery systems, but antigen loading efficiency can be low. Moreover, adsorbed antigen may rapidly desorb under physiological conditions. Encapsulation of antigens overcomes the latter problem but results in significant antigen loss during preparation and purification of the liposomes. Here, we propose an alternative attachment method, based on a complementary heterodimeric coiled coil peptide pair pepK and pepE. PepK was conjugated to cholesterol (yielding CPK) and pepE was covalently linked to model antigen OVA323 (yielding pepE-OVA323). CPK was incorporated in the lipid bilayer of cationic liposomes (180 nm in size). Antigen was associated more efficiently to functionalized liposomes (Kd 166 nM) than to cationic liposomes (Kd not detectable). In vivo co-localization of antigen and liposomes was strongly increased upon CPK-functionalization (35% -> 80%). CPK-functionalized liposomes induced 5-fold stronger CD4+ T-cell proliferation than non-functionalized liposomes in vitro. Both formulations were able to induce strong CD4+ T-cell expansion in mice, but more IFN-y and IL-10 production was observed after immunization with functionalized liposomes. In conclusion, antigen association via coiled coil peptide pair increased co-localization of antigen and liposomes, increased CD4+ T-cell proliferation in vitro and induced a stronger CD4+ T-cell response in vivo.

Much of what we know about the early stages of T cell activation has been obtained from studies of T cells interacting with glass-supported lipid bilayers that favor imaging but are orders of magnitude stiffer than typical cells. We developed a method for attaching lipid bilayers to polydimethylsiloxane polymer supports, producing “soft bilayers” with physiological levels of mechanical resistance (Young’s modulus of 4 kPa). Comparisons of T cell behavior on soft and glass-supported bilayers revealed that whereas late stages of T cell activation are thought to be substrate-stiffness dependent, early calcium signaling was unaffected by substrate rigidity, implying that early steps in T cell receptor triggering are not mechanosensitive. The exclusion of large receptor-type phosphatases was observed on the soft bilayers, however, even though it is yet to be demonstrated at authentic cell-cell contacts. This work sets the stage for an imaging-based exploration of receptor signaling under conditions closely mimicking physiological cell-cell contact.

RalA is a small GTPase and a member of the Ras family. This molecular switch is activated downstream of Ras and is widely implicated in tumor formation and growth. Previous work has shown that the ubiquitous Ca(2+)-sensor calmodulin (CaM) binds to small GTPases such as RalA and K-Ras4B, but a lack of structural information has obscured the functional consequences of these interactions. Here, we have investigated the binding of CaM to RalA and found that CaM interacts exclusively with the C terminus of RalA, which is lipidated with a prenyl group in vivo to aid membrane attachment. Biophysical and structural analyses show that the two RalA membrane-targeting motifs (the prenyl anchor and the polybasic motif) are engaged by distinct lobes of CaM and that CaM binding leads to removal of RalA from its membrane environment. The structure of this complex, along with a biophysical investigation into membrane removal, provides a framework with which to understand how CaM regulates the function of RalA and sheds light on the interaction of CaM with other small GTPases, including K-Ras4B.

In the industrial l-glutamate production established on the use of Corynebacterium glutamicum, l-glutamate synthesized intracellularly is exported through mechanosensitive transmembrane channel proteins (MscCG and MscCG2) activated by the force-from-lipids. The involvement of MscCG2 in l-glutamate export by C. glutamicum was demonstrated in 2018; however, MscCG was previously found to be the major exporter of l-glutamate. Recent advances in research methods, such as development of the microbial patch clamp, revealed unique characteristics of MscCG, including its conductance, opening and closing thresholds, and gating hysteresis, as well as the significant effect of membrane lipids on the channel properties. In addition, the cryoelectron microscopic structure of Escherichia coli MscS, the canonical representative of the mechanosensitive channel family to which MscCG and MscCG2 belong, revealed its new membrane-interacting region, new position within the lipid bilayer, and hook lipids in a newly defined cavity between subunits. In this short review, the applications of bacterial mechanosensitive channels in the development of effective microbial cell factories, which will contribute to sustainable development, are discussed.

Integrating molecular components into active synthetic cells is arising as a major goal of bioengineering and, by and large, multidisciplinary research. The encapsulation of cell-free transcription–translation systems into liposomes stands out of the current approaches because it enables programming synthetic cells with genes, as in real living cells, at the relevant biological scale and within the natural physical boundary. Although recapitulating certain biological functions in such minimal settings has been successful, serious barriers are still hard to overcome. In particular, implementing biological functions located at the lipid membrane remains one of the most challenging elements in bottom-up synthetic cell engineering. In this work, we review the current state-of-the-art of the membrane functions that have been achieved by cell-free expression, either on supported lipid bilayers, in the presence of membranes added to reactions or inside liposomes. We extend the considerations to the membrane functions sought in other types of artificial compartments.

We hypothesised that synthetic HDL nanoparticles carrying a gemcitabine prodrug and apolipoprotein A-II (sHDLGemA2) would target scavenger receptor-B1 (SR-B1) to preferentially and safely deliver gemcitabine into pancreatic ductal adenocarcinoma (PDAC). We designed, manufactured and characterised sHDLGemA2 nanoparticles sized ~130 nm, incorporating 20 mol% of a gemcitabine prodrug within the lipid bilayer, which strengthens on adding ApoA-II. We measured their ability to inhibit growth in cell lines and cell-derived and patient-derived murine PDAC xenografts. Fluorescent-labelled sHDLGemA2 delivered gemcitabine inside xenografts. Xenograft levels of active gemcitabine after sHDLGemA2 were similar to levels after high-dose free gemcitabine. Growth inhibition in mice receiving 4.5 mg gemcitabine/kg/d, carried in sHDLGemA2, was equivalent to inhibition after high-dose (75 mg/kg/d) free gemcitabine, and greater than inhibition after low-dose (4.5 mg/kg/d) free gemcitabine. sHDLGemA2 slowed growth in semi-resistant cells and a resistant human xenograft. sHDLGemA2 targeted xenografts more effectively than sHDLGemA1. SR-B1 was over-expressed in PDAC cells and xenografts. Targeting by ApoA-II was suppressed by anti-SR-B1. Because sHDLGemA2 provided only ~6% of the free gemcitabine dose for an equivalent response, patient side effects can be greatly reduced, and the sHDLGemA2 concept should be developed through clinical trials.

β-Amyloid (Aβ) is a 39–43 residue peptide involved in the pathogenesis of Alzheimer's disease. Aβ deposits onto the cells and gives rise to the plaques that are characteristic of the disease. In an effort to understand the molecular mechanism of plaque formation, we have examined the interaction of Aβ42, considered to be the most pathogenic of the peptides, with lipid bilayers consisting of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) to which small amounts of GM1 ganglioside (1–5 mol%) were incorporated. POPC bilayers exist in the fluid, or liquid-disordered state at room temperature, mimicking the fluidity of cell membranes. An Aβ42 preparation consisting essentially of peptide monomers was used. A combination of molecular dynamics (MD), isothermal calorimetry and Langmuir balance measurements was applied. Our results show that Aβ binds POPC bilayers, and that binding increases (ΔG of binding decreases) with GM1, but only up to 3 mol% of the ganglioside, larger concentrations appearing to have a lower effect. MD and Langmuir balance measurements concur in showing that the peptide adsorbs onto the bilayer surface, but does not become inserted into it at surface pressures compatible with the cell membrane conditions. Thioflavin T measurements agree with MD in revealing a very low degree of peptide oligomerization/aggregation under our conditions. This is in contrast with previous studies showing peptide aggregation and insertion when interacting with membranes in the liquid-ordered state. The present contribution underlines the importance of bilayer lipid composition and properties for Aβ plaque formation.

Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of oligosaccharide chains from lipid-linked oligosaccharides (LLO) to asparagine residues in polypeptide chains. Using high-speed atomic force microscopy (AFM), we investigated the dynamic properties of OST molecules embedded in biomembranes. An archaeal single-subunit OST protein was immobilized on a mica support via biotin–avidin interactions and reconstituted in a lipid bilayer. The distance between the top of the protein molecule and the upper surface of the lipid bilayer was monitored in real-time. The height of the extramembranous part exhibited a two-step variation with a difference of 1.8 nm. The high and low states are designated as state 1 and state 2, respectively. The transition processes between the two states fit well to single exponential functions, suggesting that the observed dynamic exchange is an intrinsic property of the archaeal OST protein. The two sets of cross peaks in the NMR spectra of the protein supported the conformational changes between the two states in detergent-solubilized conditions. Considering the height values measured in the AFM measurements, state 1 is closer to the crystal structure, and state 2 has a more compact form. Subsequent AFM experiments indicated that the binding of the sugar donor LLO decreased the structural fluctuation and shifted the equilibrium almost completely to state 1. This dynamic behavior is likely necessary for efficient catalytic turnover. Presumably, state 2 facilitates the immediate release of the bulky glycosylated polypeptide product, thus allowing OST to quickly prepare for the next catalytic cycle.

Formation of biomolecular condensates that are not enclosed by membranes via liquid-liquid phase separation (LLPS) is a general strategy that cells adopt to organize membraneless subcellular compartments for diverse functions. Neurons are highly polarized with elaborate branching and functional compartmentalization of their neurites, thus, raising additional demand for the proper subcellular localization of both membraneless and membrane-based organelles. Recent studies have provided evidence that several protein assemblies involved in the establishment of neuronal stem cell (NSC) polarity and in the asymmetric division of NSCs form distinct molecular condensates via LLPS. In synapses of adult neurons, molecular apparatuses controlling presynaptic neurotransmitter release and postsynaptic signaling transmission are also likely formed via LLPS. These molecular condensates, though not enclosed by lipid bilayers, directly associate with plasma membranes or membrane-based organelles, indicating that direct communication between membraneless and membrane-based organelles is a common theme in neurons and other types of cells.

Microfluidics has been used to process self-assembling liposomal systems that are commonly considered for drug delivery applications. However, it has been found that the parameters of the process are not universally suited for all lipid types. We hypothesize here that size aggregation and instability of microfluidic liposomes are a direct consequence of the presence of interdigitation in these liposomes. Interdigitation refers to the phenomenon where two opposing leaflets of a bilayer interpenetrate into one another and form a single layer. When this happens, aggregation results as the single layer is not thermodynamically stable. Such interdigitation can be induced by pressure, chemicals or by the type of lipid structure. In this study, we systematically investigate the role of lipid composition on membrane interdigitation in order to understand the dependency of lipid interdigitation on liposome formation by microfluidics. By doing so, we use nano DSC and SAXS to probe the extent of lipid interdigitation by measuring the changes in thermodynamics and membrane thickness of the lipid bilayers. Our results show that microfluidic-fabricated liposomes undergo chemical interdigitation in the presence of ethanol, in particular saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Strategies to prevent interdigitation is to either remove ethanol above the lipid's main transition temperature (Tm), preventing the formation of interdigitated structures and subsequent aggregated states or by the incorporation of the inhibiting additives, such as cholesterol. The author thanks Liew Wen Jie Melvin for the contribution to the cryo-EM images. Funding was kindly provided by NTUNorthwestern Institute for Nanomedicine.

Extracellular vesicles (EVs) are phospholipid bilayer membrane-enclosed structures containing RNAs, proteins, lipids, metabolites, and other molecules, secreted by various cells into physiological fluids. EV-mediated transfer of biomolecules is a critical component of a variety of physiological and pathological processes. Potential applications of EVs in novel diagnostic and therapeutic strategies have brought increasing attention. However, EV research remains highly challenging due to the inherently complex biogenesis of EVs and their vast heterogeneity in size, composition, and origin. There is a need for the establishment of standardized methods that address EV heterogeneity and sources of pre-analytical and analytical variability in EV studies. Here, we review technologies developed for EV isolation and characterization and discuss paths toward standardization in EV research., Highlights Despite the substantial recent progress made in extracellular vesicle (EV) research, our understanding of the functional and mechanistic biology of EVs and their relevance to specific pathophysiological states remains limited. Detailed characterization of the molecular composition of EVs and EV subpopulations remains a challenge. Alternative, similar, or identical experimental approaches may often lead to substantially different EV profiling results in different laboratories. Standard protocols for specimen procurement, collection, preprocessing, EV isolation, analytical characterization, and data analysis/interpretation need to be developed for specialized applications and analytical workflows, optimized, documented, cross-evaluated by several laboratories, and disseminated to further accelerate progress toward further understanding of EV biology and development of novel EV-based diagnostic and therapeutic approaches.