Disordered regions in the IRE1α ER lumenal domain mediate its stress-induced clustering.

Conserved signaling cascades monitor protein-folding homeostasis to ensure proper cellular function. One of the evolutionary conserved key players is IRE1, which maintains endoplasmic reticulum (ER) homeostasis through the unfolded protein response (UPR). Upon accumulation of misfolded proteins in the ER, IRE1 forms clusters on the ER membrane to initiate UPR signaling. What regulates IRE1 cluster formation is not fully understood. Here, we show that the ER lumenal domain (LD) of human IRE1α forms biomolecular condensates in vitro. IRE1α LD condensates were stabilized both by binding to unfolded polypeptides as well as by tethering to model membranes, suggesting their role in assembling IRE1α into signaling-competent stable clusters. Molecular dynamics simulations indicated that weak multivalent interactions drive IRE1α LD clustering. Mutagenesis experiments identified disordered regions in IRE1α LD to control its clustering in vitro and in cells. Importantly, dysregulated clustering of IRE1α mutants led to defects in IRE1α signaling. Our results revealed that disordered regions in IRE1α LD control its clustering and suggest their role as a common strategy in regulating protein assembly on membranes.

Fluid nanoporous microinterface enables multiscale-enhanced affinity interaction for tumor-derived extracellular vesicle detection.

Tumor-derived extracellular vesicles (T-EVs) represent valuable markers for tumor diagnosis and treatment guidance. However, nanoscale sizes and the low abundance of marker proteins of T-EVs restrict interfacial affinity reaction, leading to low isolation efficiency and detection sensitivity. Here, we engineer a fluid nanoporous microinterface (FluidporeFace) in a microfluidic chip by decorating supported lipid bilayers (SLBs) on nanoporous herringbone microstructures with a multiscale-enhanced affinity reaction for efficient isolation of T-EVs. At the microscale level, the herringbone micropattern promotes the mass transfer of T-EVs to the surface. At the nanoscale level, nanoporousity can overcome boundary effects for close contact between T-EVs and the interface. At the molecular level, fluid SLBs afford clustering of recognition molecules at the binding site, enabling multivalent binding with an ∼83-fold increase of affinity compared with the nonfluid interface. With the synergetic enhanced mass transfer, interface contact, and binding affinity, FluidporeFace affords ultrasensitive detection of T-EVs with a limit of detection of 10 T-EVs µL-1, whose PD-L1 expression levels successfully distinguish cancer patients from healthy donors. We expect this multiscale enhanced interfacial reaction strategy will inspire the biosensor design and expand liquid biopsy applications, especially for low-abundant targets in clinical samples.

Soft Synthetic Cells with Mobile Membrane Ligands for Ex Vivo Expansion of Therapy-Relevant T Cell Phenotypes.

The expansion of T cells ex vivo is crucial for effective immunotherapy but currently limited by a lack of expansion approaches that closely mimic in vivo T cell activation. Taking inspiration from bottom-up synthetic biology, a new synthetic cell technology is introduced based on dispersed liquid-liquid phase-separated droplet-supported lipid bilayers (dsLBs) with tunable biochemical and biophysical characteristics, as artificial antigen presenting cells (aAPCs) for ex vivo T cell expansion. These findings obtained with the dsLB technology reveal three key insights: first, introducing laterally mobile stimulatory ligands on soft aAPCs promotes expansion of IL-4/IL-10 secreting regulatory CD8+ T cells, with a PD-1 negative phenotype, less prone to immune suppression. Second, it is demonstrated that lateral ligand mobility can mask differential T cell activation observed on substrates of varying stiffness. Third, dsLBs are applied to reveal a mechanosensitive component in bispecific Her2/CD3 T cell engager-mediated T cell activation. Based on these three insights, lateral ligand mobility, alongside receptor- and mechanosignaling, is proposed to be considered as a third crucial dimension for the design of ex vivo T cell expansion technologies.

Translocation of Antimicrobial Peptides across Model Membranes: The Role of Peptide Chain Length.

Cushioned lipid bilayers are structures consisting of a lipid bilayer supported on a solid substrate with an intervening layer of soft material. They offer possibilities for studying the behavior and interactions of biological membranes more accurately under physiological conditions. In this work, we continue our studies of cushion formation induced by histatin 5 (24Hst5), focusing on the effect of the length of the peptide chain. 24Hst5 is a short, positively charged, intrinsically disordered saliva peptide, and here, both a shorter (14Hst5) and a longer (48Hst5) peptide variant were evaluated. Experimental surface active techniques were combined with coarse-grained Monte Carlo simulations to obtain information about these peptides. Results show that at 10 mM NaCl, both the shorter and the longer peptide variants behave like 24Hst5 and a cushion below the bilayer is formed. At 150 mM NaCl, however, no interaction is observed for 24Hst5. On the contrary, a cushion is formed both in the case of 14Hst5 and 48Hst5, and in the latter, an additional thick, diffuse, and highly hydrated layer of peptide and lipid molecules is formed, on top of the bilayer. Similar trends were observed from the simulations, which allowed us to hypothesize that positively charged patches of the amino acids lysine and arginine in all three peptides are essential for them to interact with and translocate over the bilayer. We therefore hypothesize that electrostatic interactions are important for the interaction between the solid-supported lipid bilayers and the peptide depending on the linear charge density through the primary sequence and the positively charged patches in the sequence. The understanding of how, why, and when the cushion is formed opens up the possibility for this system to be used in the research and development of new drugs and pharmaceuticals.

Recruitment of Receptors and Ligands in a Weakly Multivalent System with Omnipresent Signatures of Superselective Binding.

Recruitment of receptors at membrane interfaces is essential in biological recognition and uptake processes. The interactions that induce recruitment are typically weak at the level of individual interaction pairs, but are strong and selective at the level of recruited ensembles. Here, a model system is demonstrated, based on the supported lipid bilayer (SLB) that mimics the recruitment process induced by weakly multivalent interactions. The weak (mm range) histidine-nickel-nitrilotriacetate (His2 -NiNTA) pair is employed owing to its ease of implementation in both synthetic and biological systems. The recruitment of receptors (and ligands) induced by the binding of His2 -functionalized vesicles on NiNTA-terminated SLBs is investigated to identify the ligand densities necessary to achieve vesicle binding and receptor recruitment. Threshold values of ligand densities appear to occur in many binding characteristics: density of bound vesicles, size and receptor density of the contact area, and vesicle deformation. Such thresholds contrast the binding of strongly multivalent systems and constitute a clear signature of the superselective binding behavior predicted for weakly multivalent interactions. This model system provides quantitative insight into the binding valency and effects of competing energetic forces, such as deformation, depletion, and entropy cost of recruitment at different length scales.

A Membrane-Associated Light-Harvesting Model is Enabled by Functionalized Assemblies of Gene-Doubled TMV Proteins.

Photosynthetic light harvesting requires efficient energy transfer within dynamic networks of light-harvesting complexes embedded within phospholipid membranes. Artificial light-harvesting models are valuable tools for understanding the structural features underpinning energy absorption and transfer within chromophore arrays. Here, a method for attaching a protein-based light-harvesting model to a planar, fluid supported lipid bilayer (SLB) is developed.  The protein model consists of the tobacco mosaic viral capsid proteins that are gene-doubled to create a tandem dimer (dTMV). Assemblies of dTMV break the facial symmetry of the double disk to allow for differentiation between the disk faces. A single reactive lysine residue is incorporated into the dTMV assemblies for the site-selective attachment of chromophores for light absorption. On the opposing dTMV face, a cysteine residue is incorporated for the bioconjugation of a peptide containing a polyhistidine tag for association with SLBs. The dual-modified dTMV complexes show significant association with SLBs and exhibit mobility on the bilayer. The techniques used herein offer a new method for protein-surface attachment and provide a platform for evaluating excited state energy transfer events in a dynamic, fully synthetic artificial light-harvesting system.

Atomic force spectroscopy with magainin 1 functionalized tips and biomimetic supported lipid membranes.

Antimicrobial peptides are molecules synthesized by living organisms as the first line of defense against bacteria, fungi, parasites, or viruses. Since their biological activity is based on destabilization of the microbial membranes, a study of direct interaction forces between antimicrobial peptides and biomimetic membranes is very important for understanding the molecular mechanisms of their action. Herein, we use atomic force spectroscopy to probe the interaction between atomic force microscopy (AFM) tips functionalized with magainin 1 and supported lipid bilayers (SLBs) mimicking electrically uncharged membranes of normal eukaryotic cells and negatively charged membranes of bacterial cells. The investigations performed on negatively charged SLBs showed that the magainin 1 functionalized AFM tips are quickly adsorbed into the SLBs when they approach, while they adhere strongly to the lipid membrane when retracted. On contrary, same investigations performed on neutral SLBs showed mechanical resistance of the lipid membrane to the tip breakthrough and negligible adhesion force at detachment.

The potential of AFM in studying the role of the nanoscale amphipathic nature of (lipo)-peptides interacting with lipid bilayers.

Antimicrobial peptides (AMPs) and lipopeptides (LPs) represent very promising molecules to fight resistant bacterial infections due to their broad-spectrum of activity, their first target, i.e. the bacterial membrane, and the rapid bactericidal action. For both types of molecules, the action mechanism starts from the membrane of the pathogen agents, producing a disorganization of their phase structure or the formation of pores of different size altering their permeability. This mechanism of action is based on physical interactions more than on a lock-and-key recognition event and it is difficult for the pathogens to rapidly develop an effective resistance. Very small differences in the sequence of both AMPs and LPs might lead to very different effects on the target membrane. Therefore, a correct understanding of their mechanism of action is required with the aim of developing new synthetic peptides, analogues of the natural ones, with specific and more powerful bactericidal activity. Atomic force microscopy (AFM), with its high resolution and the associated force spectroscopy resource, provides a valuable technique to investigate the reorganization of lipid bilayers exposed to antimicrobial or lipopeptides. Here, we present AFM results obtained by ours and other groups on the action of AMPs and LPs on supported lipid bilayers (SLBs) of different composition. We also consider data obtained by fluorescence microscopy to compare the AFM data with another technique which can be used on different lipid bilayer model systems such as SLBs and giant unilamellar vesicles. The outcomes here presented highlight the powerful of AFM-based techniques in detecting nanoscale peptide-membrane interactions and strengthen their use as an exceptional complementary tool toin vivoinvestigations. Indeed, the combination of these approaches can help decipher the mechanisms of action of different antimicrobials and lipopeptides at both the micro and nanoscale levels, and to design new and more efficient antimicrobial compounds.

Endothelial cell spreading on lipid bilayers with combined integrin and cadherin binding ligands.

Endothelial cells play a central role in the vascular system, where their function is tightly regulated by both cell-extracellular matrix (e.g., via integrins) and cell-cell interactions (e.g., via cadherins). In this study, we incorporated cholesterol-modified integrin and N-cadherin peptide binding ligands in fluid supported lipid bilayers. Human umbilical vein endothelial cell adhesion, spreading and vinculin localization in these cells were dependent on ligand density. One composition led to observe a higher extent of cell spreading, where cells exhibited extensive lamellipodia formation and a qualitatively more distinct N-cadherin localization at the cell periphery, which is indicative of N-cadherin clustering and a mimic of cell-cell contact formation. The results can be used to reconstitute the endothelial-pericyte interface on biomedical devices and materials.

Insight into the Impact of Oxidative Stress on the Barrier Properties of Lipid Bilayer Models.

As a new field of oxidative stress-based therapy, cold physical plasma is a promising tool for several biomedical applications due to its potential to create a broad diversity of reactive oxygen and nitrogen species (RONS). Although proposed, the impact of plasma-derived RONS on the cell membrane lipids and properties is not fully understood. For this purpose, the changes in the lipid bilayer functionality under oxidative stress generated by an argon plasma jet (kINPen) were investigated by electrochemical techniques. In addition, liquid chromatography-tandem mass spectrometry was employed to analyze the plasma-induced modifications on the model lipids. Various asymmetric bilayers mimicking the structure and properties of the erythrocyte cell membrane were transferred onto a gold electrode surface by Langmuir-Blodgett/Langmuir-Schaefer deposition techniques. A strong impact of cholesterol on membrane permeabilization by plasma-derived species was revealed. Moreover, the maintenance of the barrier properties is influenced by the chemical composition of the head group. Mainly the head group size and its hydrogen bonding capacities are relevant, and phosphatidylcholines are significantly more susceptible than phosphatidylserines and other lipid classes, underlining the high relevance of this lipid class in membrane dynamics and cell physiology.

Model Lipid Membranes Assembled from Natural Plant Thylakoids into 2D Microarray Patterns as a Platform to Assess the Organization and Photophysics of Light-Harvesting Proteins.

Natural photosynthetic "thylakoid" membranes found in green plants contain a large network of light-harvesting (LH) protein complexes. Rearrangement of this photosynthetic machinery, laterally within stacked membranes called "grana", alters protein-protein interactions leading to changes in the energy balance within the system. Preparation of an experimentally accessible model system that allows the detailed investigation of these complex interactions can be achieved by interfacing thylakoid membranes and synthetic lipids into a template comprised of polymerized lipids in a 2D microarray pattern on glass surfaces. This paper uses this system to interrogate the behavior of LH proteins at the micro- and nanoscale and assesses the efficacy of this model. A combination of fluorescence lifetime imaging and atomic force microscopy reveals the differences in photophysical state and lateral organization between native thylakoid and hybrid membranes, the mechanism of LH protein incorporation into the developing hybrid membranes, and the nanoscale structure of the system. The resulting model system within each corral is a high-quality supported lipid bilayer that incorporates laterally mobile LH proteins. Photosynthetic activity is assessed in the hybrid membranes versus proteoliposomes, revealing that commonly used photochemical assays to test the electron transfer activity of photosystem II may actually produce false-positive results.

25-Hydroxycholesterol Effect on Membrane Structure and Mechanical Properties.

Cholesterol is responsible for the plasticity of plasma membranes and is involved in physiological and pathophysiological responses. Cholesterol homeostasis is regulated by oxysterols, such as 25-hydroxycholesterol. The presence of 25-hydroxycholesterol at the membrane level has been shown to interfere with several viruses' entry into their target cells. We used atomic force microscopy to assess the effect of 25-hydroxycholesterol on different properties of supported lipid bilayers with controlled lipid compositions. In particular, we showed that 25-hydroxycholesterol inhibits the lipid-condensing effects of cholesterol, rendering the bilayers less rigid. This study indicates that the inclusion of 25-hydroxycholesterol in plasma membranes or the conversion of part of their cholesterol content into 25-hydroxycholesterol leads to morphological alterations of the sphingomyelin (SM)-enriched domains and promotes lipid packing inhomogeneities. These changes culminate in membrane stiffness variations.

The disordered plant dehydrin Lti30 protects the membrane during water-related stress by cross-linking lipids.

Dehydrins are intrinsically disordered proteins, generally expressed in plants as a response to embryogenesis and water-related stress. Their suggested functions are in membrane stabilization and cell protection. All dehydrins contain at least one copy of the highly conserved K-segment, proposed to be a membrane-binding motif. The dehydrin Lti30 (Arabidopsis thaliana) is up-regulated during cold and drought stress conditions and comprises six K-segments, each with two adjacent histidines. Lti30 interacts with the membrane electrostatically via pH-dependent protonation of the histidines. In this work, we seek a molecular understanding of the membrane interaction mechanism of Lti30 by determining the diffusion and molecular organization of Lti30 on model membrane systems by imaging total internal reflection- fluorescence correlation spectroscopy (ITIR-FCS) and molecular dynamics (MD) simulations. The dependence of the diffusion coefficient explored by ITIR-FCS together with MD simulations yields insights into Lti30 binding, domain partitioning, and aggregation. The effect of Lti30 on membrane lipid diffusion was studied on fluorescently labeled supported lipid bilayers of different lipid compositions at mechanistically important pH conditions. In parallel, we compared the mode of diffusion for short individual K-segment peptides. The results indicate that Lti30 binds the lipid bilayer via electrostatics, which restricts the mobility of lipids and bound protein molecules. At low pH, Lti30 binding induced lipid microdomain formation as well as protein aggregation, which could be correlated with one another. Moreover, at physiological pH, Lti30 forms nanoscale aggregates when proximal to the membrane suggesting that Lti30 may protect the cell by "cross-linking" the membrane lipids.

Gold nanoparticles interacting with synthetic lipid rafts: an AFM investigation.

Inorganic nanoparticles (NPs) represent promising examples of engineered nanomaterials, providing interesting biomedical solutions in several fields, like therapeutics and diagnostics. Despite the extensive number of investigations motivated by their remarkable potential for nanomedicinal applications, the interactions of NPs with biological interfaces are still poorly understood. The effect of NPs on living organisms is mediated by biological barriers, such as the cell plasma membrane, whose lateral heterogeneity is thought to play a prominent role in NPs adsorption and uptake pathways. In particular, biological membranes feature the presence of rafts, that is segregated lipid micro and/or nanodomains in the so-called liquid ordered phase (Lo ), immiscible with the surrounding liquid disordered phase (Ld ). Rafts are involved in various biological functions and act as sites for the selective adsorption of materials on the membrane. Indeed, the thickness mismatch present along their boundaries generates energetically favourable conditions for the adsorption of NPs. Despite its clear implications in NPs internalisation processes and cytotoxicity, a direct proof of the selective adsorption of NPs along the rafts' boundaries is still missing to date. Here we use multicomponent supported lipid bilayers (SLBs) as reliable synthetic models, reproducing the nanometric lateral heterogeneity of cell membranes. After being characterised by atomic force microscopy (AFM) and neutron reflectivity (NR), multidomain SLBs are challenged by prototypical inorganic nanoparticles, that is citrated gold nanoparticles (AuNPs), under simplified and highly controlled conditions. By exploiting AFM, we demonstrate that AuNPs preferentially target lipid phase boundaries as adsorption sites. The herein reported study consolidates and extends the fundamental knowledge on NPs-membrane interactions, which constitute a key aspect to consider when designing NPs-related biomedical applications. LAY DESCRIPTION: Inorganic nanoparticles (NPs) represent promising examples of engineered nanomaterials, offering interesting biomedical solutions in multiple fields like therapeutics and diagnostics. Despite being extensively investigated due to their remarkable potential for nanomedicinal applications, the interaction of NPs with biological systems is in several cases still poorly understood. The interaction of NPs with living organisms is mediated by biological barriers, such as the cell plasma membrane. Supported lipid bilayers (SLBs) represent suitable synthetic membrane models for studying the physicochemical properties of natural interfaces and their interaction with inorganic nanomaterials under simplified and controlled conditions. Recently, multicomponent SLBs were developed in order to mimic the lateral heterogeneity of most biological membranes. In particular, biological membranes feature the presence of rafts, that is segregated lipid micro and/or nanodomains, enriched in cholesterol, sphingomyelin, saturated glycerophospholipids and glycosphingolipids: these lipids segregate in the so-called liquid-ordered phase (Lo ), characterised by a high molecular packing degree, which promotes the phase separation from the surrounding liquid-crystalline (disordered, Ld ) phase, where the intermolecular mobility is increased. Rafts are thought to participate in the formation and targeting of nano-sized biogenic lipid vesicles and are also actively involved in multiple membrane processes. Indeed, Lo -Ld phase boundaries represent high energy areas, providing active sites for the preferential adsorption of external material. The selective adsorption of NPs along the phase boundaries of rafted membranes has been theorised and indirectly probed by different research groups; however, a direct proof of this phenomenon is still missing to date. We herein exploit atomic force microscopy (AFM) to directly visualise the preferential adsorption of gold nanoparticles (AuNPs) along the phase boundaries of multicomponent SLBs (previously characterised by neutron reflectivity), obtained from synthetic vesicles containing both an Ld and an Lo phase. The quantitative localisation and morphometry of AuNPs adsorbed on the SLB reveal important information on their interaction with the lipid matrix and directly prove the already theorised differential NPs-lipid interaction at the phase boundaries. The presented results could help the development of future NP-based applications, involving NPs adsorption on membranes with nanoscale phase segregations.

Interaction of Aß42 with Membranes Triggers the Self-Assembly into Oligomers.

The self-assembly of amyloid ß (Aß) proteins into oligomers is the major pathogenic event leading to Alzheimer's disease (AD). Typical in vitro experiments require high protein concentrations, whereas the physiological concentration of Aß is in the picomolar to low nanomolar range. This complicates the translation of results obtained in vitro to understanding the aggregation process in vivo. Here, we demonstrate that Aß42 self-assembles into aggregates on membrane bilayers at low nanomolar concentrations - a pathway in which the membrane plays the role of a catalyst. Additionally, physiological ionic conditions (150 mM NaCl) significantly enhance on-membrane aggregation, leading to the rapid formation of oligomers. The self-assembly process is reversible, so assembled aggregates can dissociate from the membrane surface into the bulk solution to further participate in the aggregation process. Molecular dynamics simulations demonstrate that the transient membrane-Aß interaction dramatically changes the protein conformation, facilitating the assembly of dimers. The results indicate peptide-membrane interaction is the critical step towards oligomer formation at physiologically low protein concentrations.

Dry Two-Step Self-Assembly of Stable Supported Lipid Bilayers on Silicon Substrates.

Artificial membranes are models for biological systems and are important for applications. We introduce a dry two-step self-assembly method consisting of the high-vacuum evaporation of phospholipid molecules over silicon, followed by a subsequent annealing step in air. We evaporate dipalmitoylphosphatidylcholine (DPPC) molecules over bare silicon without the use of polymer cushions or solvents. High-resolution ellipsometry and AFM temperature-dependent measurements are performed in air to detect the characteristic phase transitions of DPPC bilayers. Complementary AFM force-spectroscopy breakthrough events are induced to detect single- and multi-bilayer formation. These combined experimental methods confirm the formation of stable non-hydrated supported lipid bilayers with phase transitions gel to ripple at 311.5 ± 0.9 K, ripple to liquid crystalline at 323.8 ± 2.5 K and liquid crystalline to fluid disordered at 330.4 ± 0.9 K, consistent with such structures reported in wet environments. We find that the AFM tip induces a restructuring or intercalation of the bilayer that is strongly related to the applied tip-force. These dry supported lipid bilayers show long-term stability. These findings are relevant for the development of functional biointerfaces, specifically for fabrication of biosensors and membrane protein platforms. The observed stability is relevant in the context of lifetimes of systems protected by bilayers in dry environments.

Transparent Nanopore Cavity Arrays Enable Highly Parallelized Optical Studies of Single Membrane Proteins on Chip.

Membrane proteins involved in transport processes are key targets for pharmaceutical research and industry. Despite continuous improvements and new developments in the field of electrical readouts for the analysis of transport kinetics, a well-suited methodology for high-throughput characterization of single transporters with nonionic substrates and slow turnover rates is still lacking. Here, we report on a novel architecture of silicon chips with embedded nanopore microcavities, based on a silicon-on-insulator technology for high-throughput optical readouts. Arrays containing more than 14 000 inverted-pyramidal cavities of 50 femtoliter volumes and 80 nm circular pore openings were constructed via high-resolution electron-beam lithography in combination with reactive ion etching and anisotropic wet etching. These cavities feature both, an optically transparent bottom and top cap. Atomic force microscopy analysis reveals an overall extremely smooth chip surface, particularly in the vicinity of the nanopores, which exhibits well-defined edges. Our unprecedented transparent chip design provides parallel and independent fluorescent readout of both cavities and buffer reservoir for unbiased single-transporter recordings. Spreading of large unilamellar vesicles with efficiencies up to 96% created nanopore-supported lipid bilayers, which are stable for more than 1 day. A high lipid mobility in the supported membrane was determined by fluorescent recovery after photobleaching. Flux kinetics of α-hemolysin were characterized at single-pore resolution with a rate constant of 0.96 ± 0.06 × 10-3 s-1. Here, we deliver an ideal chip platform for pharmaceutical research, which features high parallelism and throughput, synergistically combined with single-transporter resolution.

[The preparation of supported lipid bilayers and its functional study].

Supported lipid bilayers (SLBs) have been widely used in biomedical and bioengineering research in vitrobecause its structure and function are similar to natural cell membrane. A fluorescence recovery after photobleaching (FRAP) technique was used to measure the lateral diffusion of the SLBs composed of 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dioleoyl-sn-glycero-3-ï¼»(N-(5-amino-1-carboxyp-entyl) iminodiacetic acid)ï¼½ (DGS-NTA) on the glass slide, and the effects of the DOPC-to-DGS-NTA ratio, small unilamellar vesicles (SUV) producing method, sizes of bleaching areas and concentrations of loading proteins on the SLBs fluidity and diffusion coefficient were studied systematically in this paper. The results demonstrated that: (1) SUV made by probe sonication exhibited more uniform and smaller size compared with that made by film extrusion, but the whole process of SLBs formation must not be exposed to air. (2) The fluorescence recovery rate and diffusion coefficient of the SLBs decreased with the increasing bleaching area size. With the mole ratio of DOPC to DGS-NTA decreasing from 98∶2 to 84∶16, the fluidity and fluorescence recovery degree decreased gradually, and the SLBs would lose its fluidity if the ratio reached to 82∶18. (3) The average fluorescence intensity of SLBs increased linearly with the loading protein concentration (10-40 nmol·L -1), and the protein showed good mobility on the SLBs. The study would provide a good platform of bio-membrane for further research on interactions among cell membrane molecules and subsequent signals response.

Temperature induced lipid membrane restructuring and changes in nanomechanics.

The naturally occurring milk sphingomyelin is of particular interest owing to its complex composition and involvement in the formation of the milk fat globule membrane (MFGM). Knowledge of membrane organization and nanomechanical stability has proved to be crucial in understanding their properties and functions. In this work, two model membrane systems composed of 1, 2 dioleoyl-sn-glycero-3-phosphocholine (DOPC), egg sphingomyelin (egg-SM) and cholesterol, and DOPC, milk sphingomyelin (milk-SM) and cholesterol were exposed to both RT and 10°C. The morphological and nanomechanical changes were investigated using atomic force microscopy (AFM) imaging and force mapping below RT using a designed liquid cell with temperature-control. In both systems, the size and shape of SM/Chol-enriched liquid ordered domains (Lo) and DOPC-enriched liquid disordered phase (Ld) were monitored at controlled temperatures. AFM based force-mapping showed that rupture forces were consistently higher for Lo domains than Ld phases and were decreased for Ld with decreasing temperature while an increase in breakthrough force was observed in Lo domains. More interestingly, dynamic changes and defect formations in the hydrated lipid bilayers were mostly detected at low temperature, suggesting a rearrangement of lipid molecules to relieve additional tension introduced upon cooling. Noteworthy, in these model membrane systems, tension-driven defects generally heal on reheating the sample. The results of this work bring new insights to low temperature induced membrane structural reorganization and mechanical stability changes which will bring us one step closer to understand more complex systems such as the MFGM.

Pro-apoptotic cBid and Bax exhibit distinct membrane remodeling activities: An AFM study.

Bcl-2 proteins are key regulators of the mitochondrial outer membrane (MOM) permeabilization that mediates apoptosis. During apoptosis, Bid is cleaved (cBid) and translocates to the MOM, where it activates Bax. Bax then oligomerizes and induces MOM permeabilization. However, little is known about how these proteins affect membrane organization aside from pore formation. In previous studies, we have shown that both cBid and Bax are able to remodel membranes and stabilize curvature. Here, we dissected the independent effects of Bax and cBid on supported lipid structures mimicking the mitochondrial composition by means of atomic force spectroscopy. We show that cBid did not permeabilize the membrane but lowered the membrane breakthrough force. On the other hand, Bax effects were dependent on its oligomeric state. Monomeric Bax did not affect the membrane properties. In contrast, oligomeric Bax lowered the breakthrough force of the membrane, which in the context of pore formation, implies a lowering of the line tension at the edge of the pore.