Artificial Lipid Bilayers Research Articles

Effects of a Serotonergic Psychedelic on the Lipid Bilayer.

Serotonergic psychedelics, known for their hallucinogenic effects, have attracted interest due to their ability to enhance neuronal plasticity and potential therapeutic benefits. Although psychedelic-enhanced neuroplasticity is believed to require activation of 5-hydroxytryptamine (serotonin) 2A receptors (5-HT2ARs), serotonin itself is less effective in promoting such plasticity. Also, the psychoplastogenic effects of these molecules correlate with their lipophilicity, leading to suggestions that they act by influencing the intracellular receptors. However, their lipophilicity also implies that a significant quantity of lipids is accumulated in the lipid bilayer, potentially altering the physical properties of the membrane. Here, we probe whether the serotonergic psychedelic 2,5-dimethoxy-4-iodoamphetamine (DOI) can affect the properties of artificial lipid bilayers and if that can potentially affect processes such as membrane fusion. Solid-state NMR spectroscopy shows that the DOI strongly induces disorder in the lipid acyl chains. Atomic force microscopy shows that it can shrink the ordered domains in a biphasic lipid bilayer and can reduce the force needed to form nanopores in the membrane. Fluorescence correlation spectroscopy shows that DOI can promote vesicle association, and total internal fluorescence microscopy shows that it enhances vesicle fusion to a supported lipid bilayer. While serotonin has also recently been shown to cause similar effects, DOI is more than two orders of magnitude more potent in evoking these. Our results suggest that the receptor-independent effects of serotonergic psychedelics on lipid membranes may contribute to their biological actions, especially those that require significant membrane remodeling, such as neuronal plasticity.

Superlarge, Rigidified DNA Tetrahedron with a Y-Shaped Backbone for Organizing Biomolecules Spatially and Maintaining Their Full Bioactivity.

A major impediment to the clinical translation of DNA tiling nanostructures is a technical bottleneck for the programmable assembly of DNA architectures with well-defined local geometry due to the inability to achieve both sufficient structural rigidity and a large framework. In this work, a Y-backbone was inserted into each face to construct a superlarge, sufficiently rigidified tetrahedral DNA nanostructure (called RDT) with extremely high efficiency. In RDT, the spatial size increased by 6.86-fold, and the structural rigidity was enhanced at least 4-fold, contributing to an ∼350-fold improvement in the resistance to nucleolytic degradation even without a protective coating. RDT can be mounted onto an artificial lipid-bilayer membrane with molecular-level precision and well-defined spatial orientation that can be validated using the fluorescence resonance energy transfer (FRET) assay. The spatial orientation of Y-shaped backbone-rigidified RDT is unachievable for conventional DNA polyhedrons and ensures a high level of precision in the geometric positioning of diverse biomolecules with an approximately homogeneous environment. In tests of RDT, surface-confined horseradish peroxidase (HRP) exhibited nearly 100% catalytic activity and targeting aptamer-immobilized gold nanoparticles showed 5.3-fold enhanced cellular internalization. Significantly, RDT exhibited a 27.5-fold enhanced structural stability in a bodily environment and did not induce detectable systemic toxicity.

Concealing Organic Neuromorphic Devices with Neuronal-Inspired Supported Lipid Bilayers.

Neurohybrid systems have gained large attention for their potential as in vitro and in vivo platform to interrogate and modulate the activity of cells and tissue within nervous system. In this scenario organic neuromorphic devices have been engineered as bioelectronic platforms to resemble characteristic neuronal functions. However, aiming to a functional communication with neuronal cells, material synthesis, and surface engineering can yet be exploited for optimizing bio-recognition processes at the neuromorphic-neuronal hybrid interface. In this work, artificial neuronal-inspired lipid bilayers have been assembled on an electrochemical neuromorphic organic device (ENODe) to resemble post-synaptic structural and functional features of living synapses. Here, synaptic conditioning has been achieved by introducing two neurotransmitter-mediated biochemical signals, to induce an irreversible change in the device conductance thus achieving Pavlovian associative learning. This new class of in vitro devices can be further exploited for assembling hybrid neuronal networks and potentially for in vivo integration within living neuronal tissues.

Design and Construction of a Multi-Tiered Minimal Actin Cortex for Structural Support in Lipid Bilayer Applications.

Artificial lipid bilayers have revolutionized biochemical and biophysical research by providing a versatile interface to study aspects of cell membranes and membrane-bound processes in a controlled environment. Artificial bilayers also play a central role in numerous biosensing applications, form the foundational interface for liposomal drug delivery, and provide a vital structure for the development of synthetic cells. But unlike the envelope in many living cells, artificial bilayers can be mechanically fragile. Here, we develop prototype scaffolds for artificial bilayers made from multiple chemically linked tiers of actin filaments that can be bonded to lipid headgroups. We call the interlinked and layered assembly a multiple minimal actin cortex (multi-MAC). Construction of multi-MACs has the potential to significantly increase the bilayer's resistance to applied stress while retaining many desirable physical and chemical properties that are characteristic of lipid bilayers. Furthermore, the linking chemistry of multi-MACs is generalizable and can be applied almost anywhere lipid bilayers are important. This work describes a filament-by-filament approach to multi-MAC assembly that produces distinct 2D and 3D architectures. The nature of the structure depends on a combination of the underlying chemical conditions. Using fluorescence imaging techniques in model planar bilayers, we explore how multi-MACs vary with electrostatic charge, assembly time, ionic strength, and type of chemical linker. We also assess how the presence of a multi-MAC alters the underlying lateral diffusion of lipids and investigate the ability of multi-MACs to withstand exposure to shear stress.

Fluorescence-Based Proteoliposome Methods to Monitor Redox-Active Transition Metal Transmembrane Translocation by Metal Transporters.

Transmembrane transition metal transporter proteins are central gatekeepers in selectively controlling vectorial metal cargo uptake and extrusion across cellular membranes in all living organisms, thus playing key roles in essential and toxic metal homeostasis. Biochemical characterization of transporter-mediated translocation events and transport kinetics of redox-active metals, such as iron and copper, is challenged by the complexity in generating reconstituted systems in which vectorial metal transport can be studied in real time. We present fluorescence-based proteoliposome methods to monitor redox-active metal transmembrane translocation upon reconstitution of purified metal transporters in artificial lipid bilayers. By encapsulating turn-on/-off iron or copper-dependent sensors in the proteoliposome lumen and conducting real-time transport assays using small unilamellar vesicles (SUVs), in which selected purified Fe(II) and Cu(I) transmembrane importer and exporter proteins have been reconstituted, we provide a platform to monitor metal translocation events across lipid bilayers in real time. The strategy is modular and expandable toward the study of different transporter families featuring diverse metal substrate selectivity and promiscuity.

Direct single-molecule detection of CoA-SH and ATP by the membrane proteins TMEM120A and TMEM120B.

Membrane proteins are vital resources for developing biosensors. TMEM120A is a membrane protein associated with human pain transmission and lipid metabolism, and recent studies have demonstrated its ability to transport ions and bind to coenzyme A (COA-SH), indicating its potential to develop into a single-molecule sensor based on electrical methods. In this study, we investigated the ion transport properties of TMEM120A and its homolog TMEM120B on an artificial lipid bilayer using single-channel recording. The results demonstrate that both proteins can fuse into the lipid bilayer and generate stable ion currents under a bias voltage. Based on the stable ion transport capabilities of TMEM120A and TMEM120B, as well as the feature of TMEM120A binding with COA-SH, we developed these two proteins into a single-molecule sensor for detecting COA-SH and structurally similar molecules. We found that both COA-SH and ATP can reversibly bind to single TMEM120A and TMEM120B proteins embedded in the lipid bilayer and temporarily block ion currents during the binding process. By analyzing the current blocking signal, COA-SH and ATP can be identified at the single-molecule level. In conclusion, our work has provided two single-molecule biosensors for detecting COA-SH and ATP, offering insights for exploring and developing bio-inspired small molecule sensors.

Use of Synchrotron Radiation Circular Dichroism to Analyze the Interaction and Insertion of Proteins into Bacterial Outer Membrane Vesicles.

Circular dichroism (CD) is a spectroscopic technique commonly used for the analysis of proteins. Particularly, it allows the determination of protein secondary structure content in various media, including the membrane environment. In this chapter, we present how CD applications can be used to analyze the interaction of proteins with bacterial outer membrane vesicles (OMVs). Most CD studies characterizing the structure of proteins inserted into membranes rely on artificial lipid bilayers, mimicking natural membranes. Nevertheless, these artificial models lack the important features of the true membrane, especially for the outer membrane of Gram-negative bacteria. These features include lipid diversity, glycosylation, and asymmetry. Here, we show how to analyze the interactions of proteins, either integral or peripheral, with OMVs in solution and with supported membranes of OMVs, using conventional CD and orientated circular dichroism (OCD). We explain how to decipher the spectroscopic signals to obtain information on the molecular structure of the protein upon its interaction with an OMV and through its potential insertion into an OMV membrane.

Metal selectivity and translocation mechanism characterization in proteoliposomes of the transmembrane NiCoT transporter NixA from Helicobacter pylori.

Essential trace metals play key roles in the survival, replication, and virulence of bacterial pathogens. Helicobacter pylori (H. pylori), the main bacterial cause of gastric ulcers, requires Ni(ii) to colonize and persist in the acidic environment inside the stomach, exploiting the nickel-containing enzyme urease to catalyze the hydrolysis of urea to ammonia and bicarbonate and create a pH-buffered microenvironment. Urease utilizes Ni(ii) as a catalytic cofactor for its activity. In ureolytic bacteria, unique transmembrane (TM) transporters evolved to guarantee the selective uptake and efflux of Ni(ii) across cellular membranes to meet the cellular requirements. NixA is an essential Ni(ii) transporter expressed by H. pylori when the extracellular environment experiences a drop in pH. This Class I nickel-cobalt transporter of the NiCoT family catalyzes the uptake of Ni(ii) across the inner membrane from the periplasm. In this study, we characterized NixA using a platform whereby, for the first time on a NiCoT transporter, recombinantly expressed and purified NixA and key mutants in the translocation pathway have been reconstituted in artificial lipid bilayer vesicles (proteoliposomes). Fluorescent sensors responsive to Ni(ii) transport (Fluozin-3-Zn(ii)), luminal pH changes (pyranine), and membrane potential (oxonol VI) were encapsulated in the proteoliposomes lumen to monitor, in real-time, NixA transport properties and translocation mechanism. Kinetic transport analysis revealed that NixA is highly selective for Ni(ii) with no substrate promiscuity towards Co(ii), the other putative metal substrate of the NiCoT family, nor Zn(ii). NixA-mediated Ni(ii) transport exhibited a Michaelis-Menten-type saturable substrate concentration dependence, with an experimental KM, Ni(ii) = 31.0 ± 1.2 μM. Ni(ii) transport by NixA was demonstrated to be electrogenic, and metal translocation did not require a proton motive force, resulting in the generation of a positive-inside transmembrane potential in the proteoliposome lumen. Mutation analysis characterized key transmembrane residues for substrate recognition, binding, and/or transport, suggesting the presence of a three-step transmembrane translocation conduit. Taken together, these investigations reveal that NixA is a Ni(ii)-selective Class I NiCoT electrogenic uniporter. The work also provides an in vitro approach to characterize the transport properties of metal transporters responsible for Ni(ii) acquisition and extrusion in prokaryotes.

Interaction of ROMK2 channel with lipid kinases DGKE and AGK: Potential channel activation by localized anionic lipid synthesis

In this study, we utilized enzyme-catalyzed proximity labeling with the engineered promiscuous biotin ligase Turbo-ID to identify the proxisome of the ROMK2 channel. This channel resides in various cellular membrane compartments of the cell including the plasma membrane, endoplasmic reticulum and mitochondria. Within mitochondria, ROMK2 has been suggested as a pore-forming subunit of mitochondrial ATP-regulated potassium channel (mitoKATP). We found that ROMK2 proxisome in addition to previously known protein partners included two lipid kinases: acylglycerol kinase (AGK) and diacylglycerol kinase ε (DGKE), which are localized in mitochondria and the endoplasmic reticulum, respectively. Through co-immunoprecipitation, we confirmed that these two kinases are present in complexes with ROMK2 channels. Additionally, we found that the products of AGK and DGKE, lysophosphatidic acid (LPA) and phosphatidic acid (PA), stimulated the activity of ROMK2 channels in artificial lipid bilayers. Our molecular docking studies revealed the presence of acidic lipid binding sites in the ROMK2 channel, similar to those previously identified in Kir2 channels. Based on these findings, we propose a model wherein localized lipid synthesis, mediated by channel-bound lipid kinases, contributes to the regulation of ROMK2 activity within distinct intracellular compartments, such as mitochondria and the endoplasmic reticulum.

The Integrin Adaptor Kindlin-3 Is a Gatekeeper for Germinal Center Responses and the Development of Chronic Lymphocytic Leukemia

Background: High affinity antibody production occurs in the germinal centers (GCs), which are segregated into a dark zone (DZ) and a light zone (LZ). GC B cells proliferate in the DZ and encounter follicular dendritic cells in the LZ. Cell trafficking between the two zones is regulated by CXCR4 (DZ) and CXCR5 (LZ) signaling. Integrins may regulate B cell positioning and B cell immune synapse formation. Recently, we have shown that the integrin adaptor kindlin-3 is not only required for integrin activation in B cells, but unexpectedly also acts as a brake on CXCR5 responsiveness. Kindlin-3 thereby prevents B cell hyperactivation and differentiation into GC-like B cells in absence of antigen. 1 Here we investigate the impact of kindlin-3 on GC responses upon antigen challenging and on malignant transformation. Methods: We developed a B cell-specific knockout mouse model (mb1Cre Fermt3fl/fl (K3ΔB)) in which kindlin-3 ( Fermt3) was knocked out in all B cells. K3ΔB and control wild-type (WT) mice were challenged with sheep red blood cells (SRBC). GC formation and immunoglobulin (Ig) production were analyzed by flow cytometry. Ex vivo B cell proliferation and Ig class switching formation assays were performed. We investigated the immune synapse formation of WT and kindlin-3-deficient B cells using a biomimetic model involving a planar artificial lipid bilayer containing GPI-linked integrin ligands and bound fluorescently labeled surrogate antigens. The formation of immune synapse and central antigen aggregation in the immune synapse were analyzed by real-time interference reflection microscopy and laser scanning confocal microscopy. To investigate the contribution of kindlin-3 in a model of mature B cell malignancy, we used TCL1 transgenic (TCL1) mice resembling chronic lymphocytic leukemia (CLL) to generate the mb1Cre Fermt3fl/fl Tcl1 (TCL1-K3ΔB) mouse model. Tumor burden in TCL1-K3ΔB and TCL1 control mice was monitored by flow cytometry over time and at sacrifice using CD19/CD5 antibodies. B cell receptor (BCR) signaling of CLL cells was analyzed using Phosphoflow. Results: K3∆B mice immunized with SRBC exhibited defective GC responses (30% reduction in GC-B cells compared to WT) and antibody production (49% reduction in SRBC-specific IgG2b production in serum compared to WT) on day 7 after immunization. Ex vivo, kindlin-3-deficient B cells were unable to form immune synapses (Figure 1A), to proliferate, and to switch antibody classes. The tumors in TCL1 mice started in the peritoneal cavity and subsequently spread to the spleen, blood and bone marrow. TCL1-K3ΔB mice presented with an almost complete abolished leukemia onset in the peritoneal cavity (92% less tumor burden in TCL1-K3ΔB compared to control TCL1 mice) in 4-month-old mice (Figure 1B i). Moreover, tumor burden in blood (Figure 1B ii), spleen, and bone marrow (Figure 1B iii) was strongly reduced at the late stage of disease (11.5-month-old mice), with an 82% lower tumor burden in spleen and a 90% lower tumor burden in bone marrow of TCL1-K3ΔB mice compared to TCL1 control mice. In addition, kindlin-3-deficient CLL cells exhibited impaired phosphoAkt and phosphoSyk signaling upon BCR engagement. Summary/Conclusion: In summary, kindlin-3 maintains the balance between a state of B cell homeostasis and an effective antigenic response by acting as a gatekeeper of GC formation, and is substantial for leukemic onset in CLL. Reference: 1. Härzschel A, Li L, Krenn PW, et al. Kindlin-3 maintains marginal zone B cells but confines follicular B cell activation and differentiation. J Leukoc Biol. 2022 Apr;111(4):745-758.

Assembly of Cell-Free Synthesized Ion Channel Molecules in Artificial Lipid Bilayer Observed by Atomic Force Microscopy.

Artificial lipid bilayer systems, such as vesicles, black membranes, and supported lipid bilayers (SLBs), are valuable platforms for studying ion channels at the molecular level. The reconstitution of the ion channels in an active form is a crucial process in studies using artificial lipid bilayer systems. In this study, we investigated the assembly of the human ether-a-go-go-related gene (hERG) channel prepared in a cell-free synthesis system. AFM topographies revealed the presence of protrusions with a uniform size in the entire SLB that was prepared with the proteoliposomes (PLs) incorporating the cell-free-synthesized hERG channel. We attributed the protrusions to hERG channel monomers, taking into consideration the AFM tip size, and identified assembled structures of the monomer that exhibited dimeric, trimeric, and tetrameric-like arrangements. We observed molecular images of the functional hERG channel reconstituted in a lipid bilayer membrane using AFM and quantitatively evaluated the association state of the cell-free synthesized hERG channel.

Dantrolene inhibition of ryanodine channels (RyR2) in artificial lipid bilayers depends on FKBP12.6.

Dantrolene is a neutral hydantoin that is clinically used as a skeletal muscle relaxant to prevent overactivation of the skeletal muscle calcium release channel (RyR1) in response to volatile anesthetics. Dantrolene has aroused considerable recent interest as a lead compound for stabilizing calcium release due to overactive cardiac calcium release channels (RyR2) in heart failure. Previously, we found that dantrolene produces up to a 45% inhibition RyR2 with an IC50 of 160 nM, and that this inhibition requires the physiological association between RyR2 and CaM. In this study, we tested the hypothesis that dantrolene inhibition of RyR2 in the presence of CaM is modulated by RyR2 phosphorylation at S2808 and S2814. Phosphorylation was altered by incubations with either exogenous phosphatase (PP1) or kinases; PKA to phosphorylate S2808 or endogenous CaMKII to phosphorylate S2814. We found that PKA caused selective dissociation of FKBP12.6 from the RyR2 complex and a loss of dantrolene inhibition. Rapamycin-induced FKBP12.6 dissociation from RyR2 also resulted in the loss of dantrolene inhibition. Subsequent incubations of RyR2 with exogenous FKBP12.6 reinstated dantrolene inhibition. These findings indicate that the inhibitory action of dantrolene on RyR2 depends on RyR2 association with FKBP12.6 in addition to CaM as previously found.

Characterizing Oscillatory and Excitability Regimes in a Protein-Free Lipid Membrane

Observations of electric potential oscillations in artificial lipid bilayers near the order-disorder transition indicate the existence of a stable limit cycle and, therefore, the possibility of producing excitable signals close to the bifurcation. We present a theoretical investigation of membrane oscillatory and excitability regimes induced by an increase in ion permeability at the order-disorder transition. The model accounts for the coupled effects of state-dependent permeability, membrane charge density, and hydrogen ion adsorption. A bifurcation diagram shows a transition between fixed-point and limit cycle solutions, enabling both oscillatory and excitability responses at different values of the acid association parameter. Oscillations are identified in terms of the membrane state, electric potential difference, and ion concentration near the membrane. The emerging voltage and time scales agree with measurements. Excitability is demonstrated by applying an external electric current stimulus, and the emerging signals display a threshold response and the appearance of repetitive signals upon using a long-lasting stimulus. The approach highlights the important role of the order-disorder transition, enabling membrane excitability in the absence of specialized proteins.

Abstract 540: c-MET mutations sensitize to antibody-drug conjugate telisotuzumab vedotin through efficient internalization and rapid intracellular drug delivery

Abstract Background: Telisotuzumab vedotin (Teliso-V) is an antibody-drug conjugate (ADC) composed of the c-MET antibody ABT-700 and the microtubule inhibitor monomethyl auristatin E (MMAE). Patients with EGFR wildtype (WT) nonsquamous non-small cell lung cancer with high c-MET expression had an overall response rate of 54% with Teliso-V. In addition to overexpression and amplification, genetic alterations in c-MET, such as exon 14 deletion (ex14del) and missense mutations in the tyrosine kinase domain (TKD), contribute to its constitutive activation. The efficacy of Teliso-V in context of these genomic backgrounds is currently unknown. Methods: Recombinant retroviruses were used to express WT-c-MET, c-MET-TKD (D1228H/V/N and Y1230C/H mutations), c-MET-Y1003F/N (CBL E3 ubiquitin ligase binding site mutation), and c-MET-ex14del (47 aa juxtamembrane deletion) mutants in NIH3T3 and Ba/F3 cells. c-MET surface expression, intracellular signaling, ADC internalization and endolysosomal (EL) trafficking (using pHrodo Red-labeled Teliso-V), intracellular MMAE by LC/MS, and Teliso-V sensitivity were studied in 2D/3D cell cultures. Full-length c-MET protein structures were created with the ColabFold program and molecular dynamics simulation software (with/without artificial lipid bilayer) was used to predict the mechanistic basis for differential function. Results: c-MET expression was required for Teliso-V cytotoxicity and intracellular payload delivery in all cell lines. Compared with WT-c-MET, various mutants showed oncogene-driven transformation evident by elevated levels of phosphorylated c-MET/AKT, cytokine-independent colony formation, oncogene-driven proliferation, and tyrosine kinase inhibitor (TKI) resistance. Interestingly, c-MET-TKD-expressing cells were the most sensitive to Teliso-V, which was associated with faster ADC internalization and EL trafficking. An AI-generated protein model and associated molecular dynamics simulation demonstrated that all oncogenic TKD variants tested stabilized the c-MET-plexin-semaphorin-integrin domain (R592-L614), known for its ability to position the extracellular ligand-binding (ELB) site for optimal activation. Conclusions: c-MET-TKD mutations induced TKI resistance and enhanced MMAE delivery in association with robust ADC internalization and EL trafficking. Previous studies have shown that the HER2 ADC fam-trastuzumab deruxtecan-nxki was highly effective in cell lines and patients with ERBB2 kinase domain-mutant lung cancer via a similar mechanism, raising the possibility that this may be a general phenomenon across receptor tyrosine kinase-targeting ADCs. c-MET-TKD mutations are predicted to stabilize the ELB conformation to activate enhanced oncogenic signaling that may contribute to rapid endocytosis-mediated drug delivery. Citation Format: Izhar S. Batth, Bijay S. Jaiswal, Dolonchampa Maji, Robert Sparks, William Glauser, Tamar Uziel, D. Ross Camidge, Athan Vasilopoulos, Peter Ansell, PK Epling-Burnette. c-MET mutations sensitize to antibody-drug conjugate telisotuzumab vedotin through efficient internalization and rapid intracellular drug delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 540.

Revealing the single-channel characteristics of OprD (OccAB1) porins from hospital strains of Acinetobacter baumannii.

Nowadays, reports of antimicrobial resistance (AMR) against many antibiotics are increasing because of their misapplication. With this rise, there is a serious decrease in the discovery and development of new types of antibiotics amid an increase in multi-drug resistance. Unfermented Acinetobacter baumannii from gram-negative bacteria, which is one of the main causes of nosocomial infections and multi-drug resistance, has 4 main kinds of antibiotic resistance mechanism: inactivating antibiotics by enzymes, reduced numbers of porins and changing of their target or cellular functions due to mutations, and efflux pumps. In this study, characterization of the possible mutations in OprD (OccAB1) porins from hospital strains of A. baumannii were investigated using single channel electrophysiology and compared with the standard OprD isolated from wild type ATCC 19,606. For this aim, 5 A. baumannii bacteria samples were obtained from patients infected with A. baumannii, after which OprD porins were isolated from these A. baumannii strains. OprD porins were then inserted in an artificial lipid bilayer and the current-voltage curves were obtained using electrical recordings through a pair of Ag/AgCl electrodes. We observed that each porin has a characteristic conductance and single channel recording, which then leads to differences in channel diameter. Finally, the single channel data have been compared with the gene sequences of each porin. It was interesting to find out that each porin isolated has a unique porin diameter and decreased anion selectivity compared to the wild type.

Bioinspired Membrane Interfaces: Controlling Actomyosin Architecture and Contractility.

The creation of biologically inspired artificial lipid bilayers on planar supports provides a unique platform to study membrane-confined processes in a well-controlled setting. At the plasma membrane of mammalian cells, the linkage of the filamentous (F)-actin network is of pivotal importance leading to cell-specific and dynamic F-actin architectures, which are essential for the cell's shape, mechanical resilience, and biological function. These networks are established through the coordinated action of diverse actin-binding proteins and the presence of the plasma membrane. Here, we established phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2)-doped supported planar lipid bilayers to which contractile actomyosin networks were bound via the membrane-actin linker ezrin. This membrane system, amenable to high-resolution fluorescence microscopy, enabled us to analyze the connectivity and contractility of the actomyosin network. We found that the network architecture and dynamics are not only a function of the PtdIns[4,5]P2 concentration but also depend on the presence of negatively charged phosphatidylserine (PS). PS drives the attached network into a regime, where low but physiologically relevant connectivity to the membrane results in strong contractility of the actomyosin network, emphasizing the importance of the lipid composition of the membrane interface.

A molecular imprint of intracellular amyloid-beta ApoE interaction correlates with its toxicity molecular imprint of intracellular amyloid beta ApoE interaction.

ApoE isoforms are major genetic risk factors related to Alzheimer's disease (AD), which is associated with the aggregation of the amyloid beta (Aβ) peptide. However, a molecular-level understanding of how ApoE is involved in AD pathology is still lacking. Here we employ a fluorescent lifetime-based assay that reports a specific ApoE-dependent modification of Aβ, both in vitro and in neuronal cells. We explore this phenomenon in several types of cells where the change of lifetime of Aβ oligomers correlates with the relative ApoE content, with the highest effect displayed by primary-cultured rat astrocytes. At the single-molecule level, ApoE-induced modification also produces distinct Aβ oligomers with increased affinity to artificial lipid bilayers. A known peptide inhibitor of Aβ-ApoE interaction, LVFFA, diminishes both the lifetime modification and toxicity of Aβ oligomers in a dose-dependent manner. The correlation between the short lifetime component and Aβ toxicity was corroborated by experiments using two nontoxic Aβ variants. Our data, therefore, establish a clear correlation between the lifetime modification of Aβ and its toxicity. Finally, neuronal stem cells derived from cells obtained from Alzheimer's patients also show the short-lifetime component, the changed oligomeric state, and the increased membrane affinity, which is likely to be relevant for AD. In summary, our assay can provide a direct readout of the toxic ApoE-Aβ interaction in vitro and in cells, which can provide a platform for AD drug discovery.

OPRLM: Orientations of proteins in realistic lipid membranes.

A new web tool and a database, OPRLM (Orientations of Proteins in Realistic Lipid Membranes), has been created based on the OPM database and the broadly used GHARMM-GUI toolset. The database provides a complete set of experimental 3D structures of integral and peripheral membrane proteins from the Protein Data Bank that are positioned in planar and curved lipid bilayers using implicit and explicit membrane representations. Membrane systems vary from single-component artificial lipid bilayers to multi-component bilayers with complex lipid composition corresponding to 18 biomembranes with either asymmetric or symmetric distribution of lipid types between two leaflets: 14 “average” eukaryotic cellular and organelle membranes (plasma membranes of mammals, plants, and fungi, endoplasmic reticulum and apparatus Golgi membranes of mammals and fungi, mammalian membranes of endosomes and lysosomes, mitochondrial outer and inner membranes, plant vacuole membranes, thylakoid membranes of plants and cyanobacteria), the outer and the inner membranes of Gram-negative bacteria (E. coli), and “average” cell membranes of Gram-positive bacteria and archaebacteria. All these membrane systems are accessible for public use in CHARMM-GUI Archive (https://www.charmm-gui.org/docs/archive/biomembrane). The web tool allows users of any level of expertise an easy setup of protein-membrane systems for atomic molecular dynamics (MD) simulations in pre-assembled realistic lipid membranes formed by mixtures of explicit lipids. OPRLM is publicly available at https://oprlm.org/.

Chromatographic Phospholipid Trapping for Automated H/D Exchange Mass Spectrometry of Membrane Protein-Lipid Assemblies.

Lipid interactions modulate the function, folding, structure, and organization of membrane proteins. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a useful tool to understand the structural dynamics of these proteins within lipid environments. Lipids, however, have proven problematic for HDX-MS analysis of membrane-embedded proteins due to their presence of impairing proteolytic digestion, causing liquid chromatography column fouling, ion suppression, and/or mass spectral overlap. Herein, we describe the integration of a chromatographic phospholipid trap column into the HDX-MS apparatus to enable online sample delipidation prior to protease digestion of deuterium-labeled protein-lipid assemblies. We demonstrate the utility of this method on membrane scaffold protein-lipid nanodisc─both empty and loaded with the ∼115 kDa transmembrane protein AcrB─proving efficient and automated phospholipid capture with minimal D-to-H back-exchange, peptide carry-over, and protein loss. Our results provide insights into the efficiency of phospholipid capture by ZrO2-coated and TiO2 beads and describe how solution conditions can be optimized to maximize not only the performance of our online but also the existing offline, delipidation workflows for HDX-MS. We envision that this HDX-MS method will significantly ease membrane protein analysis, allowing to better interrogate their dynamics in artificial lipid bilayers or even native cell membranes.

Inhibition of Tolaasin Cytotoxicity Causing Brown Blotch Disease in Cultivated Mushrooms Using Tolaasin Inhibitory Factors.

Tolaasin, a pore-forming bacterial peptide toxin secreted by Pseudomonas tolaasii, causes brown blotch disease in cultivated mushrooms by forming membrane pores and collapsing the membrane structures. Tolaasin is a lipodepsipeptide, MW 1985, and pore formation by tolaasin molecules is accomplished by hydrophobic interactions and multimerizations. Compounds that inhibit tolaasin toxicity have been isolated from various food additives. Food detergents, sucrose esters of fatty acids, and polyglycerol esters of fatty acids can effectively inhibit tolaasin cytotoxicity. These chemicals, named tolaasin-inhibitory factors (TIF), were effective at concentrations ranging from 10-4 to 10-5 M. The most effective compound, TIF 16, inhibited tolaasin-induced hemolysis independent of temperature and pH, while tolaasin toxicity increased at higher temperatures. When TIF 16 was added to tolaasin-pretreated erythrocytes, the cytotoxic activity of tolaasin immediately stopped, and no further hemolysis was observed. In the artificial lipid bilayer, the single-channel activity of the tolaasin channel was completely and irreversibly blocked by TIF 16. When TIF 16 was sprayed onto pathogen-treated oyster mushrooms growing on the shelves of cultivation houses, the development of disease was completely suppressed, and normal growth of oyster mushrooms was observed. Furthermore, the treatment with TIF 16 did not show any adverse effect on the growth of oyster mushrooms. These results indicate that TIF 16 is a good candidate for the biochemical control of brown blotch disease.