CaV1.3 channel clusters characterized by live-cell and isolated plasma membrane nanoscopy.

A key player of excitable cells in the heart and brain is the L-type calcium channel CaV1.3. In the heart, it is required for voltage-dependent Ca2+-signaling, i.e., for controlling and modulating atrial cardiomyocyte excitation-contraction coupling. The clustering of CaV1.3 in functionally relevant channel multimers has not been addressed due to a lack of stoichiometric labeling combined with high-resolution imaging. Here, we developed a HaloTag-labeling strategy to visualize and quantify CaV1.3 clusters using STED nanoscopy to address the questions of cluster size and intra-cluster channel density. Channel clusters were identified in the plasma membrane of transfected live HEK293 cells as well as in giant plasma membrane vesicles derived from these cells that were spread on modified glass support to obtain supported plasma membrane bilayers (SPMBs). A small fraction of the channel clusters was colocalized with early and recycling endosomes at the membranes. STED nanoscopy in conjunction with live-cell and SPMB imaging enabled us to quantify CaV1.3 cluster sizes and their molecular density revealing significantly lower channel densities than expected for dense channel packing. CaV1.3 channel cluster size and molecular density were increased in SPMBs after treatment of the cells with the sympathomimetic compound isoprenaline, suggesting a regulated channel cluster condensation mechanism.

The antimicrobial fibupeptide lugdunin forms water-filled channel structures in lipid membranes.

Recently, a novel cyclo-heptapeptide composed of alternating D,L-amino acids and a unique thiazolidine heterocycle, called lugdunin, was discovered, which is produced by the nasal and skin commensal Staphylococcus lugdunensis. Lugdunin displays potent antimicrobial activity against a broad spectrum of Gram-positive bacteria, including challenging-to-treat methicillin-resistant Staphylococcus aureus (MRSA). Lugdunin specifically inhibits target bacteria by dissipating their membrane potential. However, the precise mode of action of this new class of fibupeptides remains largely elusive. Here, we disclose the mechanism by which lugdunin rapidly destabilizes the bacterial membrane potential using an in vitro approach. The peptide strongly partitions into lipid compositions resembling Gram-positive bacterial membranes but less in those harboring the eukaryotic membrane component cholesterol. Upon insertion, lugdunin forms hydrogen-bonded antiparallel ß-sheets by the formation of peptide nanotubes, as demonstrated by ATR-FTIR spectroscopy and molecular dynamics simulations. These hydrophilic nanotubes filled with a water wire facilitate not only the translocation of protons but also of monovalent cations as demonstrated by voltage-clamp experiments on black lipid membranes. Collectively, our results provide evidence that the natural fibupeptide lugdunin acts as a peptidic channel that is spontaneously formed by an intricate stacking mechanism, leading to the dissipation of a bacterial cell's membrane potential.

Chemical synthesis of site-selective advanced glycation end products in α-synuclein and its fragments.

Advanced glycation end products (AGEs) arise from the Maillard reaction between dicarbonyls and proteins, nucleic acids, or specific lipids. Notably, AGEs are linked to aging and implicated in various disorders, spanning from cancer to neurodegenerative diseases. While dicarbonyls like methylglyoxal preferentially target arginine residues, lysine-derived AGEs, such as N(6)-(1-carboxymethyl)lysine (CML) and N(6)-(1-carboxyethyl)lysine (CEL), are also abundant. Predicting protein glycation in vivo proves challenging due to the intricate nature of glycation reactions. In vitro, glycation is difficult to control, especially in proteins that harbor multiple glycation-prone amino acids. α-Synuclein (aSyn), pivotal in Parkinson's disease and synucleinopathies, has 15 lysine residues and is known to become glycated at multiple lysine sites. To understand the influence of glycation in specific regions of aSyn on its behavior, a strategy for site-specific glycated protein production is imperative. To fulfill this demand, we devised a synthetic route integrating solid-phase peptide synthesis, orthogonal protection of amino acid side-chain functionalities, and reductive amination strategies. This methodology yielded two disease-related N-terminal peptide fragments, each featuring five and six CML and CEL modifications, alongside a full-length aSyn protein containing a site-selective E46CEL modification. Our synthetic approach facilitates the broad introduction of glycation motifs at specific sites, providing a foundation for generating glycated forms of synucleinopathy-related and other disease-relevant proteins.

Fluorophore position of headgroup-labeled Gb3 glycosphingolipids in lipid bilayers.

Fluorescent lipid probes are an invaluable tool for investigating lipid membranes. In particular, localizing certain receptor lipids such as glycosphingolipids within phase-separated membranes is of pivotal interest to understanding the influence of protein-receptor lipid binding on membrane organization. However, fluorescent labeling can readily alter the phase behavior of a lipid membrane because of the interaction of the fluorescent moiety with the membrane interface. Here, we investigated Gb3 glycosphingolipids, serving as receptor lipids for the protein Shiga toxin, with a headgroup attached BODIPY fluorophore separated by a polyethylene glycol (PEG) spacer of different lengths. We found that the diffusion coefficients of the fluorescently labeled Gb3 species in 1,2-dioleoyl-sn-glycero-3-phosphocholine/Gb3 (98:2, n/n) supported lipid bilayers are unaltered by the PEG spacer length. However, quenching as well as graphene-induced energy transfer experiments indicated that the length of the PEG spacer (n = 3 and n = 13) alters the position of the BODIPY fluorophore. In particular, the graphene-induced energy transfer technique provided accurate end-to-end distances between the fluorophores in the two leaflets of the bilayer thus enabling us to quantify the distance between the membrane interface and the fluorophore with sub-nanometer resolution. The spacer with three oligo ethylene glycol groups positioned the BODIPY fluorophore directly at the membrane interface favoring its interaction with the bilayer and thus may disturb lipid packing. However, the longer PEG spacer (n = 13) separated the BODIPY moiety from the membrane surface by 1.5 nm.

Photo-Lipids: Light-Sensitive Nano-Switches to Control Membrane Properties.

Biological membranes are described as a complex mixture of lipids and proteins organized according to thermodynamic principles. This chemical and spatial complexity can lead to specialized functional membrane domains enriched with specific lipids and proteins. The interaction between lipids and proteins restricts their lateral diffusion and range of motion, thus altering their function. One approach to investigating these membrane properties is to use chemically accessible probes. In particular, photo-lipids, which contain a light-sensitive azobenzene moiety that changes its configuration from trans- to cis- upon light irradiation, have recently gained popularity for modifying membrane properties. These azobenzene-derived lipids serve as nanotools for manipulating lipid membranes in vitro and in vivo. Here, we will discuss the use of these compounds in artificial and biological membranes as well as their application in drug delivery. We will focus mainly on changes in the membrane's physical properties as well as lipid membrane domains in phase-separated liquid-ordered/liquid-disordered bilayers driven by light, and how these changes in membrane physical properties alter transmembrane protein function.

Membrane-Bound Vimentin Filaments Reorganize and Elongate under Strain.

Within a cell, intermediate filaments interact with other cytoskeletal components, altogether providing the cell's mechanical stability. However, little attention has been drawn to intermediate filaments close to the plasma membrane. In this cortex configuration, the filaments are coupled and arranged in parallel to the membrane, and the question arises of how they react to the mechanical stretching of the membrane. To address this question, we set out to establish an in vitro system composed of a polydimethylsiloxane-supported lipid bilayer. With a uniaxial stretching device, the supported membrane was stretched up to 34% in the presence of a lipid reservoir that was provided by adding small unilamellar vesicles in the solution. After vimentin attachment to the membrane, we observed structural changes of the vimentin filaments in networks of different densities by fluorescence microscopy and atomic force microscopy. We found that individual filaments respond to the membrane stretching with a reorganization along the stretching direction as well as an intrinsic elongation, while in a dense network, mainly filament reorganization was observed.

HAV-Peptides Attached to Colloidal Probes Faithfully Detect E-Cadherins Displayed on Living Cells.

Cell adhesion molecules are crucial for a variety of biological processes, including wound healing, barrier formation and tissue homeostasis. One of them is E-cadherin which is generally found at adherent junctions between epithelial cells. To identify this molecule on the surface of cells, E-cadherin mimetic peptides with a critical amino acid sequence of HAV (histidine-alanine-valine) were synthesized and attached to solid-supported membranes covering colloidal probes. Two different functionalization strategies were established, one based on the complexation of DOGS-NTA(Ni) with a polyhistidine-tagged HAV-peptide and the other one relying on the formation of a HAV-lipopeptide using in situ maleimide-thiol coupling. Binding studies were performed to verify the ability of the peptides to attach to the membrane surface. Compared to the non-covalent attachment via the His-tag, we achieved a higher yield by lipopeptide formation. Colloidal probes functionalized with HAV-peptides were employed to measure the presence of E-cadherins on living cells either using video particle tracking or force spectroscopy. Here, human HaCaT cells were examined confirming the specific interaction of the HAV-peptide with the E-cadherin of the cells. Statistical methods were also used to determine the number of single-bond ruptures and the force of a single bond. These findings may be essential for the development of novel biosynthetic materials given their potential to become increasingly relevant in medical applications.

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.

FERM domains recruit ample PI(4,5)P2s to form extensive protein-membrane attachments.

The four-point-one ezrin-radixin-moesin homology (FERM) protein domain is a multifunctional protein-lipid binding site, constituting an integral part of numerous membrane-associated proteins. Its interaction with the lipid phosphatidylinositol-4,5-bisphosphate (PIP2), located at the inner leaflet of eukaryotic plasma membranes, is important for localization, anchorage, and activation of FERM-containing proteins. FERM-PIP2 complexes structurally determined so far exclusively feature a 1:1 binding stoichiometry of protein and lipid, with a few basic FERM residues neutralizing the -4 charge of the bound PIP2. Whether this picture from static crystal structures also applies to the dynamic interaction of FERM domains on PIP2 membranes is unknown. We here quantified the stoichiometry of FERM-PIP2 binding in a lipid bilayer using atomistic molecular dynamics simulations and experiments on solid supported membranes for the FERM domains of focal adhesion kinase and ezrin. In contrast to the structural data, we find much higher average stoichiometries of FERM-PIP2 binding, amounting to 1:3 or 1:4 ratios, respectively. In simulations, the full set of basic residues at the membrane interface, 7 and 15 residues for focal adhesion kinase and ezrin, respectively, engages in PIP2 interactions. In addition, Na ions enter the FERM-membrane binding interface, compensating negative PIP2 charges in case of high charge surpluses from bound PIP2. We propose the multivalent binding of FERM domains to PIP2 in lipid bilayers to significantly enhance the stability of FERM-membrane binding and to render the FERM-membrane linkage highly adjustable.

Optical Manipulation of Gb3 Enriched Lipid Domains: Impact of Isomerization on Gb3 -Shiga Toxin B Interaction.

The plasma membrane is a complex assembly of proteins and lipids that can self-assemble in submicroscopic domains commonly termed "lipid rafts", which are implicated in membrane signaling and trafficking. Recently, photo-sensitive lipids were introduced to study membrane domain organization, and photo-isomerization was shown to trigger the mixing and de-mixing of liquid-ordered (lo ) domains in artificial phase-separated membranes. Here, we synthesized globotriaosylceramide (Gb3 ) glycosphingolipids that harbor an azobenzene moiety at different positions of the fatty acid to investigate light-induced membrane domain reorganization, and that serve as specific receptors for the protein Shiga toxin (STx). Using phase-separated supported lipid bilayers on mica surfaces doped with four different photo-Gb3 molecules, we found by fluorescence microscopy and atomic force microscopy that liquid disordered (ld ) domains were formed within lo domains upon trans-cis photo-isomerization. The fraction and size of these ld domains were largest for Gb3 molecules with the azobenzene group at the end of the fatty acid. We further investigated the impact of domain reorganization on the interaction of the B-subunits of STx with the photo-Gb3 . Fluorescence and atomic force micrographs clearly demonstrated that STxB binds to the lo phase if Gb3 is in the trans-configuration, whereas two STxB populations are formed if the photo-Gb3 is switched to the cis-configuration highlighting the idea of manipulating lipid-protein interactions with a light stimulus.

From LUVs to GUVs─How to Cover Micrometer-Sized Pores with Membranes.

Pore-spanning membranes (PSMs) are a versatile tool to investigate membrane-confined processes in a bottom-up approach. Pore sizes in the micrometer range are most suited to visualize PSMs using fluorescence microscopy. However, the preparation of these PSMs relies on the spreading of giant unilamellar vesicles (GUVs). GUV production faces several limitations. Thus, alternative ways to generate PSMs starting from large or small unilamellar vesicles that are more reproducibly prepared are highly desirable. Here we describe a method to produce PSMs obtained from large unilamellar vesicles, making use of droplet-stabilized GUVs generated in a microfluidic device. We analyzed the lipid diffusion in the free-standing and supported parts of the PSMs using z-scan fluorescence correlation spectroscopy and fluorescence recovery after photobleaching experiments in combination with finite element simulations. Employing atomic force indentation experiments, we also investigated the mechanical properties of the PSMs. Both lipid diffusion constants and lateral membrane tension were compared to those obtained on PSMs derived from electroformed GUVs, which are known to be solvent- and detergent-free, under otherwise identical conditions. Our results demonstrate that the lipid diffusion, as well as the mechanical properties of the resulting PSMs, is almost unaffected by the GUV formation procedure but depends on the chosen substrate functionalization. With the new method in hand, we were able to reconstitute the syntaxin-1A transmembrane domain in microfluidic GUVs and PSMs, which was visualized by fluorescence microscopy.

The Interaction of Gb3 Glycosphingolipids with ld and lo Phase Lipids in Lipid Monolayers Is a Function of Their Fatty Acids.

The glycosphingolipid Gb3 is a specific receptor of the bacterial Shiga toxin (STx). Binding of STx to Gb3 is a prerequisite for its internalization into the host cells, and the ceramide's fatty acid of Gb3 has been shown to influence STx binding. In in vitro studies on liquid ordered (lo)/liquid disordered (ld) coexisting artificial membranes, Shiga toxin B (STxB) binds solely to lo domains, thus harboring Gb3 concomitant with an observed lipid redistribution process. These findings raise the question of how the molecular structure of the fatty acid of Gb3 influences the interaction of Gb3 with the different lipids preferentially either found in the lo phase, namely, sphingomyelin and cholesterol, or in the ld phase. We addressed this question by using a series of synthetically available and unlabeled Gb3 glycosphingolipids carrying different long chain C24 fatty acids (saturated, monounsaturated, and α-hydroxylated). In conjunction with surface tension experiments on Langmuir monolayers, we quantified the excess of free energy of mixing of the different Gb3 species in monolayers composed of either sphingomyelin or cholesterol or composed of a fluid phase lipid (DOPC). From a calculation of the total free energy of mixing, we conclude that mixing of the saturated Gb3 species with the ld lipid DOPC is energetically less favorable than all other combinations, while the unsaturated species mix equally well with the lo phase lipids sphingomyelin and cholesterol and the ld phase lipid DOPC. Furthermore, we found that STxB partially penetrates in mixed lipid monolayers (DOPC/sphingomyelin/cholesterol) containing the Gb3 sphingolipid with a saturated or a monounsaturated C24 fatty acid. The maximum insertion pressure, as a measure for protein insertion, is >30 mN/m for both Gb3 molecules and is not significantly different for the two Gb3 species.

The role of the transmembrane domain of silicanin-1: Reconstitution of the full-length protein in artificial membranes.

Biosilica formation in diatoms is a membrane-confined process that occurs in so-called silica deposition vesicles (SDVs). As SDVs have as yet not been successfully isolated, the impact of the SDV membrane on silica morphogenesis is not well understood. However, recently the first SDV transmembrane protein, silicanin-1 (Sin1) has been identified that appears to be involved in biosilica formation. In this study, we recombinantly expressed and isolated full-length Sin1 from E. coli and investigated its reconstitution behavior in artificial membranes. A reconstitution efficiency in vesicles of up to 80% was achieved by a co-micellization method. By using a chymotrypsin digest, the orientation of Sin1 in unilamellar vesicles was analyzed indicating a positioning of the large N-terminal domain to the outside of the vesicles. These proteoliposomes were capable of precipitating silica in the presence of long-chain polyamines. Supported lipid bilayers were produced by proteoliposome spreading on lipid monolayers to form continuous lipid bilayers with Sin1 confined to the membrane. Successful Sin1 reconstitution into these planar membranes was shown by means of immunostaining with purified primary anti-Sin1 and secondary fluorescent antibodies. The established planar model membrane system, amenable for surface sensitive and microscopy techniques, will pave the way to investigate SDV-membrane interactions with other SDV associated biomolecules and its role in silica biogenesis.

TAT-RHIM: a more complex issue than expected.

Murine cytomegalovirus protein M45 contains a RIP homotypic interaction motif (RHIM) that is sufficient to confer protection of infected cells against necroptotic cell death. Mechanistically, the N-terminal region of M45 drives rapid self-assembly into homo-oligomeric amyloid fibrils, and interacts with the endogenous RHIM domains of receptor-interacting serine/threonine protein kinases (RIPK) 1, RIPK3, Z-DNA-binding protein 1, and Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-ß. Remarkably, all four aforementioned mammalian proteins harbouring such a RHIM domain are key components of inflammatory signalling and regulated cell death (RCD) processes. Immunogenic cell death by regulated necrosis causes extensive tissue damage in a wide range of diseases, including ischaemia reperfusion injury, myocardial infarction, sepsis, stroke, and solid organ transplantation. To harness the cell death suppression properties of M45 protein in a therapeutically usable manner, we developed a synthetic peptide encompassing only the RHIM domain of M45. To trigger delivery of RHIM into target cells, we fused the transactivator protein transduction domain of human immunodeficiency virus 1 to the N-terminus of the peptide. The fused peptide could efficiently penetrate eukaryotic cells, but unexpectedly it eradicated or destroyed all tested cancer cell lines and primary cells irrespective of species without further stimulus through a necrosis-like cell death. Typical inhibitors of different forms of RCD cannot impede this process, which appears to involve a direct disruption of biomembranes. Nevertheless, our finding has potential clinical relevance; reliable induction of a necrotic form of cell death distinct from all known forms of RCD may offer a novel therapeutic approach to combat resistant tumour cells.

Forces, Kinetics, and Fusion Efficiency Altered by the Full-Length Synaptotagmin-1 -PI(4,5)P2 Interaction in Constrained Geometries.

A mechanism for full-length synaptotagmin-1 (syt-1) to interact with anionic bilayers and to promote fusion in the presence of SNAREs is proposed. Colloidal probe force spectroscopy in conjunction with tethered particle motion monitoring showed that in the absence of Ca2+ the binding of syt-1 to membranes depends on the presence and content of PI(4,5)P2. Addition of Ca2+ switches the interaction forces from weak to strong, eventually exceeding the cohesion of the C2A domain of syt-1 leading to partial unfolding of the protein. Fusion of single unilamellar vesicles equipped with syt-1 and synaptobrevin 2 with planar pore-spanning target membranes containing PS and PI(4,5)P2 shows an almost complete suppression of stalled intermediate fusion states and an accelerated fusion kinetics in the presence of Ca2+, which is further enhanced upon addition of ATP.

Pore-Spanning Plasma Membranes Derived from Giant Plasma Membrane Vesicles.

Giant plasma membrane vesicles (GPMVs) are a highly promising model system for the eukaryotic plasma membrane. The unresolved challenge, however, is a path to surface-based structures that allows accessibility to both sides of the plasma membrane through high-resolution techniques. Such an approach would pave the way to advanced chip-based technologies for the analysis of complex cell surfaces to study the roles of membrane proteins, host-pathogen interactions, and many other bioanalytical and sensing applications. This study reports the generation of planar supported plasma membranes and for the first-time pore-spanning plasma membranes (PSPMs) derived from pure GPMVs that are spread on activated solid and highly ordered porous silicon substrates. GPMVs were produced by two different vesiculation agents and were first investigated with respect to their growth behavior and phase separation. Second, these GPMVs were spread onto silicon substrates to form planar supported plasma membrane patches. PSPMs were obtained by spreading of pure GPMVs on oxygen-plasma activated porous substrates with pore diameters of 3.5 µm. Fluorescence micrographs unambiguously showed that the PSPMs partially phase separate in a mobile ordered phase surrounded by a disordered phase, which was supported by cholesterol extraction using methyl-ß-cyclodextrin.

Insights into the molecular mechanism of amyloid filament formation: Segmental folding of α-synuclein on lipid membranes.

Recent advances in the structural biology of disease-relevant α-synuclein fibrils have revealed a variety of structures, yet little is known about the process of fibril aggregate formation. Characterization of intermediate species that form during aggregation is crucial; however, this has proven very challenging because of their transient nature, heterogeneity, and low population. Here, we investigate the aggregation of α-synuclein bound to negatively charged phospholipid small unilamellar vesicles. Through a combination of kinetic and structural studies, we identify key time points in the aggregation process that enable targeted isolation of prefibrillar and early fibrillar intermediates. By using solid-state nuclear magnetic resonance, we show the gradual buildup of structural features in an α-synuclein fibril filament, revealing a segmental folding process. We identify distinct membrane-binding domains in α-synuclein aggregates, and the combined data are used to present a comprehensive mechanism of the folding of α-synuclein on lipid membranes.

Membrane fusion mediated by peptidic SNARE protein analogues: Evaluation of FRET-based bulk leaflet mixing assays.

Peptide-mediated membrane fusion is frequently studied with in vitro bulk leaflet mixing assays based on Förster resonance energy transfer (FRET). In these, customized liposomes with fusogenic peptides are equipped with lipids which are labeled with fluorophores that form a FRET pair. Since FRET is dependent on distance and membrane fusion comes along with lipid mixing, the assays allow for conclusions on the membrane fusion process. The experimental outcome of these assays, however, greatly depends on the applied parameters. In the present study, the influence of the peptides, the size of liposomes, their lipid composition and the liposome stoichiometry on the fusogenicity of liposomes are evaluated. As fusogenic peptides, soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) protein analogues featuring artificial recognition units attached to the native SNARE transmembrane domains are used. The work shows that it is important to control these parameters in order to be able to properly investigate the fusion process and to prevent undesired effects of aggregation.

Structure, gating and interactions of the voltage-dependent anion channel.

The voltage-dependent anion channel (VDAC) is one of the most highly abundant proteins found in the outer mitochondrial membrane, and was one of the earliest discovered. Here we review progress in understanding VDAC function with a focus on its structure, discussing various models proposed for voltage gating as well as potential drug targets to modulate the channel's function. In addition, we explore the sensitivity of VDAC structure to variations in the membrane environment, comparing DMPC-only, DMPC with cholesterol, and near-native lipid compositions, and use magic-angle spinning NMR spectroscopy to locate cholesterol on the outside of the ß-barrel. We find that the VDAC protein structure remains unchanged in different membrane compositions, including conditions with cholesterol.