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2024 – Triplet–Triplet Annihilation Upconverting Liposomes: Mechanistic Insights into the Role of Membranes in Two-Dimensional TTA-UC
Vesicle Prep Pro Publication in Appl. Mater. Interfaces (2024) Authors: Prabhakaran A., Jha K.K., Sia R.C., Arellano Reyes R.A., Sarangi N.K, Kogut M., Guthmuller J., Czub J., Dietzek-Ivanšić B., Keyes T.E.

Triplet–triplet annihilation upconversion (TTA-UC) implemented in nanoparticle assemblies is of emerging interest in biomedical applications, including in drug delivery and imaging. As it is a bimolecular process, ensuring sufficient mobility of the sensitizer and annihilator to facilitate effective collision in the nanoparticle is key. Liposomes can provide the benefits of two-dimensional confinement and condensed concentration of the sensitizer and annihilator along with superior fluidity compared to other nanoparticle assemblies. They are also biocompatible and widely applied across drug delivery modalities. However, there are relatively few liposomal TTA-UC systems reported to date, so systematic studies of the influence of the liposomal environment on TTA-UC are currently lacking. Here, we report the first example of a BODIPY-based sensitizer TTA-UC system within liposomes and use this system to study TTA-UC generation and compare the relative intensity of the anti-Stokes signal for this system as a function of liposome composition and membrane fluidity. We report for the first time on time-resolved spectroscopic studies of TTA-UC in membranes. Nanosecond transient absorption data reveal the BODIPY-perylene dyad sensitizer has a long triplet lifetime in liposome with contributions from three triplet excited states, whose lifetimes are reduced upon coinclusion of the annihilator due to triplet–triplet energy transfer, to a greater extent than in solution. This indicates triplet energy transfer between the sensitizer and the annihilator is enhanced in the membrane system. Molecular dynamics simulations of the sensitizer and annihilator TTA collision complex are modeled in the membrane and confirm the co-orientation of the pair within the membrane structure and that the persistence time of the bound complex exceeds the TTA kinetics. Modeling also reliably predicted the diffusion coefficient for the sensitizer which matches closely with the experimental values from fluorescence correlation spectroscopy. The relative intensity of the TTA-UC output across nine liposomal systems of different lipid compositions was explored to examine the influence of membrane viscosity on upconversion (UC). UC showed the highest relative intensity for the most fluidic membranes and the weakest intensity for highly viscous membrane compositions, including a phase separation membrane. Overall, our study reveals that the co-orientation of the UC pair within the membrane is crucial for effective TTA-UC within a biomembrane and that the intensity of the TTA-UC output can be tuned in liposomal nanoparticles by modifying the phase and fluidity of the liposome. These new insights will aid in the design of liposomal TTA-UC systems for biomedical applications.

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2024 – The interaction of plant flavones with amphotericin B: Consequences for its pore-forming ability
Vesicle Prep Pro Publication in Biomedicine & Pharmacotherapy (2024) Authors: Malykhina A., Efimova S, Andriianov V., Ostroumova O.

The growth of antibiotic resistance to antifungal drugs contributes to the search for new ways to enhance their effectiveness and reduce toxicity. The undeniable advantage of polyene macrolide antibiotic amphotericin B (AmB) which ensures low pathogen resistance is its mechanism of action related to the formation of transmembrane pores in target lipid membranes. Here, we investigated the effects of plant flavones, chrysin, wogonin, baicalein, apigenin, scutellarein, luteolin, morin and fisetin on the pore-forming activity of AmB in the sterol-enriched membranes by electrophysiological assays. Сhrysin, wogonin, baicalein, apigenin, scutellarein, and luteolin were shown to decrease the AmB pore-forming activity in the bilayers composed of palmitoyloleylphosphocholine independently of their sterol composition. Morin and fisetin led to the increase and decrease in the AmB pore-forming activity in the ergosterol- and cholesterol-containing bilayers respectively. Differential scanning microcalorimetry of the gel-to-liquid crystalline phase transition of membrane forming lipids, molecular dynamics simulations, and absorbance spectroscopy revealed the possibility of direct interactions between AmB and some flavones in the water and/or in the lipid bilayer. The influence of these interactions on the antibiotic partitioning between aqueous solution and membrane and/or its transition between different states in the bilayer was discussed.

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2024 – Interaction of selected alkoxy naringenin oximes with model and bacterial membranes
Vesicle Prep Pro Publication in Biomedicine & Pharmacotherapy (2024) Authors: Wesołowska O., Duda-Madej A., Błaszczyk M., Środa-Pomianek K., Kozłowska J., Anioł M.

Naringenin is a flavonoid found in many fruits and herbs, most notably in grapefruits. In recent years, this compound and its derivatives have been of great interest due to their high biological activity, including fungicidal and bactericidal effects, also in relation to multidrug-resistant bacteria. Membrane interactions of naringenin oxime (NO) and its 7-O-alkyl (7-alkoxy) derivatives, such as methyl (7MENO), ethyl (7ETNO), isopropyl (7IPNO), n-butyl (7BUNO) and n-pentyl (7PENO) were studied. Thermotropic properties of model membranes were investigated via differential scanning calorimetry (DSC), the influence on lipid raft mimicking giant unilamellar vesicles (GUVs) via fluorescence microscopy, and membrane permeability via measuring calcein leakage from liposomes. Molecular calculations supplemented the study. The influence of naringenin oximes on two strains of multidrug resistant bacteria: Staphylococcus aureus KJ and Enterococcus faecalis 37VRE was also investigated. In DSC studies all compounds reduced the temperature and enthalpy of main phase transition and caused disappearing of the pretransition. NO was the least active. The reduction in the area of surface domains in GUVs was observed for NO. Compounds NO and 7BUNO resulted in very low secretion of calcein from liposomes (permeability < 3 %). The highest results were observed for 7MENO (88.4 %) and 7IPNO (78.5 %). When bacterial membrane permeability was investigated all compounds caused significant release of propidium iodide from S. aureus (31.6–87.0 % for concentration 128 μg/mL). In the case of E. faecalis, 7ETNO (75.7 %) and NO (28.8 %) were the most active. The rest of the tested compounds showed less activity (permeability < 13.9 %). The strong evidence was observed that antibacterial activity of the tested compounds may be associated with their interaction with bacterial membrane.

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2024 – Methoxychalcones as potential anticancer agents for colon cancer: Is membrane perturbing potency relevant?
Vesicle Prep Pro Publication in Biochimica et Biophysica Acta (BBA) - General Subjects (2024) Authors: Palko-Łabuz A., Wesołowska O., Błaszczyk M., Uryga A., Sobieszczańska B., Skonieczna M., Kostrzewa-Susłow E., Janeczko T., Środa-Pomianek K.

Chalcones are naturally produced by many plants, and constitute precursors for the synthesis of flavons and flavanons. They were shown to possess antibacterial, antifungal, anti-cancer, and anti- inflammatory properties. The goal of the study was to assess the suitability of three synthetic methoxychalcones as potential anticancer agents. In a panel of colon cancer cell lines they were demonstrated to be cytotoxic, proapoptotic, causing cell cycle arrest, and increasing intracellular level of reactive oxygen species. Anticancer activity of the compounds was not diminished in the presence of stool extract containing microbial enzymes that could change the structure of chalcones. Moreover, methoxychalcones interacted strongly with model phosphatidylcholine membranes as detected by differential scanning calorimetry. Metohoxychalcones particularly affected the properties of lipid domains in giant unilamellar liposomes formed from raft-mimicking lipid composition. This may be of importance since many molecular targets for therapy of metastatic colon cancer are raft-associated receptors (e.g., receptor tyrosine kinases). The importance of membrane perturbing potency of methoxychalcones for their biological activity was additionally corroborated by the results obtained by molecular modelling.

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2024 – A dynamically gated triangular DNA nanopore for molecular sensing and cross-membrane transport
Vesicle Prep Pro and Orbit mini Preprint Publication in Research Square (2024) Authors: Liu X., Liu F., Chhabra H., Maffeo C., Huang Q., Aksimentiev A., Arai T.

Synthetic membrane nanopores made of DNA are promising systems to sense and control molecular transport in biosensing, sequencing, and synthetic cells. Dynamically gating cargo transport like the natural ion channels and systematically increasing the lumen size have become long-standing desires in developing nanopores. Here, we design a triangular DNA nanopore with a large dynamically-gated lumen. It can switch between expanded and contracted states without changing its stable triangular shape, whereby specific DNA bindings as stimuli mechanically pinch and release the three corners of the triangular frame. Transmission electron microscopy images and molecular dynamics simulations illustrated the large lumen up to 539 nm2, the stable architectures, and the high shape retention. Single channel current recordings and fluorescence influx studies demonstrated the low-noise repeatable readouts and the controllable cross-membrane macromolecular transport. We envision that the proposed DNA nanopores could offer powerful tools in molecular sensing, drug delivery, and the creation of synthetic cells.

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2024 – Solid-phase synthesis as a tool to create exactly defined, branched polymer vectors for cell membrane targeting
Vesicle Prep Pro Publication in Macromolecules (2024) Authors: Elter J., Liščáková V., Moravec O., Vragović M., Filipová M., Štěpánek P., Šácha P., Hrubý M.

Modern drug formulations often require, besides the active drug molecule, auxiliaries to enhance their pharmacological properties. Tailor-made, biocompatible polymers covalently connected to the drug molecule can fulfill this function by increasing its solubility, reducing its toxicity, and guiding it to a specific target. If targeting membrane-bound proteins, localization of the drug close to the cell membrane and its target is beneficial to increase drug efficiency and residence time. In this study, we present the synthesis of highly defined, branched polymeric structures with membrane-binding properties. One to three hydrophilic poly(ethylene oxide) or poly(2-ethyloxazoline) side chains were connected via a peptoid backbone using a two-step iterative protocol for solid-phase peptoid synthesis. Additional groups, e.g., a hydrophobic anchor for membrane attachment, were introduced. Due to the nature of solid-phase synthesis, the number and order of the side chains and additional units can be precisely defined. The method proved to be versatile for the generation of multifunctional, branched polymeric structures of molecular weights up to approximately 7000 g mol–1. The behavior of all compounds towards biological membranes and cells was investigated using liposomes as cell membrane models, HEK293 and U251-MG cell lines, and red blood cells, thereby demonstrating their potential value as drug auxiliaries with cell membrane affinity.

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2024 – Dynamic conformational changes of a tardigrade group-3 late embryogenesis abundant protein modulate membrane biophysical properties
Vesicle Prep Pro Publication in PNAS Nexus (2024) Authors: Li X., Yu C., Gomez-Navarro N., Stancheva V., Zhu H., Murthy A., Wozny M., Malhotra K., Johnson C., Blackledge M., Santhanam B., Liu W., Huang J., Freund S., Miller E., Babu M.

A number of Intrinsically disordered proteins (IDPs) encoded in stress-tolerant organisms such as tardigrade, can confer fitness advantage and abiotic stress tolerance when heterologously expressed. Tardigrade-specific disordered proteins including the cytosolic abundant heat soluble (CAHS) proteins are proposed to confer stress tolerance through vitrification or gelation, whereas evolutionarily conserved IDPs in tardigrades may contribute to stress tolerance through other biophysical mechanisms. Here we characterized the mechanism of action of an evolutionarily conserved, tardigrade IDP, HeLEA1, which belongs to the group-3 late embryogenesis abundant (LEA) protein family. HeLEA1 homologs are found across different kingdoms of life. HeLEA1 is intrinsically disordered in solution but shows a propensity for helical structure across its entire sequence. HeLEA1 interacts with negatively charged membranes via dynamic disorder-to-helical transition, mainly driven by electrostatic interactions. Membrane interaction of HeLEA1 is shown to ameliorate excess surface tension and lipid packing defects. HeLEA1 localizes to the mitochondrial matrix when expressed in yeast and interacts with model membranes mimicking inner mitochondrial membrane. Yeast expressing HeLEA1 show enhanced tolerance to hyperosmotic stress under non-fermentative growth and increased mitochondrial membrane potential. Evolutionary analysis suggests that although HeLEA1 homologs have diverged their sequences to localize to different subcellular organelles, all homologs maintain a weak hydrophobic moment that is characteristic of weak and reversible membrane interaction. We suggest that such a dynamic and weak protein-membrane interaction buffering alterations in lipid packing could be a conserved strategy for regulating membrane properties and represents a general biophysical solution for stress tolerance across the domains of life.

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2024 – Cryo-electron tomography reveals how COPII assembles on cargo-containing membranes
Vesicle Prep Pro Pre-Print Publication in BioRxiv (2024) Authors: Pyle E., Zanetti G.

Proteins traverse the eukaryotic secretory pathway via membrane trafficking between organelles. The COPII coat mediates the anterograde transport of newly synthesised proteins from the endoplasmic reticulum, engaging cargoes with wide ranges of sizes and biophysical properties. The native architecture of the COPII coat and the cargo-dependent regulation of its assembly remain poorly understood. Here, we have reconstituted COPII-coated membrane carriers using purified S. cerevisiae proteins and cell-derived microsomes as a native membrane source. Using cryo-electron tomography with subtomogram averaging, we demonstrate that the COPII coat binds cargo and forms largely spherical vesicles from native membranes. We reveal the architecture of the inner and outer coat layers and shed light on how spherical carriers are formed. Our results provide novel insights into the architecture and regulation of the COPII coat and challenge our current understanding of how membrane curvature is generated.

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2023 – Electric Potential at the Interface of Membraneless Organelles Gauged by Graphene
Vesicle Prep Pro Publication in Nano Lett. (2023) Authors: Hoffmann C., Murastov G., Tromm J., Moog J., Aslam M., Matkovic A., Milovanovic D.

Eukaryotic cells contain membrane-bound and membrane-less organelles that are often in contact with each other. How the interface properties of membrane-less organelles regulate their interactions with membranes remains challenging to assess. Here, we employ graphene-based sensors to investigate the electrostatic properties of synapsin 1, a major synaptic phosphoprotein, either in a single phase or after undergoing phase separation to form synapsin condensates. Using these graphene-based sensors, we discover that synapsin condensates generate strong electrical responses that are otherwise absent when synapsin is present as a single phase. By introducing atomically thin dielectric barriers, we show that the electrical response originates in an electric double layer whose formation governs the interaction between synapsin condensates and graphene. Our data indicate that the interface properties of the same protein are substantially different when the protein is in a single phase versus within a biomolecular condensate, unraveling that condensates can harbor ion potential differences at their interface.

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2023 – A BODIPY-Based Molecular Rotor in Giant Unilamellar Vesicles: A Case Study by Polarization-Resolved TimeResolved Emission and Transient Absorption Spectroscopy
Vesicle Prep Pro Publication in ChemPhotoChem (2023) Authors: Jha K., Prabhakaran A., Spantzel L., Sia R., Pérez I., Arellano-Reyes R., Elmanova A., Dasgupta A., Eggeling C., Börsch M., Guthmuller J., Presselt M., Keyes T., Dietzek-Ivanšić B.

BODIPY and BODIPY-derived systems are widely applied as fluorophores and as probes for viscosity detection in solvents and biological media. Their orientational and rotational dynamics in biological media are thus of vital mechanistic importance and extensively investigated. In this contribution, polarization-resolved confocal microscopy is used to determine the orientation of an amphiphilic BODIPY-cholesterol derivative in homogeneous giant unilamellar vesicles (GUV) made from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The BODIPY-moiety of the molecule is placed near the polar headgroups, and the cholesterol moiety is embedded in the membrane along the acyl chain of the lipids. The rotational relaxation of fluorophore is conventionally investigated by time-resolved emission anisotropy (TEA); and this method is also used here. However, TEA depends on the emission of the fluorophore and may not be useful to probe rotational dynamics of the non-emissive triplet states. Thus, we employ femtosecond transient absorption anisotropy (TAA), that relies on the absorption of the molecule to complement the studies of the amphiphilic BODIPY in DCM and GUV. The photoinduced anisotropy of the BODIPY molecule in DCM decays tri-exponentially, the decay components (sub-5 ps, 43 ps and 440 ps) of anisotropy are associated with the non-spherical shape of the BODIPY molecule. However, the anisotropy decay in homogenous GUVs follows a biexponential decay; which arises from the wobbling-in-a-cone motion of the non-spherical molecule in the high viscous lipid bilayer media. The observations for the BODIPY-chol molecule in the GUV environment by TAA will extend to the investigation of non-emissive molecules in cellular environment since GUV structure and size resembles the membrane of a biological cell.

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2023 – Mechanistic Insight into the Early Stages of Toroidal Pore Formation by the Antimicrobial Peptide Smp24
Port-a-Patch, Vesicle Prep Pro Publication in Pharmaceutics (2023) Authors: Bertelsen M., Lacey M., Nichol T., Miller, K.

The antimicrobial peptide Smp24, originally derived from the venom of Scorpio maurus palmatus, is a promising candidate for further drug development. However, before doing so, greater insight into the mechanism of action is needed to construct a reliable structure–activity relationship. The aim of this study was to specifically investigate the critical early stages of peptide-induced membrane disruption. Single-channel current traces were obtained via planar patch-clamp electrophysiology, with multiple types of pore-forming events observed, unlike those expected from the traditional, more rigid mechanistic models. To better understand the molecular-level structures of the peptide-pore assemblies underlying these observed conductance events, molecular dynamics simulations were used to investigate the peptide structure and orientation both before and during pore formation. The transition of the peptides to transmembrane-like states within disordered toroidal pores occurred due to a peptide-induced bilayer-leaflet asymmetry, explaining why pore stabilization does not always follow pore nucleation in the experimental observations. To fully grasp the structure–activity relationship of antimicrobial peptides, a more nuanced view of the complex and dynamic mechanistic behaviour must be adopted

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2023 – Membrane Tension Inhibits Lipid Mixing by Increasing the Hemifusion Stalk Energy
Vesicle Prep Pro Publication in ACS Nano (2023) Authors: Shendrick P., Golani G., Dharan R., Schwarz U., Sorkin R.

Fusion of biological membranes is fundamental in various physiological events. The fusion process involves several intermediate stages with energy barriers that are tightly dependent on the mechanical and physical properties of the system, one of which is membrane tension. As previously established, the late stages of fusion, including hemifusion diaphragm and pore expansions, are favored by membrane tension. However, a current understanding of how the energy barrier of earlier fusion stages is affected by membrane tension is lacking. Here, we apply a newly developed experimental approach combining micropipette-aspirated giant unilamellar vesicles and optically trapped membrane-coated beads, revealing that membrane tension inhibits lipid mixing. We show that lipid mixing is 6 times slower under a tension of 0.12 mN/m compared with tension-free membranes. Furthermore, using continuum elastic theory, we calculate the dependence of the hemifusion stalk formation energy on membrane tension and intermembrane distance and find the increase in the corresponding energy barrier to be 1.6 kBT in our setting, which can explain the increase in lipid mixing time delay. Finally, we show that tension can be a significant factor in the stalk energy if the pre-fusion intermembrane distance is on the order of several nanometers, while for membranes that are tightly docked, tension has a negligible effect.

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2023 – Synergy between Winter Flounder antimicrobial peptides
Vesicle Prep Pro, Port-a-Patch Publication in Nature npj Antimicrobials and Resistance (2023) Authors: Clarke M., Hind C., Ferguson P., Manzo G., Mistry B., Yue B., Romanopulos J., Clifford M., Bui T., Drake A., Lorenz C., Sutton M., Mason J.

Some antimicrobial peptides (AMPs) have potent bactericidal activity and are being considered as potential alternatives to classical antibiotics. In response to an infection, such AMPs are often produced in animals alongside other peptides with low or no perceivable antimicrobial activity, whose role is unclear. Here we show that six AMPs from the Winter Flounder (WF) act in synergy against a range of bacterial pathogens and provide mechanistic insights into how this increases the cooperativity of the dose-dependent bactericidal activity and potency that enable therapy. Only two WF AMPs have potent antimicrobial activity when used alone but we find a series of two-way combinations, involving peptides which otherwise have low or no activity, yield potent antimicrobial activity. Weakly active WF AMPs modulate the membrane interactions of the more potent WF AMPs and enable therapy in a model of Acinetobacter baumannii burn wound infection. The observed synergy and emergent behaviour may explain the evolutionary benefits of producing a family of related peptides and are attractive properties to consider when developing AMPs towards clinical applications.

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2023 – CRAFTing Delivery of Membrane Proteins into Protocells using Nanodiscs
Vesicle Prep Pro, Port-a-Patch Publication in ACS Appl. Mater. Interfaces (2023) Authors: Stępień P., Świątek S., Robles M.Y.Y., Markiewicz-Mizera J., Balakrishnan D., Inaba-Inoue S., De Vries A.H., Beis K., Marrink S.J., HeddleJ.G.

For the successful generative engineering of functional artificial cells, a convenient and controllable means of delivering membrane proteins into membrane lipid bilayers is necessary. Here we report a delivery system that achieves this by employing membrane protein-carrying nanodiscs and the calcium-dependent fusion of phosphatidylserine lipid membranes. We show that lipid nanodiscs can fuse a transported lipid bilayer with the lipid bilayers of small unilamellar vesicles (SUVs) or giant unilamellar vesicles (GUVs) while avoiding recipient vesicles aggregation. This is triggered by a simple, transient increase in calcium concentration, which results in efficient and rapid fusion in a one-pot reaction. Furthermore, nanodiscs can be loaded with membrane proteins that can be delivered into target SUV or GUV membranes in a detergent-independent fashion while retaining their functionality. Nanodiscs have a proven ability to carry a wide range of membrane proteins, control their oligomeric state, and are highly adaptable. Given this, our approach may be the basis for the development of useful tools that will allow bespoke delivery of membrane proteins to protocells, equipping them with the cell-like ability to exchange material across outer/subcellular membranes.

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2023 – A new look at Hsp70 activity in phosphatidylserine-enriched membranes: chaperone-induced quasi-interdigitated lipid phase.
Vesicle Prep Pro Publication in Scientific Reports (2023) Authors: Tagaeva R., Efmova S., Ischenko A., Zhakhov A., Shevtsov M., Ostroumova O.

70 kDa heat shock protein Hsp70 (also termed HSP70A1A) is the major stress-inducible member of the HSP70 chaperone family, which is present on the plasma membranes of various tumor cells, but not on the membranes of the corresponding normal cells. The exact mechanisms of Hsp70 anchoring in the membrane and its membrane-related functions are still under debate, since the protein does not contain consensus signal sequence responsible for translocation from the cytosol to the lipid bilayer. The present study was focused on the analysis of the interaction of recombinant human Hsp70 with the model phospholipid membranes. We have confirmed that Hsp70 has strong specificity toward membranes composed of negatively charged phosphatidylserine (PS), compared to neutral phosphatidylcholine membranes. Using differential scanning calorimetry, we have shown for the first time that Hsp70 affects the thermotropic behavior of saturated PS and leads to the interdigitation that controls membrane thickness and rigidity. Hsp70-PS interaction depended on the lipid phase state; the protein stabilized ordered domains enriched with high-melting PS, increasing their area, probably due to formation of quasi-interdigitated phase. Moreover, the ability of Hsp70 to form ion-permeable pores in PS membranes may also be determined by the bilayer thickness. These observations contribute to a better understanding of Hsp70-PS interaction and biological functions of membrane-bound Hsp70 in cancer cells.

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2023 – Formation of Giant Unilamellar Vesicles Assisted byFluorinated Nanoparticles
Vesicle Prep Pro Publication in Advanced Science (2023) Authors: Waeterschoot J., Gosselé W., Zeinabad H.A., Lammertyn J., Koos E., Casadevall i Solvas X.

In the quest to produce artificial cells, one key challenge that remains to besolved is the recreation of a complex cellular membrane. Among the existingmodels, giant unilamellar vesicles (GUVs) are particularly interesting due totheir intrinsic compartmentalisation ability and their resemblance in size andshape to eukaryotic cells. Many techniques have been developed to produceGUVs all having inherent advantages and disadvantages. Here, the authorsshow that fluorinated silica nanoparticles (FNPs) used to form Pickeringemulsions in a fluorinated oil can destabilise lipid nanosystems to templatethe formation of GUVs. This technique enables GUV production across abroad spectrum of buffer conditions, while preventing the leakage of theencapsulated components into the oil phase. Furthermore, a simplecentrifugation process is sufficient for the release of the emulsion-trappedGUVs, bypassing the need to use emulsion-destabilising chemicals. Withfluorescent FNPs and transmission electron microscopy, the authors confirmthat FNPs are efficiently removed, producing contaminant-free GUVs. Furtherexperiments assessing the lateral diffusion of lipids and unilamellarity of theGUVs demonstrate that they are comparable to GUVs produced viaelectroformation. Finally, the ability of incorporating transmembrane proteinsis demonstrated, highlighting the potential of this method for the productionof GUVs for artificial cell applications.

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2023 – Photophysics of Conjugated Oligoelectrolytes Relevant to Two-Photon Fluorescence-Lifetime Imaging Microscopy
Vesicle Prep Pro Publication in Advanced Functional Materials (2023) Authors: Zhu J-Y., Mikhailovsky A., Wei S.C.J., Moreland A., Limwongyut J., Guarrotxena N., Bazan G.C.

Conjugated oligoelectrolytes (COEs) comprise a class of cell-membrane intercalating molecules that serve as effective optical reporters. However, little is known about the photophysical properties of COEs in biological environments such as buffers, cell membranes, and intracellular organelles, which is critical to optimize performance. Herein, how COE self-assembly depends on the dielectric environment (polarity and ion content) is explored based on the representative molecule 6-ring phenylenevinylene (PV) conjugated oligoelectrolyte (COE-S6), and its optical properties within mammalian cells are subsequently studied. Two-photon fluorescence lifetime imaging microscopy (FLIM), confocal laser scanning microscopy, and optical properties in solutions are brought together to obtain information about the location, accumulation, and characteristics of the local surroundings. FLIM imaging lifetime phasor plots, decays, and fluorescence spectra on stained mammalian cells provide evidence of successful COE-S6 internalization via endocytosis. The fluorescence lifetime of COE-S6 is identical when in A549 mammalian cells and in giant unilamellar vesicle model membranes, thereby providing a correlation between living system and artificial constructs.

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2023 – Triggering the Amphotericin B Pore-Forming Activity by Phytochemicals
VPP Publication in Membranes (2023) Authors: Efimova S., Malykhina A., Ostroumova O.

The macrolide polyene antibiotic amphotericin B (AmB), remains a valuable drug to treat systemic mycoses due to its wide antifungal activity and low probability of developing resistance. The high toxicity of AmB, expressed in nephropathy and hemolysis, could be partially resolved by lowering therapeutic AmB concentration while maintaining efficacy. This work discusses the possibility of using plant polyphenols and alkaloids to enhance the pore-forming and consequently antifungal activity of AmB. We demonstrated that phloretin, phlorizin, naringenin, taxifolin, quercetin, biochanin A, genistein, resveratrol, and quinine led to an increase in the integral AmB-induced transmembrane current in the bilayers composed of palmitoyloleoylphosphocholine and ergosterol, while catechin, colchicine, and dihydrocapsaicin did not practically change the AmB activity. Cardamonin, 4′-hydroxychalcone, licochalcone A, butein, curcumin, and piperine inhibited AmB-induced transmembrane current. Absorbance spectroscopy revealed no changes in AmB membrane concentration with phloretin addition. A possible explanation of the potentiation is related to the phytochemical-produced changes in the elastic membrane properties and the decrease in the energy of formation of the lipid mouth of AmB pores, which is partially confirmed by differential scanning microcalorimetry. The possibility of AmB interaction with cholesterol in the mammalian cell membranes instead of ergosterol in fungal membranes, determines its high toxicity. The replacement of ergosterol with cholesterol in the membrane lipid composition led to a complete loss or a significant decrease in the potentiating effects of tested phytochemicals, indicating low potential toxicity of these compounds and high therapeutic potential of their combinations with the antibiotic. The discovered combinations of AmB with plant molecules that enhance its pore-forming ability in ergosterol-enriched membranes, seem to be promising for further drug development in terms of the toxicity decrease and efficacy improvement.

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2023 – Metamorphic proteins at the basis of human autophagy initiation and lipid transfer
Vesicle Prep Pro Publication in Mol. Cell (2023) Authors: Nguyen A., Lugarini F., David C., Hosnani P., Alagöz Ç., Friedrich A., Schlütermann D., Knotkova B., Patel A., Parfentev I., Urlaub H., Meinecke M., Stork B., Faesen A.C.

Autophagy is a conserved intracellular degradation pathway that generates de novo double-membrane autophagosomes to target a wide range of material for lysosomal degradation. In multicellular organisms, autophagy initiation requires the timely assembly of a contact site between the ER and the nascent autophagosome. Here, we report the in vitro reconstitution of a full-length seven-subunit human autophagy initiation supercomplex built on a core complex of ATG13-101 and ATG9. Assembly of this core complex requires the rare ability of ATG13 and ATG101 to switch between distinct folds. The slow spontaneous metamorphic conversion is rate limiting for the self-assembly of the supercomplex. The interaction of the core complex with ATG2-WIPI4 enhances tethering of membrane vesicles and accelerates lipid transfer of ATG2 by both ATG9 and ATG13-101. Our work uncovers the molecular basis of the contact site and its assembly mechanisms imposed by the metamorphosis of ATG13-101 to regulate autophagosome biogenesis in space and time.

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2023 – DNA Origami Signaling Units Transduce Chemical and Mechanical Signals in Synthetic Cells
Vesicle Prep Pro Publication in Advanced Functional Materials (2023) Authors: Jahnke K., Illig M., Scheffold M., Tran M., Mersdorf U., Göpfrich K.

Transmembrane proteins transmit chemical signals as well as mechanical cues. The latter is often achieved by coupling to the cytoskeleton. The incorporation of fully engineerable membrane-spanning structures for the transduction of chemical and, in particular, mechanical signals is therefore a critical aim for bottom-up synthetic biology. Here, a membrane-spanning DNA origami signaling units (DOSUs) is designed and mechanically coupled to DNA cytoskeletons encapsulated within giant unilamellar vesicles (GUVs). The incorporation of the DOSUs into the GUV membranes is verified and clustering upon external stimulation is achieved. Dye-influx assays reveal that clustering increases the insertion efficiency. The transmembrane-spanning DOSUs act as pores to allow for the transport of single-stranded DNA into the GUVs. This is employed to trigger the reconfiguration of DNA cytoskeletons within GUVs. In addition to chemical signaling, mechanical coupling of the DOSUs to the internal DNA cytoskeletons is induced. With chemical cues from the environment, clustering of the DOSUs is induced, which triggers a symmetry break in the organization of the DNA cytoskeleton inside of the GUV. DNA-based transmembrane structures are engineered that transduce signals without transporting the signaling molecule itself—providing a route toward signal processing and adaptive synthetic cells.

Read more in the publication here.

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2023 – Molecular orientation and optimization of membrane dyes based on conjugated oligoelectrolytes
Vesicle Prep Pro Publication in Cell Reports Physical Science (2023) Authors: Zhu J., Bazan G.

Conjugated oligoelectrolytes (COEs) are amphiphilic, fluorogenic molecules that spontaneously associate with lipid bilayer membranes and are gaining attention as molecular reporters, particularly for exosome detection by flow cytometry. Questions nonetheless remain on how to best design COEs for optimal performance and on the geometry of lipid bilayer intercalation. In response, we designed a series of oligo-phenylenevinylene COEs with varying lengths and numbers of charged groups to address these uncertainties. Examination of the organization within lipid bilayers through polarized fluorescence microscopy shows that the optical transition moments are perpendicular to the bilayer plane, with the conjugated segment flanked by hydrophobic phospholipid tails. COEs initially form a disorganized layer on the vesicle periphery, reflecting electrostatic association before intercalation. Uptake experiments show that longer dimensions and increased numbers of charges allow for a higher degree of cellular association. Both shorter core length and increased number of charges accelerate the rate needed to achieve emission saturation.

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2023 – Calcium-induced compaction and clustering of vesicles tracked with molecular resolution
Vesicle Prep Pro Publication in Biophys. J (2023) Authors: Saldanha O., Schiller L., Hauser K.

Theory and simulations predict the complex nature of calcium interaction with the lipid membrane. By maintaining the calcium concentrations at physiological conditions, herein we demonstrate experimentally the effect of Ca2+ in a minimalistic cell-like model. For this purpose, giant unilamellar vesicles (GUVs) with a neutral lipid DOPC are generated, and the ion-lipid interaction is observed with attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy providing molecular resolution. Firstly, Ca2+ encapsulated within the vesicle binds to the phosphate head groups of the inner leaflets and triggers vesicle compaction. This is tracked by changes in vibrational modes of the lipid groups. As the calcium  concentration within the GUV increases, IR intensities change indicating vesicle dehydration and lateral compression of the membrane. Secondly, by inducing a calcium gradient across the membrane up to a ratio of 1:20, interaction between several vesicles occurs as Ca2+ can bind to the outer leaflets leading to vesicle clustering. It is observed that larger calcium gradients induce stronger interactions. These findings with an exemplary biomimetic model reveal that divalent calcium ions not only cause local changes to the lipid packing but also have macroscopic implications to initiate vesicle-vesicle interaction.

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2023 – Modulators of cellular cholesterol homeostasis as antiproliferative and model membranes perturbing agents
Vesicle Prep Pro Publication in BBA - Biomembranes (2023) Authors: Błaszczyk M., Kozioł A., Palko-Łabuz A., Środa-Pomianek K., Wesołowska O.

Cholesterol is an important component of mammalian cell membranes affecting their fluidity and permeability. Together with sphingomyelin, cholesterol forms microdomains, called lipid rafts. They play important role in signal transduction forming platforms for interaction of signal proteins. Altered levels of cholesterol are known to be strongly associated with the development of various pathologies (e.g., cancer, atherosclerosis and cardiovascular diseases). In the present work, the group of compounds that share the property of affecting cellular homeostasis of cholesterol was studied. It contained antipsychotic and antidepressant drugs, as well as the inhibitors of cholesterol biosynthesis, simvastatin, betulin, and its derivatives. All compounds were demonstrated to be cytotoxic to colon cancer cells but not to non-cancerous cells. Moreover, the most active compounds decreased the level of free cellular cholesterol. The interaction of drugs with raft-mimicking model membranes was visualized. All compounds reduced the size of lipid domains, however, only some affected their number and shape. Membrane interactions of betulin and its novel derivatives were characterized in detail. Molecular modeling indicated that high dipole moment and significant lipophilicity were characteristic for the most potent antiproliferative agents. The importance of membrane interactions of cholesterol homeostasis-affecting compounds, especially betulin derivatives, for their anticancer potency was suggested.

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2023 – Systematic comparison of unilamellar vesicles reveals that archaeal core lipid membranes are more permeable than bacterial membranes
Vesicle Prep Pro Publication in PLoS Biol (2023) Authors: Łapińska U., Glover G., Kahveci Z., Irwin N., Milner D., Tourte M., Albers S., Santoro A., Richards T., Pagliara S.

One of the deepest branches in the tree of life separates the Archaea from the Bacteria. These prokaryotic groups have distinct cellular systems including fundamentally different phospholipid membrane bilayers. This dichotomy has been termed the lipid divide and possibly bestows different biophysical and biochemical characteristics on each cell type. Classic experiments suggest that bacterial membranes (formed from lipids extracted from Escherichia coli, for example) show permeability to key metabolites comparable to archaeal membranes (formed from lipids extracted from Halobacterium salinarum), yet systematic analyses based on direct measurements of membrane permeability are absent. Here, we develop a new approach for assessing the membrane permeability of approximately 10 μm unilamellar vesicles, consisting of an aqueous medium enclosed by a single lipid bilayer. Comparing the permeability of 18 metabolites demonstrates that diether glycerol-1-phosphate lipids with methyl branches, often the most abundant membrane lipids of sampled archaea, are permeable to a wide range of compounds useful for core metabolic networks, including amino acids, sugars, and nucleobases. Permeability is significantly lower in diester glycerol-3-phosphate lipids without methyl branches, the common building block of bacterial membranes. To identify the membrane characteristics that determine permeability, we use this experimental platform to test a variety of lipid forms bearing a diversity of intermediate characteristics. We found that increased membrane permeability is dependent on both the methyl branches on the lipid tails and the ether bond between the tails and the head group, both of which are present on the archaeal phospholipids. These permeability differences must have had profound effects on the cell physiology and proteome evolution of early prokaryotic forms. To explore this further, we compare the abundance and distribution of transmembrane transporter-encoding protein families present on genomes sampled from across the prokaryotic tree of life. These data demonstrate that archaea tend to have a reduced repertoire of transporter gene families, consistent with increased membrane permeation. These results demonstrate that the lipid divide demarcates a clear difference in permeability function with implications for understanding some of the earliest transitions in cell origins and evolution.

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2023 – The Specific Effect of Grapefruit Seed, Sea-Buckthorn Leaves, and Chaga Extracts on the Properties of Model Lipid Membranes
Vesicle Prep Pro Publication in Cell Tiss. Biol. (2023). Authors: Efimova S, Zakharova A., Chernyshova D., Ostroumova O.

The effect of extracts of grapefruit seeds (EGSs), sea-buckthorn leaves (EBLs), and chaga (ECs) on model lipid membranes has been investigated. It has been shown that the threshold concentrations of EGSs and ECs that induced destabilization of phosphatidylglycerol-enriched bilayers are 1.3–1.4 times lower than for phosphatidylcholine-containing membranes. It has been established that EGSs and EBLs reduce the boundary potential of membranes formed from a mixture of phosphatadylcholine and cholesterol (the changes reach 45 and 40 mV at concentrations of 60 and 800 μg/mL, respectively). ECs did not produce pronounced potential-modifying effect. It was shown that changes in the boundary potential in the presence of EBLs were due to the presence of flavonols, quercetin, and myricetin in its composition. Using the method of differential scanning calorimetry, it was also found that quercetin and myricetin were able to influence the thermotropic behavior of membrane lipids and, consequently, their packing density. The potentiation of the pore-forming activity of the antifungal polyene macrolide nystatin and antibacterial lipopeptide polymyxin B was shown with introduction of an EBLs. These data indicate a possible synergism of the antimicrobial action of the tested antibiotics and EBLs, which can be used to generate combined broad-spectrum antimicrobial agents.

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2023 – Transmembrane signaling by a synthetic receptor in artificial cells
Vesicle Prep Pro Publication in Nat. Commun (2023) Authors: Bretschneider Søgaard A., Bøtker Pedersen A., Borup Løvschall K., Monge P., Hammer Jakobsen J., Džabbarova L., Friis Nielsen L., Stevanovic S., Walther R., Zelikin A.

Signal transduction across biological membranes is among the most important evolutionary achievements. Herein, for the design of artificial cells, we engineer fully synthetic receptors with the capacity of transmembrane signaling, using tools of chemistry. Our receptors exhibit similarity with their natural counterparts in having an exofacial ligand for signal capture, being membrane anchored, and featuring a releasable messenger molecule that performs enzyme activation as a downstream signaling event. The main difference from natural receptors is the mechanism of signal transduction, which is achieved using a self-immolative linker. The receptor scaffold is modular and can readily be re-designed to respond to diverse activation signals including biological or chemical stimuli. We demonstrate an artificial signaling cascade that achieves transmembrane enzyme activation, a hallmark of natural signaling receptors. Results of this work are relevant for engineering responsive artificial cells and interfacing them and/or biological counterparts in co-cultures.

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2023 – Amphipathic peptide–phospholipid nanofibers: Kinetics of fiber formation and molecular transfer between assemblies
Vesicle Prep Pro Publication in Biophysical Chemistry (2023) Authors: Shimizu C., Ikeda K., Nakao H., Nakano M.

Understanding the kinetics of nano-assembly formation is important to elucidate the biological processes involved and develop novel nanomaterials with biological functions. In the present study, we report the kinetic mechanisms of nanofiber formation from a mixture of phospholipids and the amphipathic peptide 18A[A11C], carrying cysteine substitution of the apolipoprotein A-I-derived peptide 18A at residue 11. 18A[A11C] with acetylated N-terminus and amidated C-terminus can associate with phosphatidylcholine to form fibrous aggregates at neutral pH and lipid-to-peptide molar ratio of ∼1, although the reaction pathways of self-assembly remain unclear. Here, the peptide was added to giant 1-palmitoyl-2-oleoyl phosphatidylcholine vesicles to monitor nanofiber formation under fluorescence microscopy. The peptide initially solubilized the lipid vesicles into particles smaller than the resolution of optical microscope, and fibrous aggregates appeared subsequently. Transmission electron microscopy and dynamic light scattering analyses revealed that the vesicle-solubilized particles were spherical or circular, measuring ∼10–20 nm in diameter. The rate of nanofiber formation of 18A with 1,2-dipalmitoyl phosphatidylcholine from the particles was proportional to the square of lipid–peptide concentration in the system, suggesting that the association of particles, accompanied by conformational changes, was the rate-limiting step. Moreover, molecules in the nanofibers could be transferred between aggregates faster than those in the lipid vesicles. These findings provide useful information for the development and control of nano-assembling structures using peptides and phospholipids.

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2023 – The Degree of Hydroxylation of Phenolic Rings Determines the Ability of Flavonoids and Stilbenes to Inhibit Calcium-Mediated Membrane Fusion
Vesicle Prep Pro Publication in Nutrients (2023) Authors: Zlodeeva P.D., Shekunov E.V., Ostroumova O.S., Efimova S.S.

This paper discusses the possibility of using plant polyphenols as viral fusion inhibitors with a lipid-mediated mechanism of action. The studied agents are promising candidates for the role of antiviral compounds due to their high lipophilicity, low toxicity, bioavailability, and relative cheapness. Fluorimetry of calcein release at the calcium-mediated fusion of liposomes, composed of a ternary mixture of dioleoyl phosphatidylcholine, dioleoyl phosphatidylglycerol, and cholesterol, in the presence of 4′-hydroxychalcone, cardamonin, isoliquiritigenin, phloretin, resveratrol, piceatannol, daidzein, biochanin A, genistein, genistin, liquiritigenin, naringenin, catechin, taxifolin, and honokiol, was performed. It was found that piceatannol significantly inhibited the calcium-induced fusion of negatively charged vesicles, while taxifolin and catechin showed medium and low antifusogenic activity, respectively. As a rule, polyphenols containing at least two OH-groups in both phenolic rings were able to inhibit the calcium-mediated fusion of liposomes. In addition, there was a correlation between the ability of the tested compounds to inhibit vesicle fusions and to perturb lipid packing. We suggest that the antifusogenic action of polyphenols was determined by the depth of immersion and the orientation of the molecules in the membrane.

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2023 – Rational design of magnetoliposomes for enhanced interaction with bacterial membrane models
Vesicle Prep Pro Publication in Biochimica & Biophysica Acta (BBA) - Biomembranes (2023) Authors: Soares F.A. Costa P., Sousa C.T., Horta M., Pereira-Leite C., Seabra C.L. Lima S.A.C., Reis S., Nunes C.

There is a growing need for alternatives to target and treat bacterial infection. Thus, the present work aims to develop and optimize the production of PEGylated magnetoliposomes (MLPs@PEG), by encapsulating superparamagnetic iron oxide nanoparticles (SPIONs) within fusogenic liposomes. A Box–Behnken design was applied to modulate size distribution variables, using lipid concentration, SPIONs amount and ultrasonication time as independent variables. As a result of the optimization, it was possible to obtain MLPs@PEG with a mean size of 182 nm, with polydispersity index (PDI) of 0.19, and SPIONs encapsulation efficiency (%EE) around 76%. Cytocompatibility assays showed that no toxicity was observed in fibroblasts, for iron concentrations up to 400 μg/ml. Also, for safe lipid and iron concentrations, no hemolytic effect was detected. The fusogenicity of the nanosystems was first evaluated through lipid mixing assays, based on Förster resonance energy transfer (FRET), using liposomal membrane models, mimicking bacterial cytoplasmic membrane and eukaryotic plasma membrane. It was shown that the hybrid nanosystems preferentially interact with the bacterial membrane model. Confocal microscopy and fluorescence lifetime measurements, using giant unilamellar vesicles (GUVs), validated these results. Overall, the developed hybrid nanosystem may represent an efficient drug delivery system with improved targetability for bacterial membrane.

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2023 – Energetics and kinetics of membrane permeation of photoresists for bioprinting
Vesicle Prep Pro Pre-Print Publication in BioRxiv (2023) Authors: Diedrich L., Brosz M., Abele T., Steinke S,, Gräter F., Göpfrich K. Aponte-Santamaría C.

Three-dimensional (3D) bioprinting is a promising technology which typically uses bioinks to pattern cells and their scaffolds. The selection of cytocompatible inks is critical for the printing success. In laserbased 3D bioprinting, photoresist molecules are used as bioinks. We propose that cytotoxicity can be a consequence of the interaction of photoresists with lipid membranes and their permeation into the cell. Here, molecular dynamics simulations and in vitro assays address this issue, retrieving partition coefficients, free energies, and permeabilities for eight commonly-used photoresists in model lipid bilayers. Crossing the hydrophobic center of the membrane constitutes the rate limiting step during permeation. In addition, three photoresists feature a preferential localization site at the acyl chain headgroup interface. Photoresist permeabilities range over eight orders of magnitude, with some molecules being membrane-permeable on bioprinting timescales. Moreover, permeation correlates well with the oil-water partition coefficients and is severely hampered by the lipid ordering imposed by the lipid saturation. Overall, the mechanism of interaction of photoresists with model lipid bilayers is provided here, helping to classify them according to their residence in the membrane and permeation through it. This is useful information to guide the selection of cytocompatible photoresists for 3D bioprinting.

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2023 – ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes
Vesicle Prep Pro Pre-Print Publication in BioRxiv (2023) Authors: Nixon-Abell J., Ruggeri F.S., Qamar S., Herling T.W., Czekalska M.A., Shen Yi., Wang G., King C., Fernandopulle M.S., Sneideris T., Watson J.L., Pillai V.V.S., Meadows W., Henderson J.W., Chambers J.E., Wagstaff J.L., Williams S.H., Coyle H., Lu Y., Zhang S., Marciniak S.J., Freund S.M.V., Derivery E., Ward M.E., Vendruscolo M., Knowles T.P.J. St George-Hyslop P.

Phase transitions of cellular proteins and lipids play a key role in governing the organisation and coordination of intracellular biology. The frequent juxtaposition of proteinaceous biomolecular condensates to cellular membranes raises the intriguing prospect that phase transitions in proteins and lipids could be co-regulated. Here we investigate this possibility in the ribonucleoprotein (RNP) granule-ANXA11-lysosome ensemble, where ANXA11 tethers RNP granule condensates to lysosomal membranes to enable their co-trafficking. We show that changes to the protein phase state within this system, driven by the low complexity ANXA11 N-terminus, induce a coupled phase state change in the lipids of the underlying membrane. We identify the ANXA11 interacting proteins ALG2 and CALC as potent regulators of ANXA11-based phase coupling and demonstrate their influence on the nanomechanical properties of the ANXA11-lysosome ensemble and its capacity to engage RNP granules. The phenomenon of protein-lipid phase coupling we observe within this system offers an important template to understand the numerous other examples across the cell whereby biomolecular condensates closely juxtapose cell membranes.

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2018 – Structures of monomeric and oligomeric forms of the Toxoplasma gondii perforin-like protein 1
Vesicle Prep Pro Publication in Science Advances (2018) Authors: Ni T., Williams S.I., Rezelj S., Anderluh G., Harlos K., Stansfeld P.J., Gilbert R.J.C.

Toxoplasma and Plasmodium are the parasitic agents of toxoplasmosis and malaria, respectively, and use perforin-like proteins (PLPs) to invade host organisms and complete their life cycles. The Toxoplasma gondii PLP1 (TgPLP1) is required for efficient exit from parasitophorous vacuoles in which proliferation occurs. We report structures of the membrane attack complex/perforin (MACPF) and Apicomplexan PLP C-terminal β-pleated sheet (APCβ) domains of TgPLP1. The MACPF domain forms hexameric assemblies, with ring and helix geometries, and the APCβ domain has a novel β-prism fold joined to the MACPF domain by a short linker. Molecular dynamics simulations suggest that the helical MACPF oligomer preserves a biologically important interface, whereas the APCβ domain binds preferentially through a hydrophobic loop to membrane phosphatidylethanolamine, enhanced by the additional presence of inositol phosphate lipids. This mode of membrane binding is supported by site-directed mutagenesis data from a liposome-based assay. Together, these structural and biophysical findings provide insights into the molecular mechanism of membrane targeting by TgPLP1.

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2022 – Unraveling the host-selective toxic interaction of cassiicolin with lipid membranes and its cytotoxicity
Vesicle Prep Pro Publication in Phytopathology (2022) Authors: Ngo K.X., Quo N.B., Nguyen P.D.N., Furusho H., Miyata M., Shimonaka T., Chau N.N.B.C., Vinh N.P., Nghia N.A., Mohammed T.O., Ichikawa T., Kodera N., Konno H., Fukuma T., Ando T.

Corynespora cassiicola is the pathogen that causes Corynespora leaf fall (CLF) disease. Cassiicolin (Cas), a toxin produced by C. cassiicola, is responsible for CLF disease in rubber trees (Hevea brasiliensis). Currently, the molecular mechanism of the cytotoxicity of Cas and its host selectivity have not been fully elucidated. To gain insight into these issues, we analyzed the binding of Cas1 and Cas2 to membranes consisting of different plant lipids and their membrane-disruption activities. Our real-time observations with high-speed atomic force microscopy (HS-AFM) and confocal microscopy reveal that the binding and disruption activities of Cas1 and Cas2 are strongly dependent on the types of membrane lipids. The mixtures of DPPC with DPPA, MGDG, DGDG, and stigmasterol are more susceptible to membrane damage caused by Cas1 and Cas2 than DPPC alone or its mixtures with sitosterol, DGTS-d9, and DGTS. This difference derives from the stronger binding of the toxins to membranes with the former lipid composition. Cytotoxicity tests on rubber leaves of RRIV 1, RRIV 4, and PB 255 clones suggest that the toxins cause necrosis of rubber leaves, except for the strong resistance of PB 255 against Cas2. Cryo-SEM analyses of necrotic leaf tissues exposed to Cas1 confirm that cytoplasmic membranes are vulnerable to the toxin. Thus, the host selectivity of Cas toxin in CLF disease is attained by the lipid-dependent binding activity of Cas to the membrane, and the cytotoxicity of Cas arises from its ability to disrupt membranes.

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2022 – The Small Heat Shock Protein, HSPB1, Interacts with and Modulates the Physical Structure of Membranes
Vesicle Prep Pro Publication in International Journal of Molecular Science (2022) Authors: Csoboz B., Gombos I., Kóta Z., Dukic B., Klement E., Varga-Zsíros V., Lipinszki Z., Páli T., László Vígh L., Török Z.

Small heat shock proteins (sHSPs) have been demonstrated to interact with lipids and modulate the physical state of membranes across species. Through these interactions, sHSPs contribute to the maintenance of membrane integrity. HSPB1 is a major sHSP in mammals, but its lipid interaction profile has so far been unexplored. In this study, we characterized the interaction between HSPB1 and phospholipids. HSPB1 not only associated with membranes via membrane-forming lipids, but also showed a strong affinity towards highly fluid membranes. It participated in the modulation of the physical properties of the interacting membranes by altering rotational and lateral lipid mobility. In addition, the in vivo expression of HSPB1 greatly affected the phase behavior of the plasma membrane under membrane fluidizing stress conditions. In light of our current findings, we propose a new function for HSPB1 as a membrane chaperone.

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2022 – The Staphylococcus aureus CidA and LrgA Proteins Are Functional Holins Involved in the Transport of By-Products of Carbohydrate Metabolism
Vesicle Prep Pro Publication in mBio (2022) Authors: Endres J. L., Chaudhari S. S., Zhang X., Prahlad J., Wang S-Q., Foley L. A., Luca S., Bose J. L., Thomas V. C., Bayles K. W.

The Staphylococcus aureus cidABC and lrgAB operons encode members of a well-conserved family of proteins thought to be involved in programmed cell death (PCD). Based on the structural similarities that CidA and LrgA share with bacteriophage holins, we have hypothesized that these proteins function by forming pores within the cytoplasmic membrane. To test this, we utilized a "lysis cassette" system that demonstrated the abilities of the cidA and lrgA genes to support bacteriophage endolysin-induced cell lysis. Typical of holins, CidA- and LrgA-induced lysis was dependent on the coexpression of endolysin, consistent with the proposed holin-like functions of these proteins. In addition, the CidA and LrgA proteins were shown to localize to the surface of membrane vesicles and cause leakage of small molecules, providing direct evidence of their hole-forming potential. Consistent with recent reports demonstrating a role for the lrgAB homologues in other bacterial and plant species in the transport of by-products of carbohydrate metabolism, we also show that lrgAB is important for S. aureus to utilize pyruvate during microaerobic and anaerobic growth, by promoting the uptake of pyruvate under these conditions. Combined, these data reveal that the CidA and LrgA membrane proteins possess holin-like properties that play an important role in the transport of small by-products of carbohydrate metabolism. IMPORTANCE The Staphylococcus aureus cidABC and lrgAB operons represent the founding members of a large, highly conserved family of genes that span multiple kingdoms of life. Despite the fact that they have been shown to be involved in bacterial PCD, very little is known about the molecular/biochemical functions of the proteins they encode. The results presented in this study reveal that the cidA and lrgA genes encode proteins with bacteriophage holin-like functions, consistent with their roles in cell death. However, these studies also demonstrate that these operons are involved in the transport of small metabolic by-products of carbohydrate metabolism, suggesting an intriguing link between these two seemingly disparate processes.

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2022 – Soybean meal peptides regulated membrane phase of giant unilamellar vesicles: A key role for bilayer amphipathic region localization
Vesicle Prep Pro Publication in Food Research International (2022) Authors: Jiang F., Liu J., Du Z., Liu X., Shang X., Yu Y., Zhang T.

Membrane phase separation forms liquid-ordered (Lo) and liquid-disordered (Ld) phases and is involved in cellular processes and functions. Our previous study has confirmed that peptides can regulate phase separation by increasing the Lo phase. However, the specific mechanisms underlying the phase separation regulation of peptides remain poorly understood. This study aimed to explore the effect of soybean meal peptides on phase separation and illustrate the correlation between phase regulation and membrane localization of the peptides. Phase separation was studied by giant unilamellar vesicles (GUVs), and membrane localization of the peptides was detected by steady-state fluorescence quenching. Our results revealed that peptides YYK, CLA, and SLW enhanced the Lo phase while WLQ decreased the Lo phase. The localization in the membrane amphiphilic region of the peptides played a crucial role in their regulation of phase separation. The more localization of the peptides (YYK, CLA, and SLW) in the membrane amphiphilic region, the stronger the capacity to increase the Lo phase.

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2022 – The nuclear egress complex of Epstein-Barr virus buds membranes through an oligomerization-driven mechanism
Vesicle Prep Pro Publication in PLOS Pathogens (2022) Authors: Thorsen M., Draganova E., Heldwein E.

During replication, herpesviral capsids are translocated from the nucleus into the cytoplasm by an unusual mechanism, termed nuclear egress, that involves capsid budding at the inner nuclear membrane. This process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid. Although the NEC is essential for capsid nuclear egress across all three subfamilies of the Herpesviridae, most studies to date have focused on the NEC homologs from alpha- and beta- but not gammaherpesviruses. Here, we report the crystal structure of the NEC from Epstein-Barr virus (EBV), a prototypical gammaherpesvirus. The structure resembles known structures of NEC homologs yet is conformationally dynamic. We also show that purified, recombinant EBV NEC buds synthetic membranes in vitro and forms membrane-bound coats of unknown geometry. However, unlike other NEC homologs, EBV NEC forms dimers in the crystals instead of hexamers. The dimeric interfaces observed in the EBV NEC crystals are similar to the hexameric interfaces observed in other NEC homologs. Moreover, mutations engineered to disrupt the dimeric interface reduce budding. Putting together these data, we propose that EBV NEC-mediated budding is driven by oligomerization into membrane-bound coats.

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2022 – On the assembly of zwitterionic block copolymers with phospholipids
Vesicle Prep Pro Publication in European Polymer Journal (2022) Authors: Spanjers J., Brodszkij E., Gal N., Pedersen J., Städler B.

Materials containing zwitterionic polymers are interesting candidates for diverse applications due to their versatile properties. The assembly of the amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-methacryloyloxyethyl phosphorylcholine) with three different phospholipids (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)) into small and giant vesicles is reported focusing on their morphology, size, and membrane properties. Giant hybrid vesicles were obtained for all types of lipids, but DOPC was more suitable to assemble small hybrid vesicles without a large fraction of hybrid micelles present. Further, the permeability of the small vesicle membranes towards 5(6)-carboxyfluorescein is very similar to comparable sized liposomes. In contrast, the permeability of the giant hybrid vesicle membranes towards 5(6)-carboxy-X-rhodamine is higher compared to only cholesterol-containing lipid giant vesicles. DOPS-containing vesicles showed pH-dependent morphology changes. Hybrid vesicles containing DOPS and DOPE in addition to the block copolymer have the highest association with HepG2 cells. In contrast, only DOPC-containing hybrid vesicles can be incorporated into alginate beads. Taken together, using these block copolymers with a zwitterionic hydrophilic extension of the chosen architecture offers fundamental insight into the possibility to assemble hybrid vesicles and their potential in bottom-up synthetic biology.

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2022 – Polymer Micelles vs Polymer–Lipid Hybrid Vesicles: A Comparison Using RAW 264.7 Cells
Vesicle Prep Pro Publication in Frontiers in Biomacromolecules (2022) Authors: Ade C., Qian X., Brodszkij E., Andres P.D.D., Spanjers J., Westensee I.N., Städler B.

Bottom-up synthetic biology aims to integrate artificial moieties with living cells and tissues. Here, two types of structural scaffolds for artificial organelles were compared in terms of their ability to interact with macrophage-like murine RAW 264.7 cells. The amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-carboxyethyl acrylate) was used to assemble micelles and polymer–lipid hybrid vesicles together with 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipids in the latter case. In addition, the pH-sensitive fusogenic peptide GALA was conjugated to the carriers to improve their lysosomal escape ability. All assemblies had low short-term toxicity toward macrophage-like murine RAW 264.7 cells, and the cells internalized both the micelles and hybrid vesicles within 24 h. Assemblies containing DOPE lipids or GALA in their building blocks could escape the lysosomes. However, the intracellular retention of the building blocks was only a few hours in all the cases. Taken together, the provided comparison between two types of potential scaffolds for artificial organelles lays out the fundamental understanding required to advance soft material-based assemblies as intracellular nanoreactors.

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2022 – Membrane composition of polymer-lipid hybrid vesicles
Vesicle Prep Pro Publication in Frontiers in Applied Materials Today (2022) Authors: Brodszkij E., Westensee I.N., Holleufer S.F., Ade C., De Dios Andres P., Pedersen J.S., Städler B.

Hybrid vesicles (HVs) assembled from phospholipids and amphiphilic block copolymers (BCPs) are a more recent alternative to liposomes and polymersomes. We aim to change the properties of the HV membranes by varying the chemical composition of the hydrophobic block in the BCPs that have poly(carboxyethyl acrylate) (PCEA) as the hydrophilic part. To this end, statistical copolymers of cholesteryl methacrylate and either butyl methacrylate (BuMA) or 2-hydroxyethyl methacrylate (HEMA) as well as the corresponding homopolymers were synthesized and used as macroinitiator for the extension with PCEA. All the BCPs allowed for the assembly of small and giant HVs with soybean L-α-phosphatidylcholine. The extend of the co-extisting micellar populations varied as shown by transmission electron microscopy and small-angle X-ray spectroscopy. Although the membrane packings derived from spectra when using Laurdan as an environmentally sensitive fluorescent probe were comparable between the different HVs, their permeability towards 5(6)-carboxy-X-rhodamine or carboxyfluorescein depended on the membrane composition, i.e., HEMA-containing membranes had higher permeability than membranes containing the other tested BCPs for small and giant HVs. Further, membranes with BuMA offered the most suitable environment for the association with β-galactosidase illustrated by the efficient substrate conversion. Taken together, the hydrophobic block is a relevant mean to control the morphologies and membrane properties of HVs.

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2022 – Membrane lipid organization and nicotinic acetylcholine receptor function: A two-way physiological relationship
Vesicle Prep Pro Publication in Archives of Biochemistry and Biophysics (2022) Authors: Fabiani C., Georgiev V., Peñalva D., Sigaut L., Pietrasanta L., Corradi J., Dimova R., Antollini S.

Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR. Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function.

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2022 – Evaluation of Polyoxazolines Insertion into the Epidermis: From Membrane Models to in Vivo Studies
Vesicle Prep Pro Pre-Print Publication in European Journal of Pharmaceutics and Biopharmaceutics (2022) Authors: Simon L., E. Bellard E., B. Jouanmiqueou B., V. Lapinte V., N. Marcotte N., J.M. Devoisselle J.M., Rols M.P., Golzio M., Bégu S.

In this study, we evaluated the potential of amphiphilic polyoxazolines (POx) to interact with biological membranes thanks to models of increasing complexity, from a simple lipid bilayer using giant unilamellar vesicles (GUV), to plasma membranes of three different cell types, fibroblasts, keratinocytes and melanocytes, which are found in human skin. Upon assessing an excellent penetration into GUV membranes and cultured cells, we addressed POx’s potential to penetrate the murine skin within an in vivo model. Exposure studies were made with native POx and with POx encapsulated within lipid nanocapsules (LNC). Our findings indicate that POx’s interactions with membranes tightly depend on the nature of the alkyl chain constituting the POx. Saturated C 16 POx insert rapidly and efficiently into GUV and plasma membranes, while unsaturated C 18:2 POx insert to a smaller extent. The high amount of membrane-inserted saturated C 16 POx impacts cell viability to a greater extent than the unsaturated C 18:2 POx. The in vivo study, performed on mice, showed an efficient accumulation of both POx types in the stratum corneum barrier, reaching the upper epidermis, independently of POx’s degree of saturation. Furthermore, the formulation of POx into lipid nanocapsules allowed delivering an encapsulated molecule, the quercetin, in the upper epidermis layers of murine skin, proving POx’s efficacy for topical delivery of active molecules. Overall, POx proved to be an excellent choice for topical delivery, which might in turn offer new possibilities for skin treatments in diseases such as psoriasis or melanomas.

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2022 – Lipid Microenvironment Modulates the Pore-Forming Ability of Polymyxin B
Vesicle Prep Pro Publication in Antibiotics (2022) Authors: Zakharova A., Efimova S., Ostroumova O.

The ability of polymyxin B, an antibiotic used to treat infections caused by multidrug-resistant Gram-negative bacteria as a last-line therapeutic option, to form ion pores in model membranes composed of various phospholipids and lipopolysaccharides was studied. Our data demonstrate that polymyxin B predominantly interacts with negatively charged lipids. Susceptibility decreases as follows: Kdo2-Lipid A >> DOPG ≈ DOPS >> DPhPG ≈ TOCL ≈ Lipid A. The dimer and hexamer of polymyxin B are involved in the pore formation in DOPG(DOPS)- and Kdo2-Lipid A-enriched bilayers, respectively. The pore-forming ability of polymyxin B significantly depends on the shape of membrane lipids, which indicates that the antibiotic produces toroidal lipopeptide-lipid pores. Small amphiphilic molecules diminishing the membrane dipole potential and inducing positive curvature stress were shown to be agonists of pore formation by polymyxin B and might be used to develop innovative lipopeptide-based formulations.

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2022 – Chromone-Containing Allylmorpholines Influence Ion Channels in Lipid Membranes via Dipole Potential and Packing Stress
Vesicle Prep Pro Publication in International Journal of Molecular Science (2022) Authors: Efimova S., Martynyuk V., Zakharova A., Yudintceva N., Chernov N., Yakovlev I., Ostroumova O.

Herein, we report that chromone-containing allylmorpholines can affect ion channels formed by pore-forming antibiotics in model lipid membranes, which correlates with their ability to influence membrane boundary potential and lipid-packing stress. At 100 µg/mL, allylmorpholines 1, 6, 7, and 8 decrease the boundary potential of the bilayers composed of palmitoyloleoylphosphocholine (POPC) by about 100 mV. At the same time, the compounds do not affect the zeta-potential of POPC liposomes, but reduce the membrane dipole potential by 80–120 mV. The allylmorpholine-induced drop in the dipole potential produce 10–30% enhancement in the conductance of gramicidin A channels. Chromone-containing allylmorpholines also affect the thermotropic behavior of dipalmytoylphosphocholine (DPPC), abolishing the pretransition, lowering melting cooperativity, and turning the main phase transition peak into a multicomponent profile. Compounds 4, 6, 7, and 8 are able to decrease DPPC’s melting temperature by about 0.5–1.9 °C. Moreover, derivative 7 is shown to increase the temperature of transition of palmitoyloleoylphosphoethanolamine from lamellar to inverted hexagonal phase. The effects on lipid-phase transitions are attributed to the changes in the spontaneous curvature stress. Alterations in lipid packing induced by allylmorpholines are believed to potentiate the pore-forming ability of amphotericin B and gramicidin A by several times.

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2022 – Egg White Peptides Increased the Membrane Liquid-Ordered Phase of Giant Unilamellar Vesicles: Visualization, Localization, and Phase Regulation Mechanism
Vesicle Prep Pro Publication in Journal of Agricultural and Food Chemistry (2022) Authors: Jiang F., Liu J., Niu X., Zhang D., Wang E., Zhang T.

Cell membranes are heterogeneous and consist of liquid-ordered (Lo) and liquid-disordered (Ld) phases due to phase separation. Membrane regulation of egg white peptides (LCAY and QVPLW) was confirmed in our previous study. However, the underlying mechanism of phase regulation by the peptides has not been elucidated. This study aimed to explore the effect of LCAY and QVPLW on the membrane phase separation and illustrate their mechanism by giant unilamellar vesicles (GUVs). Based on phase separation visualization, LCAY and QVPLW were found to increase the Lo phase by rearranging lipids and ordering the Ld phase. LCAY and QVPLW can bind to the GUVs and localize in the amphiphilic region of the membrane. By hydrogen bonds and hydrophobic interactions, LCAY and QVPLW may play a cholesterol-like role in regulating phase separation.

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2021 – Unexpected Gating Behaviour of an Engineered Potassium Channel Kir
Vesicle Prep Pro Publication in Frontiers in Molecular Biosciences (2021) Authors: Fagnen, C., Bannwarth, L., Zuniga, D., Oubella, I., De Zorzi, R., Forest, E., Scala, R., Guilbault, S., Bendahhou, S., Perahia, D., & Vénien-Bryan, C.

In this study, we investigated the dynamics and functional characteristics of the KirBac3.1 S129R, a mutated bacterial potassium channel for which the inner pore-lining helix (TM2) was engineered so that the bundle crossing is trapped in an open conformation. The structure of this channel has been previously determined at high atomic resolution. We explored the dynamical characteristics of this open state channel using an in silico method MDeNM that combines molecular dynamics simulations and normal modes. We captured the global and local motions at the mutation level and compared these data with HDX-MS experiments. MDeNM provided also an estimation of the probability of the different opening states that are in agreement with our electrophysiological experiments. In the S129R mutant, the Arg129 mutation releases the two constriction points in the channel that existed in the wild type but interestingly creates another restriction point.

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2022 – Astrocytes in Paper Chips and Their Interaction with Hybrid Vesicles
Vesicle Prep Pro Publication in Advanced Biology (2022) Authors: Meyer C., De Dios Andres P., Brodszkij E., Westensee I., Lyons J., Vaz S., Städler B.

The role of astrocytes in brain function has received increased attention lately due to their critical role in brain development and function under physiological and pathophysiological conditions. However, the biological evaluation of soft material nanoparticles in astrocytes remains unexplored. Here, the interaction of crosslinked hybrid vesicles (HVs) and either C8-D1A astrocytes or primary astrocytes cultured in polystyrene tissue culture or floatable paper-based chips is investigated. The amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-carboxyethyl acrylate) (P1) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine lipids are used for the assembly of HVs with crosslinked membranes. The assemblies show no short-term toxicity towards the C8-D1A astrocytes and the primary astrocytes, and both cell types internalize the HVs when cultured in 2D cell culture. Further, it is demonstrated that both the C8-D1A astrocytes and the primary astrocytes could mature in paper-based chips with preserved calcium signaling and glial fibrillary acidic protein expression. Last, it is confirmed that both types of astrocytes could internalize the HVs when cultured in paper-based chips. These findings lay out a fundamental understanding of the interaction between soft material nanoparticles and astrocytes, even when primary astrocytes are cultured in paper-based chips offering a 3D environment.

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2021 – Proton gradients from light-harvesting E. coli control DNA assemblies for synthetic cells
Vesicle Prep Pro Publication in Nature Communications (2021) Authors: Jahnke K., Ritzmann N., Fichtler J., Nitschke A., Dreher Y., Abele T., Hofhaus G., Platzman I., Schröder R. R., Müller D. J., Spatz J. P., Göpfrich K.

Bottom-up and top-down approaches to synthetic biology each employ distinct methodologies with the common aim to harness living systems. Here, we realize a strategic merger of both approaches to convert light into proton gradients for the actuation of synthetic cellular systems. We genetically engineer E. coli to overexpress the light-driven inward-directed proton pump xenorhodopsin and encapsulate them in artificial cell-sized compartments. Exposing the compartments to light-dark cycles, we reversibly switch the pH by almost one pH unit and employ these pH gradients to trigger the attachment of DNA structures to the compartment periphery. For this purpose, a DNA triplex motif serves as a nanomechanical switch responding to the pH-trigger of the E. coli. When DNA origami plates are modified with the pH-sensitive triplex motif, the proton-pumping E. coli can trigger their attachment to giant unilamellar lipid vesicles (GUVs) upon illumination. A DNA cortex is formed upon DNA origami polymerization, which sculpts and deforms the GUVs. We foresee that the combination of bottom-up and top down approaches is an efficient way to engineer synthetic cells.

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2021 – Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes
Vesicle Prep Pro Publication in Nature Communications (2021) Authors: Tremel S., Ohashi Y., Morado D.R., Bertram J., Perisic O., Brandt L.T.L., von Wrisberg M-K., Chen Z.A., Maslen S.L., Kovtun O., Skehel M., Rappsilber J., Lang K., Munro S., Briggs J.A.G., Williams R.L.

The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a–GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.

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2021 – Phosphodiesterase Type 5 Inhibitors Greatly Affect Physicochemical Properties of Model Lipid Membranes
Vesicle Prep Pro Publication in Membranes (2021) Authors: Zakharova A.A., Efimova S.S.,Ostroumova O.S.

Although phosphodiesterase type 5 inhibitors are widely used and well-studied drugs, the potential benefits of their application in the treatment of various diseases and new drug delivery systems, including liposome forms, are still being discussed. In this regard, the role of the lipid matrix of cell membranes in the pharmacological action of the inhibitors is of special interest. It was shown that sildenafil, vardenafil, and tadalafil caused a significant decrease in the boundary potential of model membranes composed of palmitoyloleoylphosphatidylcholine or its mixture with cholesterol, by 70–80 mV. The reduction in the membrane dipole potential induced by inhibitors led to a 20–25% increase in the conductance of cation-selective pores formed by the antimicrobial peptide gramicidin A. The addition of sildenafil or vardenafil also led to a significant decrease in the temperature of the main phase transition of dipalmytoylphosphatidylcholine, by about 1.5 °C, while tadalafil did not change the melting temperature. Sildenafil, vardenafil, and tadalafil enhanced the pore-forming activity of the antifungal polyene antibiotic nystatin by 11, 13, and 2 times, respectively. This fact might indicate the induction of membrane curvature stress by the inhibitors. The data obtained might be of special interest for the development of lipid-mediated forms of drugs.

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2021 – Pore-forming Esx proteins mediate toxin secretion by Mycobacterium tuberculosis
Vesicle Prep Pro Publication in Nature Communications (2021) Authors: Tak U., Dokland T. and Niederweis M.

Mycobacterium tuberculosis secretes the tuberculosis necrotizing toxin (TNT) to kill host cells. Here, we show that the WXG100 proteins EsxE and EsxF are essential for TNT secretion. EsxE and EsxF form a water-soluble heterodimer (EsxEF) that assembles into oligomers and long filaments, binds to membranes, and forms stable membrane-spanning channels. Electron microscopy of EsxEF reveals mainly pentameric structures with a central pore. Mutations of both WXG motifs and of a GXW motif do not affect dimerization, but abolish pore formation, membrane deformation and TNT secretion. The WXG/GXW mutants are locked in conformations with altered thermostability and solvent exposure, indicating that the WXG/GXW motifs are molecular switches controlling membrane interaction and pore formation. EsxF is accessible on the bacterial cell surface, suggesting that EsxEF form an outer membrane channel for toxin export. Thus, our study reveals a protein secretion mechanism in bacteria that relies on pore formation by small WXG proteins.

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2021 – Is the Membrane Lipid Matrix a Key Target for Action of Pharmacologically Active Plant Saponins?
Vesicle Prep Pro Publication in International Journal of Molecular Sciences  (2021) Authors: Efimova S.S., Ostroumova O.S.

This study was focused on the molecular mechanisms of action of saponins and related compounds (sapogenins and alkaloids) on model lipid membranes. Steroids and triterpenes were tested. A systematic analysis of the effects of these chemicals on the physicochemical properties of the lipid bilayers and on the formation and functionality of the reconstituted ion channels induced by antimicrobial agents was performed. It was found that digitonin, tribulosin, and dioscin substantially reduced the boundary potential of the phosphatidylcholine membranes. We concluded that saponins might affect the membrane boundary potential by restructuring the membrane hydration layer. Moreover, an increase in the conductance and lifetime of gramicidin A channels in the presence of tribulosin was due to an alteration in the membrane dipole potential. Differential scanning microcalorimetry data indicated the key role of the sapogenin core structure (steroid or triterpenic) in affecting lipid melting and disordering. We showed that an alteration in pore forming activity of syringomycin E by dioscin might be due to amendments in the lipid packing. We also found that the ability of saponins to disengage the fluorescent marker calcein from lipid vesicles might be also determined by their ability to induce a positive curvature stress.

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2021 – Membrane interaction and disulphide-bridge formation in the unconventional secretion of Tau
Vesicle Prep Pro Publication in Bioscience Reports (2021) Authors: Hellen M., Bhattacharjee A., Uronen R-L., Huttunen H.J.

Misfolded, pathological tau protein propagates from cell to cell causing neuronal degeneration in Alzheimer’s disease and other tauopathies. The molecular mechanisms of this process have remained elusive. Unconventional secretion of tau takes place via several different routes, including direct penetration through the plasma membrane. Here, we show that tau secretion requires membrane interaction via disulphide bridge formation. Mutating residues that reduce tau interaction with membranes or formation of disulphide bridges decrease both tau secretion from cells, and penetration through artificial lipid membranes. Our results demonstrate that tau is indeed able to penetrate protein-free membranes in a process independent of active cellular processes and that both membrane interaction and disulphide bridge formation are needed for this process. QUARK-based de novo modelling of the second and third microtubule-binding repeat domains (MTBDs), in which the two cysteine residues of 4R isoforms of tau are located, supports the concept that this region of tau could form transient amphipathic helices for membrane interaction.

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2021 – EPA and DHA differentially modulate membrane elasticity in the presence of cholesterol
Vesicle Prep Pro Publication in Biophysical Journal (2021) Authors: Jacobs M.L., Faizi H.A., Peruzzi J.A., Vlahovska P.M., Kamat N.P.

Polyunsaturated fatty acids (PUFAs) modify the activity of a wide range of membrane proteins and are increasingly hypothesized to modulate protein activity by indirectly altering membrane physical properties. Among the various physical properties affected by PUFAs, the membrane area expansion modulus (Ka), which measures membrane strain in response to applied force, is expected to be a significant controller of channel activity. Yet the impact of PUFAs on membrane Ka has not been measured previously. Through a series of micropipette aspiration studies, we measured the apparent Ka (Kapp) of phospholipid model membranes containing non-esterified fatty acids. First, we measured membrane Kapp as a function of the location of the unsaturated bond and degree of unsaturation in the incorporated fatty acids and found that Kapp generally decreases in the presence of fatty acids with three or more unsaturated bonds. Next, we assessed how select ω-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), affect the Kapp of membranes containing cholesterol. In vesicles prepared with high amounts of cholesterol, which should increase the propensity of the membrane to phase segregate, we found that inclusion of DHA decreases the Kapp in comparison to EPA. We also measured how these ω-3 PUFAs affect membrane fluidity and bending rigidity to determine how membrane Kapp changes in relation to these other physical properties. Our study shows that PUFAs generally decrease Kapp of membranes and that EPA and DHA have differential effects on Kapp when membranes contain higher levels of cholesterol. Our results suggest membrane phase behavior, and the distribution of membrane elasticizing amphiphiles, impacts the ability of a membrane to stretch.

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2021 – Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering
Vesicle Prep Pro Publication in mBio (2021) Authors: Thorsen M.K., Lai A., Lee M.W., Hoogerheide D.P., Wong G.C.L., Freed J.H., Heldweina E.E

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding.

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2021 – Dynamic remodeling of giant unilamellar vesicles induced by monoglyceride nano-micelles: Insights into supramolecular organization
Vesicle Prep Pro Publication in Applied Materials Today (2021) Authors: Yoon B.K., Kim M.C., Jackman J.A., Cho N-J

There is broad interest in developing nanostructured assemblies composed of fatty acids and monoglycerides to inhibit membrane-enveloped pathogens and modulate immune cell behavior. Herein, we investigated the interactions of micellar nanostructures composed of a biologically active monoglyceride, glycerol monolaurate (GML), or its ether-bonded equivalent, 1-O-dodecyl-rac-glycerol (DDG), with cell-membrane-mimicking giant unilamellar vesicles (GUVs). Our findings revealed that GML nanostructures induced fission or fusion depending on the GML concentration and corresponding degree of supramolecular organization, while DDG nanostructures only caused aggregation-like disruption of the GUV outer surface. In specific conditions, the GML nanostructures also triggered pearling instability, which led to dynamic membrane remodeling behavior and the pattern of GML interactions was consistent across simplified and complex membrane compositions. Notably, the spectrum of membrane morphological changes induced by GML nanostructures, including fission, fusion, and pearling behaviors, is appreciably wider than the fission behavior exhibited by fatty acid nanostructures in past studies. Collectively, these findings demonstrate how controlling the supramolecular organization of monoglycerides within nanostructured assemblies can be useful to modulate the type and degree of membrane interactions relevant to biophysical and nanomedicine applications.

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2021 – Electroformation of Giant Unilamellar Vesicles and A Case Study of the Specific Host-Toxin Interaction of Lipid Membranes with Cassiicolin
Vesicle Prep Pro Protocol in Bio-101 (2021) Authors: Ngo KX., Weichbrodt C., Quoc NB

An alternating electric field is applied to induce swelling of thin lipid films and generation of giant unilamellar vesicles (GUVs) on an indium tin oxide (ITO)-coated glass surface. The process is, hereafter, referred to as the electroformation of GUVs. Several important parameters such as lipid manipulation, temperature, osmolarity and ionic strength of the solutions involved, and the electric field (current (DC, AC), amplitude, frequency) should be optimal for the successful electroformation of GUVs. In our case study, GUVs composed of lipid mixtures available in plant cells provide many benefits for studying the lipid-dependent pathogenicity of cassiicolin (Cas) toxins and thereby deciphering the host-selective toxin interaction of Cas toxins with the specific lipid membranes of plant cells. GUVs gently maintained in the solution furnish perfectly suspended and intact lipid membranes similar to cytoplasmic membranes enabling us to examine the selective binding of GFP-Cas1 and GFP-Cas2 to the specific lipid membranes. In this protocol, we briefly explain the principle of electroformation method and provide the experimental conditions and the manipulation for successfully making GUVs composed of plant lipids (DPPC, DPPC/DPPA, DPPC/MGDG, DPPC/DGDG, DPPC/stigmasterol, DPPC/sitosterol, DPPC/DGTS-d9, and DPPC/DGTS). 

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2021 – Binding of DNA origami to lipids: maximizing yield and switching via strand displacement
Vesicle Prep Pro Publication in Nucleic Acids Research (2021) Authors: Daljit Singh J. K., Darley E., Ridone P., Gaston J. P., Abbas A., Wickham S. F J., Baker M. A B

Liposomes are widely used as synthetic analogues of cell membranes and for drug delivery. Lipid-binding DNA nanostructures can modify the shape, porosity and reactivity of liposomes, mediated by cholesterol modifications. DNA nanostructures can also be designed to switch conformations by DNA strand displacement. However, the optimal conditions to facilitate stable, high-yield DNA–lipid binding while allowing controlled switching by strand displacement are not known. Here, we characterized the effect of cholesterol arrangement, DNA structure, buffer and lipid composition on DNA–lipid binding and strand displacement. We observed that binding was inhibited below pH 4, and above 200 mM NaCl or 40 mM MgCl2, was independent of lipid type, and increased with membrane cholesterol content. For simple motifs, binding yield was slightly higher for double-stranded DNA than single-stranded DNA. For larger DNA origami tiles, four to eight cholesterol modifications were optimal, while edge positions and longer spacers increased yield of lipid binding. Strand displacement achieved controlled removal of DNA tiles from membranes, but was inhibited by overhang domains, which are used to prevent cholesterol aggregation. These findings provide design guidelines for integrating strand displacement switching with lipid-binding DNA nanostructures. This paves the way for achieving dynamic control of membrane morphology, enabling broader applications in nanomedicine and biophysics.

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2021 – Design of biologically active binary protein 2D materials
Vesicle Prep Pro Publication in Nature (2021) Authors: Ben-Sasson A.J., Watson J.L., Sheffler W., Camp Johnson M., Bittleston A., Somasundaram L., Decarreau J., Jiao F., Chen J., Mela I., Drabek A.A., Jarrett S.M., Blacklow S.C., Kaminski C.F., Hura G.L., De Yoreo J.J., Kollman J.M., Ruohola-Baker H., Derivery E., Baker D.

Ordered two-dimensional arrays such as S-layers and designed analogues have intrigued bioengineers, but with the exception of a single lattice formed with flexible linkers, they are constituted from just one protein component. Materials composed of two components have considerable potential advantages for modulating assembly dynamics and incorporating more complex functionality. Here we describe a computational method to generate co-assembling binary layers by designing rigid interfaces between pairs of dihedral protein building blocks, and use it to design a p6m lattice. The designed array components are soluble at millimolar concentrations, but when combined at nanomolar concentrations, they rapidly assemble into nearly crystalline micrometre-scale arrays nearly identical to the computational design model in vitro and in cells without the need for a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces, which we demonstrate can drive extensive receptor clustering, downstream protein recruitment and signalling. Using atomic force microscopy on supported bilayers and quantitative microscopy on living cells, we show that arrays assembled on membranes have component stoichiometry and structure similar to arrays formed in vitro, and that our material can therefore impose order onto fundamentally disordered substrates such as cell membranes. In contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, we find that large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work provides a foundation for a synthetic cell biology in which multi-protein macroscale materials are designed to modulate cell responses and reshape synthetic and living systems.

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2021 – Alamethicin Channels as a Signal Transduction Element in an Immobilized Single Giant Unilamellar Vesicle
Vesicle Prep Pro Publication in Sensors and Materials (2021) Authors: Fukuda H., Nitta M., Sakamoto M., Shoji A., Sugawara M.

A single giant unilamellar vesicle (GUV) functionalized with an anti-bovine serum albumin (BSA) antibody was immobilized on an avidin slip, and alamethicin channels were embedded as a signal transduction element for creating a channel-based molecular sensing system. The GUV sensor based on the membrane-bound anti-BSA antibody receptor exhibited alamethicin activities that reflected the binding of BSA (an analyte) at the membrane/solution interface. The normalized integrated current at −60 mV was able to be used as a measure of the amount of BSA in a solution. The quantification of BSA at pg/mL level was demonstrated.

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2021 – Binding of DNA origami to lipids: maximising yield and switching via strand-displacement
Vesicle Prep Pro Publication in Nucleic Acids Research (2021) Authors: Singh J.KD., Darley E., Ridone P., Gaston J. P., Abbas A., Wickham S.FJ., Baker M.AB.

iposomes are widely used as synthetic analogues of cell membranes and for drug delivery. Lipid-binding DNA nanostructures can modify the shape, porosity and reactivity of liposomes, mediated by cholesterol modifications. DNA nanostructures can also be designed to switch conformations by DNA strand displacement. However, the optimal conditions to facilitate stable, high-yield DNA–lipid binding while allowing controlled switching by strand displacement are not known. Here, we characterized the effect of cholesterol arrangement, DNA structure, buffer and lipid composition on DNA–lipid binding and strand displacement. We observed that binding was inhibited below pH 4, and above 200 mM NaCl or 40 mM MgCl2, was independent of lipid type, and increased with membrane cholesterol content. For simple motifs, binding yield was slightly higher for double-stranded DNA than single-stranded DNA. For larger DNA origami tiles, four to eight cholesterol modifications were optimal, while edge positions and longer spacers increased yield of lipid binding. Strand displacement achieved controlled removal of DNA tiles from membranes, but was inhibited by overhang domains, which are used to prevent cholesterol aggregation. These findings provide design guidelines for integrating strand displacement switching with lipid-binding DNA nanostructures. This paves the way for achieving dynamic control of membrane morphology, enabling broader applications in nanomedicine and biophysics.

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2020 – The mechanisms of action of water-soluble aminohexanoic and malonic adducts of fullerene C60 with hexamethonium on model lipid membranes
Vesicle Prep Pro Publication found in Biochimica et Biophysica Acta (BBA) -  Biomembranes (2020) Authors: Efimova S.S., Khaleneva D.A., Litasova E.V., Piotrovskiy L.B., Ostroumova O.S.

In an attempt to understand the possibility of applications of the fullerene-based systems for transporting various polar compounds like hexamethonium through the blood–brain barrier, we studied the influence of a series of derivatives of fullerene C60 in the form of salts with hexamethonium bis-anion, namely the adducts of fullerenols with 6-aminohexanoic acid (IEM-2197), and two bis-adduct malonic acid derivatives of fullerene with addents bound in two hemispheres (IEM-2143) and in equatorial positions (IEM-2144), on model membranes. We showed that IEM-2197 induced the disintegration of the bilayers composed of DOPC at the concentrations more than 2 mg/ml. IEM-2144 and IEM-2143-induced ion-permeable pores at concentrations of 0.3 and 0.02 mg/ml, respectively; herewith, IEM-2143 was characterized by the greater efficiency than IEM-2144. IEM-2197 did not significantly affect the phase behavior of DPPC, while the melting temperature significantly decreased with addition of IEM-2144 and IEM-2143. The increase in the half-width of the main transition peaks by more than 2.0 °C in the presence of IEM-2144 and IEM-2143 was observed, along with the pronounced peak deconvolution. We proposed that the immersion of IEM-2144 and IEM-2143 into the polar region of the DOPC or DPPC bilayers led to an increase in the relative mobility of tails and formation of ion-permeable defects. IEM-2197 demonstrated the more pronounced effects on the melting and ion permeability of PG- and PS-containing bilayers compared to PC-enriched membranes. These results indicated that IEM-2197 preferentially interacts with the negatively charged lipids compared to neutral species.

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2021 – A microfluidic platform for sequential assembly and separation of synthetic cell models
Vesicle Prep Pro Pre-Print Publication in ACS Synthetic Biology (2021) Authors: Tivony R., Fletcher M., Al Nahas K., Keyser U. F.

Cell-sized vesicles like giant unilamellar vesicles (GUVs) are established as a promising biomimetic model for studying cellular phenomena in isolation. However, the presence of residual components and by-products, generated during vesicles preparation and manipulation, severely limits the utility of GUVs in applications like synthetic cells. Therefore, with the rapidly growing field of synthetic biology, there is an emergent demand for techniques that can continuously purify cell-like vesicles from diverse residues, while GUVs are being simultaneously synthesized and manipulated. We developed a microfluidic platform capable of purifying GUVs through stream bifurcation, where a stream of vesicles suspension is partitioned into three fractions - purified GUVs, residual components, and a washing solution. Using our purification approach, we showed that giant vesicles can be separated from various residues – that range in size and chemical composition – with a very high efficiency (e = 0.99), based on size and deformability of the filtered objects. In addition, by incorporating the purification module with a microfluidic-based GUV-formation method, octanol-assisted liposome assembly (OLA), we established an integrated production-purification microfluidic unit that sequentially produces, manipulates, and purifies GUVs. We demonstrate the applicability of the integrated device to synthetic biology through sequentially fusing SUVs with freshly prepared GUVs and separating the fused GUVs from extraneous SUVs and oil droplets at the same time.

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2020 – Soluble cyanobacterial carotenoprotein as a robust antioxidant nanocarrier and delivery module
Vesicle Prep Pro Publication in Antioxidants (Basel) (2020) Authors: Maksimov E.G., Zamaraev A.V., Parshina E.Y., Slonimskiy Y.B., Slastnikova T.A., Abdrakhmanov A.A., Babaev P.A., Efimova, S.S., Ostroumova O.S., Stepanov A.V., Ryabova, A.V., Friedrich T., Sluchanko N.N.

To counteract oxidative stress, antioxidants including carotenoids are highly promising, yet their exploitation is drastically limited by the poor bioavailability and fast photodestruction, whereas current delivery systems are far from being efficient. Here we demonstrate that the recently discovered nanometer-sized water-soluble carotenoprotein from Anabaena (termed CTDH) transiently interacts with liposomes to efficiently extract carotenoids via carotenoid-mediated homodimerization, yielding violet-purple protein samples amenable to lyophilization and long-term storage. We characterize spectroscopic properties of the pigment-protein complexes and thermodynamics of liposome-protein carotenoid transfer and demonstrate the highly efficient delivery of echinenone form CTDH into liposomes. Most importantly, we show carotenoid delivery to membranes of mammalian cells, which provides protection from reactive oxygen species. The described carotenoprotein may be considered as part of modular systems for the targeted antioxidant delivery. Significance statement: Carotenoids are excellent natural antioxidants but their delivery to vulnerable cells is challenging due to their hydrophobic nature and susceptibility to degradation. Thus, systems securing antioxidant stability and facilitating targeted delivery are of great interest for the design of medical agents. In this work, we have demonstrated that soluble cyanobacterial carotenoprotein can deliver echinenone into membranes of liposomes and mammalian cells with almost 70 % efficiency, which alleviates the induced oxidative stress. Our findings warrant the robustness of the protein-based carotenoid delivery for studies of carotenoid activities and effects on cell models.

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2020 – The Disordering Effect of Plant Metabolites on Model Lipid Membranes of Various Thickness
Vesicle Prep Pro Publication in Cell and Tissue Biology (2020) Authors: Efimova S.S., Ostroumova O.S.

This study was focused on the effect of plant metabolites (phloretin, capsaicin, digitonin, diosgeninThis study was focused on the effect of plant metabolites (phloretin, capsaicin, digitonin, diosgeninand betulin) on the model lipid membranes. The methods of assessing the permeability of lipid bilayers basedon measuring the leakage of a fluorescent marker (calcein) from liposomes and differential scanning microcalorimetryof vesicle suspension were used. It was found that the release rate of calcein from 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) liposomes with test compounds added to the suspension at aratio with lipid 1 : 50 decreased in the order capsaicin > phloretin >> betulin ≈ diosgenin ≈ digitonin. In thecase of cholesterol- and ergosterol-containing POPC liposomes, the activity decreased as diosgenin ≈ digitonin> betulin > capsaicin > phloretin. It was demonstrated that phloretin and capsaicin significantly reducethe melting temperature (Tm) and to increase the half-width of the main peak on the endotherm (T1/2) ofdipalmitoylphosphocholine (DPPC), distearoylphosphocholine (DSPC) and diarachidoylphosphocholine(DAPC). These results show that the incorporation of these small molecules between the polar “heads” ofphosphocholine. It was found that the increase in the length of saturated chains of membrane-forming lipids(from 16 to 20 hydrocarbon units), the absolute values of ΔTm and ΔT1/2 decreased in the presence of phloretinand increased with capsaicin. This may be the result of differences in the localization of phloretin and capsaicinin the membrane. Steroid saponins exhibited a weak effect on the thermotropic behavior of phosphocholines.The absolute values of ΔTm and ΔT1/2 decreased in the order DPPC, DSPC, and DAPC and increasedin the order betulin, diosgenin, and digitonin. Steroid saponins are characterized by a more pronouncedeffect on the thermotropic behavior of the sterol–phospholipid mixture. The findings are consistent with theassumption of a high affinity of the tested saponins for sterol-containing membranes.

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2020 – Possible Mechanisms of Toxicity of Local Aminoamide Anesthetics: Lipid-Mediated Action of Ropivacaine
Vesicle Prep Pro Publication in Cell and Tissue Biology (2020) Authors: Zakharova A. A., Efimova S.S., Koryachkin V.A., Zabolotskii D.V., Ostroumova O.S. 

This work is devoted to the identification of molecular mechanisms of action of local anesthetic ropivacaine and other aminoamides (mepivacaine and bupivacaine) on the membrane physicochemical properties and formation and functioning of various ion channels in model lipid bilayers. The boundary membrane potential and its components, permeability for fluorescent markers, and the temperature and cooperativity of the melting of membrane lipid, as well as the mosaic organization of the bilayer, were studied. It was found that ropivacaine, as well as mepivacaine and bupivacaine, changed the surface charge of the bilayer and increased the membrane boundary potential. It was demonstrated that the permeability of lipid vesicles for calcein increased with the introduction of aminoamides, while the temperature and cooperativity of the melting of saturated phosphocholines decreased. The effect of anesthetics on the packing density of lipids in the membrane correlated with the hydrophobicity of their molecules. A comparison of the effects of aminoamides allowed three mechanisms of anesthetics action on the functioning of ion channels to be determined: increasing the surface potential of the membrane, decreasing the packing density of lipids in the membrane, and blocking ion channels.

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2020 – Romo1-Derived Antimicrobial Peptide Is a New Antimicrobial Agent against Multidrug-Resistant Bacteria in a Murine Model of Sepsis
Vesicle Prep Pro Publication in mBio (2020) Authors: Lee H.-R., You D.-G., Kim H.K., Sohn J.W., Kim M.J., Park J.K., Lee G.Y., Yoo Y.D.

To overcome increasing bacterial resistance to conventional antibiotics, many antimicrobial peptides (AMPs) derived from host defense proteins have been developed. However, there are considerable obstacles to their application to systemic infections because of their low bioavailability. In the present study, we developed an AMP derived from Romo1 (AMPR-11) that exhibits a broad spectrum of antimicrobial activity. AMPR-11 showed remarkable efficacy against sepsis-causing bacteria, including multidrug-resistant strains, with low toxicity in a murine model of sepsis after intravenous administration. It seems that AMPR-11 disrupts bacterial membranes by interacting with cardiolipin and lipid A. From the results of this study, we suggest that AMPR-11 is a new class of agent for overcoming low efficacy in the intravenous application of AMPs and is a promising candidate to overcome multidrug resistance.

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2020 – Membrane characteristics tune activities of endosomal and autophagic human VPS34 complexes
Vesicle Prep Pro Publication in eLife (2020) Authors: Ohashi Y., Tremel S., Masson G.R., McGinney L., Boulanger J., Rostislavleva K., Johnson C.M., Niewczas I., Clark J., Williams R.J.

The lipid kinase VPS34 orchestrates diverse processes, including autophagy, endocytic sorting, phagocytosis, anabolic responses and cell division. VPS34 forms various complexes that help adapt it to specific pathways, with complexes I and II being the most prominent ones. We found that physicochemical properties of membranes strongly modulate VPS34 activity. Greater unsaturation of both substrate and non-substrate lipids, negative charge and curvature activate VPS34 complexes, adapting them to their cellular compartments. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) of complexes I and II on membranes elucidated structural determinants that enable them to bind membranes. Among these are the Barkor/ATG14L autophagosome targeting sequence (BATS), which makes autophagy-specific complex I more active than the endocytic complex II, and the Beclin1 BARA domain. Interestingly, even though Beclin1 BARA is common to both complexes, its membrane-interacting loops are critical for complex II, but have only a minor role for complex I.

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2020 – Polymer–Lipid Hybrid Vesicles and Their Interaction with HepG2 Cells
Vesicle Prep Pro Publication in Small (2020) Authors: Brodszkij E., Westensee I.N., Bertelsen M., Gal N., Boesen T.,Städler B.

Polymer–lipid hybrid vesicles are an emerging type of nano‐assemblies that show potential as artificial organelles among others. Phospholipids and poly(cholesteryl methacrylate)‐block‐poly(methionine methacryloyloxyethyl ester (METMA)—random– 2‐carboxyethyl acrylate (CEA)) labeled with a Förster resonance energy transfer (FRET) reporter pair are used for the assembly of small and giant hybrid vesicles with homogenous distribution of both building blocks in the membrane as confirmed by the FRET effect. These hybrid vesicles have no inherent cytotoxicity when incubated with HepG2 cells up to 1.1 × 1011 hybrid vesicles per mL, and they are internalized by the cells. In contrast to the fluorescent signal originating from the block copolymer, the fluorescent signal coming from the lipids is barely detectable in cells incubated with hybrid vesicles for 6 h followed by 24 h in cell media, suggesting that the two building blocks have a different intracellular fate. These findings provide important insight into the design criteria of artificial organelles with potential structural integrity.

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2020 – Hybrid giant lipid vesicles incorporating a PMMA-based copolymer
Vesicle Prep Pro Publication found in Biochimica et Biophysica Acta (BBA) - General Subjects (2020) Authors: Miele Y., Mingotaud A.F., Caruso E., Malacarne M. C., Izzo L., Lonetti B., Rossi F.

Background - In recent years, there has been a growing interest in the formation of copolymer-lipid hybrid self-assemblies, which allow combining and improving the main features of pure lipid-based and copolymer-based systems known for their potential applications in the biomedical field. As the most common method used to obtain giant vesicles is electroformation, most systems so far used low Tg polymers for their flexibility at room temperature.

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2020 – Identification of a Membrane Binding Peptide in the Envelope Protein of MHV CoroNaVirus
Vesicle Prep Pro Publication in Viruses (2020) Authors: Alsaadi E.A.J., Neuman B.W., Jones I.M.

CoroNaViruses (CoVs) are enveloped, positive sense, single strand RNA viruses that cause respiratory, intestinal and neurological diseases in mammals and birds. Following replication, CoVs assemble on intracellular membranes including the endoplasmic reticulum Golgi intermediate compartment (ERGIC) where the envelope protein (E) functions in virus assembly and release. In consequence, E potentially contains membrane-modifying peptides. To search for such peptides, the E coding sequence of Mouse Hepatitis Virus (MHV) was inspected for its amino acid conservation, proximity to the membrane and/or predicted amphipathic helices. Peptides identified in silico were synthesized and tested for membrane-modifying activity in the presence of giant unilamellar vesicles (GUVs) consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), sphingomyelin and cholesterol. To confirm the presence of membrane binding peptides identified in the context of a full-length E protein, the wild type and a number of mutants in the putative membrane binding peptide were expressed in Lenti-X-293T mammalian and insect cells, and the distribution of E antigen within the expressing cell was assessed. Our data identify a role for the post-transmembrane region of MHV E in membrane binding.

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2020 – Exploring the Relationship between BODIPY Structure and Spectroscopic Properties to Design Fluorophores for Bioimaging
Vesicle Prep Pro Publication in Chemistry a European Journal (2020) Authors: Donnelly J.L., Offenbartl-Stiegert D., Marín-Beloqui J.M., Rizzello L., Battaglia G., Clarke T.M., Howorka S., Wilden J.D.

Designing chromophores for biological applications requires a fundamental understanding of how the chemical structure of a chromophore influences its photophysical properties. We here describe the synthesis of a library of BODIPY dyes, exploring diversity at various positions around the BODIPY core. The results show that the nature and position of substituents have a dramatic effect on the spectroscopic properties. Substituting in a heavy atom or adjusting the size and orientation of a conjugated system provides a means of altering the spectroscopic profiles with high precision. The insight from the structure–activity relationship was applied to devise a new BODIPY dye with rationally designed photochemical properties including absorption towards the near‐infrared region. The dye also exhibited switch‐on fluorescence to enable visualisation of cells with high signal‐to‐noise ratio without washing‐out of unbound dye. The BODIPY‐based probe is non‐cytotoxic and compatible with staining procedures including cell fixation and immunofluorescence microscopy.

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2020 – Facile Mixing of Phospholipids Promotes Self-Assembly of Low-Molecular-Weight Biodegradable Block Co-Polymers into Functional Vesicular Architectures
Vesicle Prep Pro Publication in Polymers (2020) Authors: Khan A., Ho J.C.S., Roy S., Bo Liedberg B., Nallani M.

In this work, we have used low-molecular-weight (PEG12-b-PCL6, PEG12-b-PCL9 or PEG16-b-PLA38; MW, 1.25-3.45 kDa) biodegradable block co-polymers to construct nano- and micron-scaled hybrid (polymer/lipid) vesicles, by solvent dispersion and electroformation methods, respectively. The hybrid vesicles exhibit physical properties (size, bilayer thickness and small molecule encapsulation) of a vesicular boundary, confirmed by cryogenic transmission electron microscopy, calcein leakage assay and dynamic light scattering. Importantly, we find that these low MW polymers, on their own, do not self-assemble into polymersomes at nano and micron scales. Using giant unilamellar vesicles (GUVs) model, their surface topographies are homogeneous, independent of cholesterol, suggesting more energetically favorable mixing of lipid and polymer. Despite this mixed topography with a bilayer thickness similar to that of a lipid bilayer, variation in surface topology is demonstrated using the interfacial sensitive phospholipase A2 (sPLA2). The biodegradable hybrid vesicles are less sensitive to the phospholipase digestion, reminiscent of PEGylated vesicles, and the degree of sensitivity is polymer-dependent, implying that the nano-scale surface topology can further be tuned by its chemical composition. Our results reveal and emphasize the role of phospholipids in promoting low MW polymers for spontaneous vesicular self-assembly, generating a functional hybrid lipid-polymer interface.

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2020 – Destructing the Plasma Membrane with Activatable Vesicular DNA Nanopores
Vesicle Prep Pro Publication in ACS Appl. Mater. Interfaces (2020) Authors: Chen L, Liang S., Chen Y., Wu M., Zhang Y.

Pore-forming proteins are an agent for attack or defense in various organisms, and its cytolytic activity has medical potential in cancer therapy. Despite recent advances in mimicking these proteins by amphipathic DNA nanopores, it remains inefficient to incorporate them into lipid bilayers. Here, we present the development of vesicular DNA nanopores that can controllably open a lipid membrane. Different from previously reported DNA nanopores that randomly insert into the planar bilayers, we design on-command fusogenic liposome-incorporated transmembrane DNA nanopores (FLIPs) that bypass the direct insertion process. By steric deshielding of fusogenic liposomal supports under low pH conditions, the embedded FLIPs are transferred and perforate lipid bilayers. We find that FLIPs depolarize the plasma membrane and thereby induce pyroptosis-like cell death. We further demonstrate the use of FLIPs to inhibit tumor growth in murine tumor models, which provides a new route to cancer nanotherapy. 

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2020 – Effect of DMSO on the Mechanical and Structural Properties of Model and Biological Membranes
Vesicle Prep Pro Publication in the Biophysical Journal (2020) Authors: Gironi B., Kahveci Z., McGill B., Lechner B.D., Pagliara S., Metz J., Morresi A., Palombo F., Sassi P., Petrov P.G.

Dimethyl sulfoxide (DMSO) is widely used in a number of biological and biotechnological applications, mainly because of its effects on the cell plasma membrane, but the molecular origins of this action are yet to be fully clarified. In this work, we used two- and three-component synthetic membranes (liposomes) and the plasma membrane of human erythrocytes to investigate the effect of DMSO when added to the membrane-solvating environment. Fourier transform infrared spectroscopy and thermal fluctuation spectroscopy revealed significant differences in the response of the two types of liposome systems to DMSO in terms of the bilayer thermotropic behavior, available free volume of the bilayer, its excess surface area, and bending elasticity. DMSO also alters the mechanical properties of the erythrocyte membrane in a concentration-dependent manner and is capable of increasing membrane permeability to ATP at even relatively low concentrations (3% v/v and above). Taken in its entirety, these results show that DMSO is likely to have a differential effect on heterogeneous biological membranes, depending on their local composition and structure, and could affect membrane-hosted biological functions.

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2020 – Design and Assembly of Membrane-Spanning DNA Nanopores
Vesicle Prep Pro Publication in Nanopore Technology (2020) Authors: Göpfrich K., Ohmann A., Keyser U.F.

Versatile lipid membrane-inserting nanopores have been made by functionalizing DNA nanostructures with hydrophobic tags. Here, we outline design and considerations to obtain DNA nanopores with the desired dimensions and conductance properties. We further provide guidance on their reconstitution into lipid membranes.

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2020 – Design of Protein Logic Gate System Operating on Lipid Membranes
Vesicle Prep Pro Publication in ACS Synth. Biol. (2020) Authors: Omersa N., Aden S., Kisovec M., Podobnik M., Anderluh G.

Lipid membranes are becoming increasingly popular in synthetic biology due to their biophysical properties and crucial role in communication between different compartments. Several alluring protein–membrane sensors have already been developed, whereas protein logic gates designs on membrane-embedded proteins are very limited. Here we demonstrate the construction of a two-level protein–membrane logic gate with an OR-AND logic. The system consists of an engineered pH-dependent pore-forming protein listeriolysin O and its DARPin-based inhibitor, conjugated to a lipid vesicle membrane. The gate responds to low pH and removal of the inhibitor from the membrane either by switching to a reducing environment, protease cleavage, or any other signal depending on the conjugation chemistry used for inhibitor attachment to the membrane. This unique protein logic gate vesicle system advances generic sensing and actuator platforms used in synthetic biology and could be utilized in drug delivery. 

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2020 – Cholesterol and desmosterol incorporation into ram sperm membrane before cryopreservation: Effects on membrane biophysical properties and sperm quality
Vesicle Prep Pro Publication in Biochimica et Biophysica Acta (BBA) - Biomembranes (2020) Authors: de las Mercedes Carro M., Peñalva D.A., Antollini S.S., Hozbora F.A., Buschiazzo J.

Ram sperm are particularly sensitive to freeze-thawing mainly due to their lipid composition, limiting their use in artificial insemination programs. We evaluated the extent of cholesterol and desmosterol incorporation into ram sperm through incubation with increasing concentrations of methyl-β-cyclodextrin (MβCD)-sterol complexes, and its effect on membrane biophysical properties, membrane lateral organization and cryopreservation outcome. Sterols were effectively incorporated into the sperm membrane at 10 and 25 mM MβCD-sterols, similarly increasing membrane lipid order at physiological temperature and during temperature decrease. Differential ordering effect of sterols in ternary-mixture model membranes revealed a reduced tendency of desmosterol of segregating into ordered domains. Live cell imaging of fluorescent cholesterol showed sterol incorporation and evidenced the presence of sperm sub-populations compatible with different sterol contents and a high concentration of sterol rich-ordered domains mainly at the acrosome plasma membrane. Lateral organization of the plasma membrane, assessed by identification of GM1-related rafts, was preserved after sterol incorporation except when high levels of sterols (25 mM MβCD-desmosterol) were incorporated. Ram sperm incubation with 10 mM MβCD-sterols prior to cryopreservation in a cholesterol-free extender improved sperm quality parameters after cooling and freezing. While treatment with 10 mM MβCD-cholesterol increased sperm motility, membrane integrity and tolerance to osmotic stress after thawing, incorporation of desmosterol increased the ability of ram sperm to overcome osmotic stress. Our research provides evidence on the effective incorporation and biophysical behavior of cholesterol and desmosterol in ram sperm membranes and on their consequences in improving functional parameters of sperm after temperature decrease and freezing.

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2020 – Coating and Stabilization of Liposomes by Clathrin-Inspired DNA Self-Assembly
Vesicle Prep Pro Publication found in ACS Nano (2020) Authors: Baumann, K.N., Piantanida L., García-Nafría J., Sobota D., Voïtchovsky K., Knowles T.P.J., Hernández-Ainsa S.

The self-assembly of the protein clathrin on biological membranes facilitates essential processes of endocytosis and has provided a source of inspiration for materials design by the highly ordered structural appearance. By mimicking the architecture of the protein building blocks and clathrin self-assemblies to coat liposomes with biomaterials,advanced hybrid carriers can be derived. Here, we present a method for fabricating DNA-coated liposomes by hydrophobically anchoring and subsequently connecting DNA-based triskelion structures on the liposome surface inspired by the assembly of the protein clathrin. Dynamic light scattering, ζ-potential, confocal microscopy, and cryo-electron microscopy measurements independently demonstrate successful DNA coating. Nanomechanical measurements conducted with atomic force microscopy show that the DNA coating enhances the mechanical stability of the liposomes relative to uncoated ones. Furthermore, we provide the possibility to reverse the coating process by triggering the disassembly of the DNA coats through a toehold-mediated displacement reaction. Our results describe a straightforward, versatile, and reversible approach for coating and stabilizing lipid vesicles through the assembly of rationally designed DNA structures. This method has potential for further development toward the ordered arrangement of tailored functionalities on the surface of liposomes and for applications as hybrid nanocarriers.

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2020 – Atg3 promotes Atg8 lipidation via altering lipid diffusion and rearrangement
Vesicle Prep Pro Publication in Protein Science (2020) Authors: Wang S., Li Y., Ma C.

Atg3‐catalyzed transferring of Atg8 to phosphatidylethanolamine (PE) in the phagophore membrane is essential for autophagy. Previous studies have demonstrated that this process requires Atg3 to interact with the phagophore membrane via its N‐terminal amphipathic helix. In this study, by using combined biochemical and biophysical approaches, our data showed that in addition to binding to the membranes, Atg3 attenuates lipid diffusion and enriches lipid molecules with smaller headgroup. Our data suggest that Atg3 promotes Atg8 lipidation via altering lipid diffusion and rearrangement.

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2020 – Characterization of lipid composition and diffusivity in OLA generated vesicles
Vesicle Prep Pro Publication in Biochimica et Biophysica Acta (BBA) - Biomembranes (2020) Authors: Schaich M., Sobota D., Sleath H., Cama J., Keyser U.F.

Giant Unilamellar Vesicles (GUVs) are a versatile tool in many branches of science, including biophysics and synthetic biology. Octanol-Assisted Liposome Assembly (OLA), a recently developed microfluidic technique enables the production and testing of GUVs within a single device under highly controlled experimental conditions. It is therefore gaining significant interest as a platform for use in drug discovery, the production of artificial cells and more generally for controlled studies of the properties of lipid membranes. In this work, we expand the capabilities of the OLA technique by forming GUVs of tunable binary lipid mixtures of DOPC, DOPG and DOPE. Using fluorescence recovery after photobleaching we investigated the lateral diffusion coefficients of lipids in OLA liposomes and found the expected values in the range of 1 μm2/s for the lipid systems tested. We studied the OLA derived GUVs under a range of conditions and compared the results with electroformed vesicles. Overall, we found the lateral diffusion coefficients of lipids in vesicles obtained with OLA to be quantitatively similar to those in vesicles obtained via traditional electroformation. Our results provide a quantitative biophysical validation of the quality of OLA derived GUVs, which will facilitate the wider use of this versatile platform.

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2019 – The Mechanisms of Action of Triindolylmethane Derivatives on Lipid Membranes
Vesicle Prep Pro Publication in Acta Naturae (2019) Authors: Efimova, S.S. Tertychnaya T.E., Lavrenov S.N., Ostroumova, O.S.

The effects of new synthetic antibacterial agents – tris(1-pentyl-1H-indol-3-yl)methylium chloride (LCTA-1975) and (1-(4-(dimethylamino)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1H-indol-3-yl)bis(1-propyl- 1H-indol-3-yl)methylium chloride (LCTA-2701 – on model lipid membranes were studied. The ability of the tested agents to form ion-conductive transmembrane pores, influence the electrical stability of lipid bilayers and the phase transition of membrane lipids, and cause the deformation and fusion of lipid vesicles was investigated. It was established that both compounds exert a strong detergent effect on model membranes. The results of differential scanning microcalorimetry and measuring of the threshold transmembrane voltage that caused membrane breakdown before and after adsorption of LCTA-1975 and LCTA-2701 indicated that both agents cause disordering of membrane lipids. Synergism of the uncoupling action of antibiotics and the alkaloid capsaicin on model lipid membranes was shown. The threshold concentration of the antibiotic that caused an increase in the ion permeability of the lipid bilayer depended on the membrane lipid composition. It was lower by an order of magnitude in the case of negatively charged lipid bilayers than for the uncharged membranes. This can be explained by the positive charge of the tested agents. At the same time, LCTA-2701 was characterized by greater efficiency than LCTA-1975. In addition to its detergent action, LCTA-2701 can induce ion-permeable transmembrane pores: step-like current fluctuations corresponding to the opening and closing of individual ion channels were observed. The difference in the mechanisms of action might be related to the structural features of the antibiotic molecules: in the LCTA-1975 molecule, all three substituents at the nitrogen atoms of the indole rings are identical and represent n-alkyl (pentyl) groups, while LCTA-2701 contains a maleimide group, along with two alkyl substituents (n-propyl). The obtained results might be relevant to our understanding of the mechanism of action of new antibacterial agents, explaining the difference in the selectivity of action of the tested agents on the target microorganisms and their toxicity to human cells. Model lipid membranes should be used in further studies of the trends in the modification and improvement of the structures of new antibacterial agents.

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2020 – Actuation of synthetic cells with proton gradients generated by light-harvesting E. coli
Vesicle Prep Pro prePublication in Research Square (2020) Authors: Jahnke K., Ritzmann N., Fichtler J., Nitschke A., Dreher Y., Abele T., Hofhaus G., Platzman I., Schröder R., Muller D., Spatz J., Göpfrich K.

Bottom-up and top-down approaches to synthetic biology each employ distinct methodologies with the common aim to harness new types of living systems. Both approaches, however, face their own challenges towards biotechnological and biomedical applications. Here, we realize a strategic merger to convert light into proton gradients for the actuation of synthetic cellular systems. We genetically engineer E. coli to overexpress the light-driven inward-directed proton pump xenorhodopsin and encapsulate them as organelle mimics in artificial cell-sized compartments. Exposing the compartments to light-dark cycles, we can reversibly switch the pH by almost one pH unit and employ these pH gradients to trigger the attachment of DNA structures to the compartment periphery. For this purpose, a DNA triplex motif serves as a nanomechanical switch responding to the pH-trigger of the E. coli. By attaching a polymerized DNA origami plate to the DNA triplex motif, we obtain a cytoskeleton mimic that considerably deforms lipid vesicles in a pH-responsive manner. We foresee that the combination of bottom-up and top down approaches is an efficient way to engineer synthetic cells as potent microreactors.

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2019 – Protein-free division of giant unilamellar vesicles controlled by enzymatic activity
Vesicle Prep Pro Pre-Publication in BioRxiv (2019) Authors: Dreher Y., Spatz J.P., Göpfrich K.

Cell division is one of the hallmarks of life. Success in the bottom-up assembly of synthetic cells will, no doubt, depend on strategies for the controlled autonomous division of protocellular compartments. Here, we describe the protein-free division of giant unilamellar lipid vesicles (GUVs) based on the combination of two physical principles – phase separation and osmosis. We visualize the division process with confocal fluorescence microscopy and derive a conceptual model based on the vesicle geometry. The model successfully predicts the shape transformations over time as well as the time point of the final pinching of the daughter vesicles. Remarkably, we show that two fundamentally distinct yet highly abundant processes – water evaporation and metabolic activity – can both regulate the autonomous division of GUVs. Our work may hint towards mechanisms that governed the division of protocells and adds to the strategic toolbox of bottom-up synthetic biology with its vision of bringing matter to life.

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2019 – Self‐Assembly of Giant Unilamellar Vesicles by Film Hydration Methodologies
Vesicle Prep Pro Publication in Advanced Biosystems (2019) Authors: Rideau E., Wurm F.R., Landfester K.

Self‐assembly of lipids or polymeric amphiphiles into vesicular structures has been achieved by various methods since the first generation of liposomes in the 1960s. Vesicles can be obtained with diameters from the nanometer to the micrometer regime. From the perspective of cell mimicking, vesicles with diameters of several micrometers are most relevant. These vesicles are called giant unilamellar vesicles (GUVs). Commonly used methods to form GUVs are solvent‐displacement techniques, especially since the development of microfluidics. These methodologies however, trap undesirable organic solvents in their membrane as well as other potentially undesired additives (surfactants, polyelectrolytes, polymers, etc.). In contrast to those strategies, summarized herein are solvent‐free approaches as suitable clean alternatives. The vesicles are formed from a dry thin layer of the lipid or amphiphilic polymers and are hydrated in aqueous media using the entropically favored self‐assembly of amphiphiles into GUVs. The rearrangement of the amphiphilic films into vesicular structures is usually aided by shear forces such as an alternative current (electroformation) or the swelling of water‐soluble polymeric supports (gel‐assisted hydration).

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2019 – Linker length in fluorophore–cholesterol conjugates directs phase selectivity and cellular localisation in GUVs and live cells
Vesicle Prep Pro Publication in RSC Advances (2019) Authors: O' Connor D., Byrnea A., Keyes T.E.

Lipid membrane fluorescent probes that are both domain-selective and compatible with demanding microscopy methods are crucial to elucidate the presence and function of rafts and domains in cells and biophysical models. Whereas targeting fluorescent probes to liquid-disordered (Ld) domains is relatively facile, it is far more difficult to direct probes with high selectivity to liquid-ordered (Lo) domains. Here, a simple, one-pot approach to probe–cholesterol conjugation is described using Steglich esterification to synthesise two identical BODIPY derivatives that differ only in the length of the aliphatic chain between the dye and cholesterol. In the first, BODIPY-Ar-Chol, the probe and cholesterol were directly ester linked and in the second BODIPY-Ahx-Chol, a hexyl linker separated probe from cholesterol. Uptake and distribution of each probe was compared in ternary, phase separated giant unilamellar vesicles (GUVs) using a commercial Ld marker as a reference. BODIPY-Ar-Chol targets almost exclusively the Ld domains with selectivity of >90% whereas by contrast introducing the C6 linker between the probe and cholesterol drove the probe to Lo with excellent selectivity (>80%). The profound impact of the linker length extended also to uptake and distribution in live mammalian cells. BODIPY-Ahx-Chol associates strongly with the plasma membrane where it partitioned preferably into opposing micron dimensioned do-mains to a commercial Ld marker and its concentration at the membrane was reduced by cyclodextrin treatment of the cells. By contrast the BODIPY-Ahx-Chol permeated the membrane and localised strongly to lipid droplets within the cell. The data demonstrates the profound influence of linker length in cholesterol bioconjugates in directing the probe.

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2019 – Mechanisms of Regulation of Amyloid-Induced Permeability of Model Lipid Membranes by Polyphenols
Vesicle Prep Pro Publication in Cell Tiss. Biol. (2019) Authors: Efimova, S.S. & Ostroumova, O.S.

This work is devoted to the study of the processes of formation and functioning of ion channels by amyloidogenic peptides, pathological aggregation and accumulation of which is a cause of neurodegenerative disorders. The effect of the plant polyphenols phloretin, butein, resveratrol, isoliquiritigenin, 4'-hydroxychalcone, and cardamonine on the pore-forming activity of β-amyloid peptide fragment 25–35 in bilayer lipid membranes from palmitoyl-phosphocholine was studied. It was demonstrated that the introduction of phloretin, butein or isoliquiritigenin in membrane-bathing solutions to a concentration of 20 µM leads to the increase of macroscopic transmembrane currents induced by peptide. At the same time, cardamonine, 4'-hydroxychalcone, and resveratrol have no effect on the activity of β-amyloid peptide fragment 25–35. The comparison of the results of studying the effect of tested polyphenols on electric and elastic properties of model membranes and pore-forming ability of β-amyloid peptide fragment 25–35 allowed it to concluded that there is no connection between the potentiating effect of phloretin, butein, or isoliquiritigenin and changes in the physicochemical properties of lipid bilayers. Results obtained by means of a confocal fluorescent microscopy indicate that the domain organization of the lipid bilayer may play a role in the pore-forming activity of amyloidogenic peptide. The results of electrophysiological measurements obtained for α-synuclein (another protein forming ion-permeable pores) do not contradict the hypothesis of binding of polyphenols, hydroxylated in the 7 position of the A cycle and in the 4'-position of the B cycle, with an open propane fragment with β-layers formed by amyloid peptides.

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2019 – Fluorescent Artificial Receptor-Based Membrane Assay (FARMA) for Spatiotemporally Resolved Monitoring of Biomembrane Permeability
Vesicle Prep Pro Pre-Publication in ChemRxiv (2019) Authors: Biedermann F., Ghale F., Hennig A., Nau W.M.

The spatiotemporally resolved monitoring of membrane translocation, e.g., of drugs or toxins, has been a long-standing goal. Herein, we introduce the fluorescent artificial receptor-based membrane assay (FARMA), a facile, label-free method. With FARMA, the permeation of more than hundred organic compounds (drugs, toxins, pesticides, neurotransmitters, peptides, etc.) through vesicular phospholipid bilayer membranes has been monitored in real time (µs-h time scale) and with high sensitivity (nM-µM concentration), affording permeability coefficients across an exceptionally large range from 10-9‑10-3 cm s-1. From a fundamental point of view, FARMA constitutes a powerful tool to assess structure-permeability relationships and to test biophysical models for membrane passage. From an applied perspective, FARMA can be extended to high-throughput screening by adaption of the microplate reader format, to spatial monitoring of membrane permeation by microscopy imaging, and to the compartmentalized monitoring of enzymatic activity.

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2019 – Hepatitis B virus X protein induces size-selective membrane permeabilization through interaction with cardiolipin
Vesicle Prep Pro Publication in Biochimica et Biophysica Acta (BBA) - Biomembranes (2019) Authors: You D.G., Cho Y.Y., Lee H.R., Lee J.H., Yu S.J., Yoon J.H., Yoo Y.D., Kim Y.J., Lee G.Y.

Hepatitis B virus X protein (HBx) functions in a variety of cellular events during the HBV life cycle. In a previous study, we reported that the HBx protein is sufficient to induce mitochondrial membrane permeabilization; however, the exact mechanism of HBx-induced mitochondrial membrane permeabilization has been not proposed. In this study, we report that HBx specifically targets cardiolipin (CL) and induces membrane permeabilization depending on CL concentration in mitochondrial outer membrane-mimic artificial liposomes. Interestingly, HBx-induced membrane permeabilization was enhanced by liposomes containing phosphatidylethanolamine, which plays a crucial role in forming a negative curvature on the membrane. We also show that the 68-117 region of HBx, which interacts with mitochondria, is necessary for membrane permeabilization. We examined the size of the pores formed by HBx and found that HBx permeates fluorescent dyes depending on the hydrodynamic diameter with a pore size of approximately 10 nm. The results of this study suggest that CL is necessary for HBx-induced membrane permeabilization and provide important information that suggests a new strategy for anti-HBV therapy.

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2019 – Barcoding biological reactions with DNA-functionalized vesicles
Vesicle Prep Pro Publication in Angew Chem Int Ed Engl. (2019) Authors: Peruzzi J.A., Jacobs, ML., Vu T.Q., Kamat N.P.

Targeted vesicle fusion is a promising approach to selectively control interactions between vesicle compartments and would enable the initiation of biological reactions in complex aqueous environments. Here, we explore how two features of vesicle membranes, DNA tethers and phase-segregated membranes, promote fusion between specific vesicle populations. We show that membrane phase-segregation provides an energetic driver for membrane fusion that increases the efficiency of DNA-mediated fusion events. Using this system, we show that orthogonality provided by DNA tethers allows us to direct fusion and delivery of DNA cargo to specific vesicle populations. We then demonstrate that vesicle fusion between DNA-tethered vesicles can be used to initiate in vitro protein expression that leads to the synthesis of model soluble and membrane proteins. The ability to engineer orthogonal fusion events between DNA-tethered vesicles will provide a new strategy to control the spatio-temporal dynamics of cell-free reactions, expanding opportunities to engineer artificial cellular systems.

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2019 – Cellular uptake of self-assembled phytantriol-based hexosomes is independent of major endocytic machineries
Vesicle Prep Pro Publication in Journal of Colloid Interface Science (2019) Authors: Rodrigues L., Schneider F. , Zhang X., Larsson E., Moodie L., Dietz H., Papadakis C., Winter G., Lundmark R., Hubert M.

Despite increasing interests in non-lamellar liquid crystalline dispersions, such as hexosomes, for drug delivery, little is known about their interactions with cells and mechanism of cell entry. Here we examine the cellular uptake of hexosomes based on phytantriol and mannide monooleate by HeLa cells using live cell microscopy in comparison to conventional liposomes. To investigate the importance of specific endocytosis pathways upon particle internalization, we silenced regulatory proteins of major endocytosis pathways using short interfering RNA. While endocytosis plays a significant role in liposome internalization, hexosomes are not taken up via endocytosis but through a mechanism that is dependent on cell membrane tension. Biophysical studies using biomembrane models highlighted that hexosomes have a high affinity for membranes and an ability to disrupt lipid layers. Our data suggest that direct biomechanical interactions of hexosomes with membrane lipids play a crucial role and that the unique morphology of hexosomes is vital for their membrane activity. Based on these results, we propose a mechanism, where hexosomes destabilize the bilayer, allowing them to “phase through” the membrane. Understanding parameters that influence the uptake of hexosomes is critical to establish them as carrier systems that can potentially deliver therapeutics efficiently to intracellular sites of action.

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2019 – A trifunctional linker for palmitoylation and peptide and protein localization in biological membranes
Vesicle Prep Pro Publication in ChemBioChem (2019) Authors: Syga L., De Vries R.H., van Oosterhout H., Bartelds R., Boersma A.J., Roelfes G., Poolman B.

Attachment of lipophilic groups is an important post‐translational modification of proteins, which involves the coupling of one or more anchors such as fatty acids, isoprenoids, phospholipids or glycosylphosphatidyl inositols. To study its impact on the membrane partitioning of hydrophobic peptides or proteins, we designed a tyrosine‐based trifunctional linker. The linker allows in a single step facile incorporation of two different functionalities at a cysteine. We determined the effect of the lipid modification on the membrane partitioning of the synthetic α‐helical model peptide WALP w/wo palmitoyl groups in giant unilamellar vesicles that contain a liquid‐ordered (Lo) and liquid‐disordered (Ld) phase. Introduction of two palmitoyl groups did not alter the localization of the membrane peptides, nor did the membrane thickness or lipid composition. In all cases, the peptide was retained in the Ld phase. These data demonstrate that the Lo domain in model membranes is highly unfavorable for a single membrane‐spanning peptide.

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2019 – An Intrinsically Disordered Region in OSBP Acts as an Entropic Barrier to Control Protein Dynamics and Orientation at Membrane Contact Sites
Vesicle Prep Pro Publication in Developmental Cell (2019) Authors: Jamecna D., Polidori J., Mesmin B., Dezi M., Levy D., Bigay J., Antonny B.

Lipid transfer proteins (LTPs) acting at membrane contact sites (MCS) between the ER and other organelles contain domains involved in heterotypic (e.g., ER to Golgi) membrane tethering as well as domains involved in lipid transfer. Here, we show that a long ≈90 aa intrinsically unfolded sequence at the N terminus of oxysterol-binding protein (OSBP) controls OSBP orientation and dynamics at MCS. This Gly-Pro-Ala-rich sequence, whose hydrodynamic radius is twice as that of folded domains, prevents the two PH domains of the OSBP dimer from homotypically tethering two Golgi-like membranes and considerably facilitates OSBP in-plane diffusion and recycling at MCS. Although quite distant in sequence, the N terminus of OSBP-related protein-4 (ORP4) has similar effects. We propose that N-terminal sequences of low complexity in ORPs form an entropic barrier that restrains protein orientation, limits protein density, and facilitates protein mobility in the narrow and crowded MCS environment.

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2018 – Visualizing Tension and Growth in Model Membranes Using Optical Dyes
Vesicle Prep Pro Publication in Biophysical Journal (2018) Authors: Boyd M.A., Kamat N.P.

Cells dynamically regulate their membrane surface area during a variety of processes critical to their survival. Recent studies with model membranes have pointed to a general mechanism for surface area regulation under tension in which cell membranes unfold or take up lipid to accommodate membrane strain. Yet we lack robust methods to simultaneously measure membrane tension and surface area changes in real time. Using lipid vesicles that contain two dyes isolated to spatially distinct parts of the membrane, we introduce, to our knowledge, a new method to monitor the processes of membrane stretching and lipid uptake in model membranes. Laurdan, located within the bilayer membrane, and Förster resonance energy transfer dyes, localized to the membrane exterior, act in concert to report changes in membrane tension and lipid uptake during osmotic stress. We use these dyes to show that membranes under tension take up lipid more quickly and in greater amounts compared to their nontensed counterparts. Finally, we show that this technique is compatible with microscopy, enabling real-time analysis of membrane dynamics on a single vesicle level. Ultimately, the combinatorial use of these probes offers a more complete picture of changing membrane morphology. Our optical method allows us to remotely track changes in membrane tension and surface area with model membranes, offering new opportunities to track morphological changes in artificial and biological membranes and providing new opportunities in fields ranging from mechanobiology to drug delivery.

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2019 – A Fusion Peptide in the Spike Protein of MERS CoroNaVirus
Vesicle Prep Pro Publication in Viruses (2019) Authors: Alsaadi E.A.J., Neuman B.W., and Jones I.M.

CoroNaViruses represent current and emerging threats for many species, including humans. Middle East respiratory syndrome-related coroNaVirus (MERS-CoV) is responsible for sporadic infections in mostly Middle Eastern countries, with occasional transfer elsewhere. A key step in the MERS-CoV replication cycle is the fusion of the virus and host cell membranes mediated by the virus spike protein, S. The location of the fusion peptide within the MERS S protein has not been precisely mapped. We used isolated peptides and giant unilamellar vesicles (GUV) to demonstrate membrane binding for a peptide located near the N-terminus of the S2 domain. Key residues required for activity were mapped by amino acid replacement and their relevance in vitro tested by their introduction into recombinant MERS S protein expressed in mammalian cells. Mutations preventing membrane binding in vitro also abolished S-mediated syncytium formation consistent with the identified peptide acting as the fusion peptide for the S protein of MERS-CoV.

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2018 – Probing Interactions between AuNPs/AgNPs and Giant Unilamellar Vesicles (GUVs) Using Hyperspectral Dark-field Microscopy
Vesicle Prep Pro Publication in the International Journal of Molecular Sciences (2018) Authors: Bhat A., Huan K., Cooks T., Boukari H., Lu Q.

Noble metallic nanoparticles (NPs) such as gold and silver nanoparticles (AuNPs and AgNPs) have been shown to exhibit anti-tumor effect in anti-angiogenesis, photothermal and radio therapeutics. On the other hand, cell membranes are critical locales for specific targeting of cancerous cells. Therefore, NP-membrane interactions need be studied at molecular level to help better understand the underlying physicochemical mechanisms for future applications in cancer nanotechnology. Herein, we report our study on the interactions between citrate stabilized colloidal AuNPs/AgNPs (10 nm in size) and giant unilamellar vesicles (GUVs) using hyperspectral dark-field microscopy. GUVs are large model vesicle systems well established for the study of membrane dynamics. GUVs used in this study were prepared with dimyristoyl phosphatidylcholine (DMPC) and doped with cholesterol at various molar concentrations. Both imaging and spectral results support that AuNPs and AgNPs interact very differently with GUVs, i.e., AuNPs tend to integrate in between the lipid bilayer and form a uniform golden-brown crust on vesicles, whereas AgNPs are bejeweled on the vesicle surface as isolated particles or clusters with much varied configurations. The more disruptive capability of AuNPs is hypothesized to be responsible for the formation of golden brown crusts in AuNP-GUV interaction. GUVs of 20 mol% CHOL:DMPC were found to be a most economical concentration for GUVs to achieve the best integrity and the least permeability, consistent with the finding from other phase studies of lipid mixture that the liquid-ordered domains have the largest area fraction of the entire membrane at around 20 mol% of cholesterol.

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2018 – Regulation of the Pore-Forming Activity of Cecropin A by Local Anesthetics
Vesicle Prep Pro Publication in Cell and Tissue Biology (2018) Authors: Efimova S.S., Medvedev R.Ya., Chulkov E.G., Schagina L.V., Ostroumova O.S.

The influence of local anesthetics on the regulation of the channel-forming activity of the antimicrobial peptide cecropin A has been investigated. The mean current flowing through the single cecropin channels isc was determined, and steady-state transmembrane current induced by cecropin AI∞ was measured. It has been shown that the introduction of 1 mM of bupivacaine, benzocaine or 0.3 mM of tetracaine into the membrane bathing solution results in a decrease in isc and I∞. At the same time, the addition of 1 mM lidocaine or procaine to the membrane-bathing solutions does not lead to a significant change in isc and I∞. Comparison of the absolute values and the sign of the change in the boundary potential of negatively charged membranes and relative changes of isc and I∞ after addition of local anesthetics shows that neither parameter correlates with the membrane boundary potential. The results of studying the effect of tested local anesthetics on the phase transition of membrane lipids allow us to conclude that the observed changes of isc and I∞ are due to modulation of the elastic properties of the membrane.

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2018 – Methods for Single-Molecule Sensing and Detection Using Bacteriophage Phi29 DNA Packaging Motor
Vesicle Prep Pro book chapter in Molecular Motors (2018) Authors: Haque F, Zhang H, Wang S, Chang C-L, Savran C, Guo P.

Bacteriophage phi29 DNA packaging motor consists of a dodecameric portal channel protein complex termed connector that allows transportation of genomic dsDNA and a hexameric packaging RNA (pRNA) ring to gear the motor. The elegant design of the portal protein has facilitated its applications for real-time single-molecule detection of biopolymers and chemicals with high sensitivity and selectivity. The robust self-assembly property of the pRNA has enabled biophysical studies of the motor complex to determine the stoichiometry and structure/folding of the pRNA at single-molecule level. This chapter focuses on biophysical and analytical methods for studying the phi29 motor components at the single-molecule level, such as single channel conductance assays of membrane-embedded connectors; single molecule photobleaching (SMPB) assay for determining the stoichiometry of phi29 motor components; fluorescence resonance energy transfer (FRET) assay for determining the structure and folding of pRNA; atomic force microscopy (AFM) for imaging pRNA nanoparticles of various size, shape, and stoichiometry; and bright-field microscopy with magnetomechanical system for direct visualization of viral DNA packaging process. The phi29 system with explicit engineering capability has incredible potentials for diverse applications in nanotechnology and nanomedicine including, but not limited to, DNA sequencing, drug delivery to diseased cells, environmental surveillance, and early disease diagnosis.

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2018 – Phase partitioning, solvent-switchable BODIPY probes for high contrast cellular imaging and FCS
Vesicle Prep Pro Publication in New Journal of Chemistry (2018) Authors: O’Connor D. Byrne A., Dolana C., Keyes T.E.

Lipophilic BODIPY fluorphores, in which the BODIPY core bears pendant dipyrido[3,2-a:2′,3′-c]phenazine (Dppz) or naphthyridyl and cholesterol substituents were designed and prepared as lipid probes for both liposomes and live cell imaging. The probes are non-emissive in water but permeate both GUV and live cell membranes and provide high contrast fluorescence and lifetime imaging of membranous structures and lipid droplets in cells and are suitable for FCS measurements on lipid membranes.

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2018 – Lipid-mediated regulation of pore-forming activity of syringomycin E by thyroid hormones and xanthene dyes
Vesicle Prep Pro Publication in Biochimica et Biophysica Acta (BBA) - Biomembranes (2018) Authors: Efimova S.S., Zakharova A.A., Ismagilov A.A., Schagina L.V., Malev V.V., Bashkirov P.V., Ostroumova O.S.

The effects of dipole modifiers, thyroid hormones (thyroxine and triiodothyronine) and xanthene dyes (Rose Bengal, phloxineB, erythrosin, eosinY and fluorescein) on the pore-forming activity of the lipopeptide syringomycin E (SRE) produced by Pseudomonas syringae were studied in a model bilayer. Thyroxine does not noticeably influence the steady-state number of open SRE channels (Nop), whereas triiodothyronine decreases it 10-fold at − 50 mV. Rose Bengal, phloxine B and erythrosin significantly increase Nop by 350, 100 and 70 times, respectively. Eosin Y and fluorescein do not practically affect the pore-forming activity of SRE. Recently, we showed that hormones decrease the dipole potential of lipid bilayers by approximately 60 mV at 50 μM, while Rose Bengal, phloxine B and erythrosin at 2.5 μM reduce the membrane dipole potential by 120, 80 and 50 mV, respectively. In the present study using differential scanning microcalorimetry, confocal fluorescence microscopy, the calcein release technique and measurements of membrane curvature elasticity, we show that triiodothyronine strongly affects the fluidity of model membranes: its addition leads to a significant decrease in the temperature and cooperativity of the main phase transition of DPPC, calcein leakage from DOPC vesicles, fluidization of solid domains in DOPC/DPPC liposomes, and promotion of lipid curvature stress. Thyroxine exerts a weaker effect. Xanthene dyes do not influence the phase transition of DPPC. Despite the decrease in the dipole potential, thyroid hormones modulate SRE channels predominantly via the elastic properties of the membrane, whereas the xanthene dyes Rose Bengal, phloxine B and erythrosine affect SRE channels via bilayer electrostatics.

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2018 – Method for immobilization of living and synthetic cells for high-resolution imaging and single-particle tracking
Vesicle Prep Pro Publication in Scientific Reports (2018) Authors: Syga Ł., Spakman D., Punter C.M., Poolman B.

Super-resolution imaging and single-particle tracking require cells to be immobile as any movement reduces the resolution of the measurements. Here, we present a method based on APTES-glutaraldehyde coating of glass surfaces to immobilize cells without compromising their growth. Our method of immobilization is compatible with Saccharomyces cerevisiae, Escherichia coli, and synthetic cells (here, giant-unilamellar vesicles). The method introduces minimal background fluorescence and is suitable for imaging of single particles at high resolution. With S. cerevisiae we benchmarked the method against the commonly used concaNaValin A approach. We show by total internal reflection fluorescence microscopy that modifying surfaces with ConA introduces artifacts close to the glass surface, which are not present when immobilizing with the APTES-glutaraldehyde method. We demonstrate validity of the method by measuring the diffusion of membrane proteins in yeast with single-particle tracking and of lipids in giant-unilamellar vesicles with fluorescence recovery after photobleaching. Importantly, the physical properties and shape of the fragile GUVs are not affected upon binding to APTES-glutaraldehyde coated glass. The APTES-glutaraldehyde is a generic method of immobilization that should work with any cell or synthetic system that has primary amines on the surface.

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2018 – High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope
Vesicle Prep Pro Publication in the Journal of Biophotonics (2018) Authors: Li Y., Montague S.J., Brüstle A., He X., Gillespie C., Gaus K., Gardiner E.E., Lee W.M.

In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2-fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub-diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 μm) within fluorescent giant unilamellar vesicles and micro-vascular dynamics after laser-induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological-imaging capacity of any polygon scanning microscope system.

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2018 – Hybrid vesicles from lipids and block copolymers: Phase behavior from the micro- to the nano-scale
Vesicle Prep Pro Publication in Colloids and Surfaces B: Biointerfaces (2018) Authors: Magnani C., Montis C., Mangiapia G., Mingotaud A.-F., Mingotaud C., Roux C., Joseph P., Berti D., Lonetti B.

In recent years, there has been a growing interest in the formation of copolymers-lipids hybrid self-assemblies, which allow combining and improving the main features of pure lipids-based and copolymer-based systems known for their potential applications in the biomedical field. In this contribution we investigate the self-assembly behavior of dipalmitoylphosphatidylcholine (DPPC) mixed with poly(butadiene-b-ethyleneoxide) (PBD-PEO), both at the micro- and at the nano-length scale. Epifluorescence microscopy and Laser Scanning Confocal microscopy are employed to characterize the morphology of micron-sized hybrid vesicles. The presence of fluid-like inhomogeneities in their membrane has been evidenced in all the investigated range of compositions. Furthermore, a microfluidic set-up characterizes the mechanical properties of the prepared assemblies by measuring their deformation upon flow: hybrids with low lipid content behave like pure polymer vesicles, whereas objects mainly composed of lipids show more variability from one vesicle to the other. Finally, the structure of the nanosized assemblies is characterized through a combination of Dynamic Light Scattering, Small Angle Neutron Scattering and Transmission Electron Microscopy. A vesicles-to-wormlike transition has been evidenced due to the intimate mixing of DPPC and PBD-PEO at the nanoscale. Combining experimental results at the micron and at the nanoscale improves the fundamental understanding on the phase behavior of copolymer-lipid hybrid assemblies, which is a necessary prerequisite to tailor efficient copolymer-lipid hybrid devices.

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2018 – Development of an on-chip antibiotic permeability assay with single molecule detection capability
Vesicle Prep Pro Publication in IEEE Transactions on NanoBioscience (2018) Authors: Guzel F.D., Citak F.

Electrophysiology is the method of choice to characterize membrane channels. In this study, we demonstrate a patch pipette based simple miniaturization that allows performing conductance measurements on a planar lipid bilayer in a microfluidic channel. Membrane proteins were reconstituted into Giant Unilamellar Vesicles (GUVs) by electroswelling, and GUVs with a single channel insertion were patched at the tip of pipette. We applied this approach to investigate the interactions of porins from E. coli with single antibiotics, and this will potentially provide information on the permeability rates. The results of this study suggest that this approach can be extended to the integration of several pipettes into the microfluidic channel from different positions, allowing the multiplexed recordings and also reducing the substrate consumption below μL volumes.

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2018 – Differential interactions of bacterial lipopolysaccharides with lipid membranes: implications for TRPA1-mediated chemosensation
Vesicle Prep Pro Publication in Scientific Reports (2018) Authors: Startek J.B., Talavera K., Voets T., Alpizar Y.A.

Bacterial lipopolysaccharides (LPS) activate the TRPA1 cation channels in sensory neurons, leading to acute pain and inflammation in mice and to aversive behaviors in fruit flies. However, the precise mechanisms underlying this effect remain elusive. Here we assessed the hypothesis that TRPA1 is activated by mechanical perturbations induced upon LPS insertion in the plasma membrane. We asked whether the effects of different LPS on TRPA1 relate to their ability to induce mechanical alterations in artificial and cellular membranes. We found that LPS from E. coli, but not from S. minnesota, activates TRPA1. We then assessed the effects of these LPS on lipid membranes using dyes whose fluorescence properties change upon alteration of the local lipid environment. E. coli LPS was more effective than S. minnesota LPS in shifting Laurdan’s emission spectrum towards lower wavelengths, increasing the fluorescence anisotropy of diphenylhexatriene and reducing the fluorescence intensity of merocyanine 540. These data indicate that E. coli LPS induces stronger changes in the local lipid environment than S. minnesota LPS, paralleling its distinct ability to activate TRPA1. Our findings indicate that LPS activate TRPA1 by producing mechanical perturbations in the plasma membrane and suggest that TRPA1-mediated chemosensation may result from primary mechanosensory mechanisms.

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2018 – Cargo induces retromer-mediated membrane remodeling on membranes
Vesicle Prep Pro Publication in ascb Molecular Biology of the Cell (2018) Authors: Purushothaman L.K., Ungermann C.

Endosomes serve as a central sorting station of lipids and proteins that arrive via vesicular carrier from the plasma membrane and the Golgi complex. At the endosome, retromer complexes sort selected receptors and membrane proteins into tubules or vesicles that bud off the endosome. The mature endosome finally fuses with the lysosome. Retromer complexes consist of a cargo selection complex (CSC) and a membrane remodeling part (SNX-BAR or Snx3 in yeast), and different assemblies of retromer mediate recycling of different cargoes. Due to this complexity, the exact order of events that results in carrier formation is not yet understood. Here, we reconstituted this process on giant unilamellar vesicles together with purified retromer complexes from yeast and selected cargoes. Our data reveal that the membrane remodeling activity of both Snx3 and the SNX-BAR complex is strongly reduced at low concentrations, which can be reactivated by CSC. At even lower concentrations, these complexes still associate with membranes, but only remodel membranes in the presence of their specific cargoes. Our data thus favor a simple model, where cargo functions as a specific trigger of retromer-mediated sorting on endosomes.

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2018 – Cholesterol Enriched Archaeosomes as a Molecular System for Studying Interactions of Cholesterol-Dependent Cytolysins with Membranes
Vesicle Prep Pro Publication in The Journal of Membrane Biology (2018) Authors: Rezelj S., Kozorog M, Švigelj T., Ulrih N.P., Žnidaršič N., Podobnik M., Anderluh G.

Archaeosomes are vesicles made of lipids from archaea. They possess many unique features in comparison to other lipid systems, with their high stability being the most prominent one, making them a promising system for biotechnological applications. Here, we report a preparation protocol of large unilamellar vesicles, giant unilamellar vesicles (GUVs), and nanodiscs from archaeal lipids with incorporated cholesterol. Incorporation of cholesterol led to additional increase in thermal stability of vesicles. Surface plasmon resonance, sedimentation assays, intrinsic tryptophan fluorescence measurements, calcein release experiments, and GUVs experiments showed that members of cholesterol-dependent cytolysins, listeriolysin O (LLO), and perfringolysin O (PFO), bind to cholesterol-rich archaeosomes and thereby retain their pore-forming activity. Interestingly, we observed specific binding of LLO, but not PFO, to archaeosomes even in the absence of cholesterol. This suggests a new capacity of LLO to bind to carbohydrate headgroups of archaeal lipids. Furthermore, we were able to express LLO inside GUVs by cell-free expression. GUVs made from archaeal lipids were highly stable, which could be beneficial for synthetic biology applications. In summary, our results describe novel model membrane systems for studying membrane interactions of proteins and their potential use in biotechnology.

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2018 – A synthetic enzyme built from DNA flips 10e7 lipids per second in biological membranes
Vesicle Prep Pro Publication in the Nature Communications (2018) Authors: Ohmann A, Li C-Y, Maffeo C, Al Nahas K, Baumann K.N, Göpfrich K, Yoo J, Keyser U.F, & Aksimentiev A.

Mimicking enzyme function and increasing performance of naturally evolved proteins is one of the most challenging and intriguing aims of nanoscience. Here, we employ DNA nanotechnology to design a synthetic enzyme that substantially outperforms its biological archetypes. Consisting of only eight strands, our DNA nanostructure spontaneously inserts into biological membranes by forming a toroidal pore that connects the membrane’s inner and outer leaflets. The membrane insertion catalyzes spontaneous transport of lipid molecules between the bilayer leaflets, rapidly equilibrating the lipid composition. Through a combination of microscopic simulations and fluorescence microscopy we find the lipid transport rate catalyzed by the DNA nanostructure exceeds 107 molecules per second, which is three orders of magnitude higher than the rate of lipid transport catalyzed by biological enzymes. Furthermore, we show that our DNA-based enzyme can control the composition of human cell membranes, which opens new avenues for applications of membrane-interacting DNA systems in medicine.

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2018 – Acyl chain asymmetry and polyunsaturation of brain phospholipids facilitate membrane vesiculation without leakage
Vesicle Prep Pro Publication in eLife (2018) Authors: Manni M.M., Tiberti M.L., Pagnotta S., Barelli H., Gautier R., Antonny B.

Phospholipid membranes form cellular barriers but need to be flexible enough to divide by fission. Phospholipids generally contain a saturated fatty acid (FA) at position sn1 whereas the sn2-FA is saturated, monounsaturated or polyunsaturated. Our understanding of the impact of phospholipid unsaturation on membrane flexibility and fission is fragmentary. Here, we provide a comprehensive view of the effects of the FA profile of phospholipids on membrane vesiculation by dynamin and endophilin. Coupled to simulations, this analysis indicates that: (i) phospholipids with two polyunsaturated FAs make membranes prone to vesiculation but highly permeable; (ii) asymmetric sn1-saturated-sn2-polyunsaturated phospholipids provide a tradeoff between efficient membrane vesiculation and low membrane permeability; (iii) When incorporated into phospholipids, docosahexaenoic acid (DHA; omega-3) makes membranes more deformable than arachidonic acid (omega-6). These results suggest an explanation for the abundance of sn1-saturated-sn2-DHA phospholipids in synaptic membranes and for the importance of the omega-6/omega-3 ratio on neuronal functions.

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2017 – Lipid phase separation in the presence of hydrocarbons in giant unilamellar vesicles
Vesicle Prep Pro Publication in AIMS Biophysics (2017) Authors: Bartelds R., Barnoud J., Boersma A.J., Marrink S.J., Poolman B.

Hydrophobic hydrocarbons are absorbed by cell membranes. The effects of hydrocarbons on biological membranes have been studied extensively, but less is known how these compounds affect lipid phase separation. Here, we show that pyrene and pyrene-like hydrocarbons can dissipate lipid domains in phase separating giant unilamellar vesicles at room temperature. In contrast, related aromatic compounds left the phase separation intact, even at high concentration. We hypothesize that this behavior is because pyrene and related compounds lack preference for either the liquid-ordered (Lo) or liquid-disordered (Ld) phase, while larger molecules prefer Lo, and smaller, less hydrophobic molecules prefer Ld. In addition, our data suggest that localization in the bilayer (depth) and the shape of the molecules might contribute to the effects of the aromatic compounds. Localization and shape of pyrene and related compounds are similar to cholesterol and therefore these molecules could behave as such.

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2018 – A direct role for hepatitis B virus X protein in inducing mitochondrial membrane permeabilization
Vesicle Prep Pro Publication in Journal of Viral Hepatitis (2018) Authors: Lee H‐R., Cho Y.Y., Lee G.Y., You D‐g., Yoo Y.D., Kim Y.J.

Hepatitis B virus X protein (HBx) acts as a multifunctional protein that regulates intracellular signalling pathways during HBV infection. It has mainly been studied in terms of its interaction with cellular proteins. Here, we show that HBx induces membrane permeabilization independently of the mitochondrial permeability transition pore complex. We generated mitochondrial outer membrane‐mimic liposomes to observe the direct effects of HBx on membranes. We found that HBx induced membrane permeabilization, and the region comprising the transmembrane domain and the mitochondrial‐targeting sequence was sufficient for this process. Membrane permeabilization was inhibited by nonselective channel blockers or by N‐(n‐nonyl)deoxynojirimycin (NN‐DNJ), a viroporin inhibitor. Moreover, NN‐DNJ inhibited HBx‐induced mitochondrial depolarization in Huh‐7 cells. Based on the results of this study, we can postulate that the HBx protein itself is sufficient to induce mitochondrial membrane permeabilization. Our finding provides important information for a strategy of HBx targeting during HBV treatment

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2017 – Dipole Modifiers Regulate Lipid Lateral Heterogeneity in Model Membranes
Vesicle Prep Pro Publication in Acta Naturae (2017) Authors: Efimova S.S., Ostroumova O.S.

In this study we report on experimental observations of giant unilamellar liposomes composed of ternary mixtures of cholesterol (Chol), phospholipids with relatively low Tmelt (DOPC, POPC, or DPoPC) and high Tmelt (sphingomyelin (SM), or tetramyristoyl cardiolipin (TMCL)) and their phase behaviors in the presence and absence of dipole modifiers. It was shown that the ratios of liposomes exhibiting noticeable phase separation decrease in the series POPC, DOPC, DPoPC regardless of any high-Tmelt lipid. Substitution of SM for TMCL led to increased lipid phase segregation. Taking into account the fact that the first and second cases corresponded to a reduction in the thickness of the lipid domains enriched in low- and high-Tmelt lipids, respectively, our findings indicate that the phase behavior depends on thickness mismatch between the ordered and disordered domains. The dipole modifiers, flavonoids and styrylpyridinium dyes, reduced the phase segregation of membranes composed of SM, Chol, and POPC (or DOPC). The other ternary lipid mixtures tested were not affected by the addition of dipole modifiers. It is suggested that dipole modifiers address the hydrophobic mismatch through fluidization of the ordered and disordered domains. The ability of a modifier to partition into the membrane and fluidize the domains was dictated by the hydrophobicity of modifier molecules, their geometric shape, and the packing density of domain-forming lipids. Phloretin, RH 421, and RH 237 proved the most potent among all the modifiers examined.

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2017 – Ion- and water-binding sites inside an occluded hourglass pore of a trimeric intracellular cation (TRIC) channel
Vesicle Prep Pro Publication in BioMed Central Biology (2017) Authors: Ou X., Guo J., Wang L., Yang H., Liu X., Sun J., Liu Z.

Background:Trimeric intracellular cation (TRIC) channels are crucial for Ca2+ handling in eukaryotes and are involved in K+ uptake in prokaryotes. Recent studies on the representative members of eukaryotic and prokaryotic TRIC channels demonstrated that they form homotrimeric units with the ion-conducting pores contained within each individual monomer.Results:Here we report detailed insights into the ion- and water-binding sites inside the pore of a TRIC channel from Sulfolobus solfataricus (SsTRIC). Like the mammalian TRIC channels, SsTRIC is permeable to both K+ and Na+ with a slight preference for K+, and is nearly impermeable to Ca2+, Mg2+, or Cl–. In the 2.2-Å resolution K+-bound structure of SsTRIC, ion/water densities have been well resolved inside the pore. At the central region, a filter-like structure is shaped by the kinks on the second and fifth transmembrane helices and two nearby phenylalanine residues. Below the filter, the cytoplasmic vestibule is occluded by a plug-like motif attached to an array of pore-lining charged residues.Conclusions:The asymmetric filter-like structure at the pore center of SsTRIC might serve as the basis for the channel to bind and select monovalent cations. A Velcro-like plug-pore interacting model has been proposed and suggests a unified framework accounting for the gating mechanisms of prokaryotic and eukaryotic TRIC channels.

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2013 – Lipid-Bilayer-Spanning DNA Nanopores with a Bifunctional Porphyrin Anchor
Vesicle Prep Pro Publication in Angewandte Chemie International Edition (2013) Authors: Burns J.R., Göpfrich K., Wood J.W.,Thacker V.V.,Stulz E., Keyser U.F., Howorka S.

An artificial membrane nanopore assembled from DNA oligonucleotides carries porphyrin tags, which anchor the nanostructure into the lipid bilayer. The porphyrin moieties also act as fluorescent dyes to aid the microscopic visualization of the DNA nanopore.

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2014 – Nanoscale phase behavior on flat and curved membranes
Vesicle Prep Pro Publication in Nanotechnology (2014) Authors: Andersen T., Bahadori A., Ott D., Kyrsting A., Reihani S.N.S., Bendix P.M.

The diverse physical properties of membranes play a critical role in many membrane associated biological processes. Proteins responsible for membrane transport can be affected by the lateral membrane order and lateral segregation of proteins is often controlled by the preference of certain membrane anchors for membrane phases having a physically ordered state. The dynamic properties of coexisting membrane phases are often studied by investigating their thermal behavior. Optical trapping of gold nanoparticles is a useful tool to generate local phase transitions in membranes. The high local temperatures surrounding an irradiated gold nanoparticle can be used to melt a part of a giant unilamellar lipid vesicle (GUV) which is then imaged using phase sensitive fluorophores embedded within the bilayer. By local melting of GUVs we reveal how a protein-free, one component lipid bilayer can mediate passive transport of fluorescent molecules by localized and transient pore formation. Also, we show how tubular membrane curvatures can be generated by optical pulling from the melted region on the GUV. This will allow us to measure the effect of membrane curvature on the phase transition temperature.

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2012 – Hydrophobic gating of mechanosensitive channel of large conductance evidenced by single-subunit resolution
Vesicle Prep Pro Publication in Proceedings of the National Academy of Sciences of the United States of America (2012) Authors: Birkner JP, Poolman B, Koçer A.

Mechanosensitive (MS) ion channels are membrane proteins that detect and respond to membrane tension in all branches of life. In bacteria, MS channels prevent cells from lysing upon sudden hypoosmotic shock by opening and releasing solutes and water. Despite the importance of MS channels and ongoing efforts to explain their functioning, the molecular mechanism of MS channel gating remains elusive and controversial. Here we report a method that allows single-subunit resolution for manipulating and monitoring “mechanosensitive channel of large conductance” from Escherichia coli. We gradually changed the hydrophobicity of the pore constriction in this homopentameric protein by modifying a critical pore residue one subunit at a time. Our experimental results suggest that both channel opening and closing are initiated by the transmembrane 1 helix of a single subunit and that the participation of each of the five identical subunits in the structural transitions between the closed and open states is asymmetrical. Such a minimal change in the pore environment seems ideal for a fast and energy-efficient response to changes in the membrane tension.

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2013 – Effect of flavonoids on the phase separation in giant unilamellar vesicles formed from binary lipid mixtures
Vesicle Prep Pro Publication in Chemistry and Physics of Lipids (2013) Authors: Ostroumova O.S., Chulkov E.G., Stepanenko O.V., Schagina L.V.

Confocal fluorescence microscopy have been employed to investigate phase separation in giant unilamellar vesicles prepared from binary mixtures of unsaturated dioleoylphosphocholine with saturated phosphocholines or brain sphingomyelin in the absence and presence of the flavonoids, biochanin A, phloretin, and myricetin. It has been demonstrated that biochanin A and phloretin make uncolored domains more circular or eliminate visible phase separation in liposomes while myricetin remains the irregular shape of fluorescence probe-excluding domains. Influence of the flavonoids on the endotherms of liposome suspension composed of dioleoylphosphocholine and dimyristoylphosphocholine was investigated by the differential scanning calorimetry. Calorimetry data do not contradict to confocal imaging results.

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2016 – Electrophysiological characterization of the archaeal transporter NCX_Mj using solid supported membrane technology
SURFE2R N1 and Vesicle Prep Pro Publication in Journal of General Physiology (2016) Authors: Barthmes M., Liao J., Jiang Y., Brüggemann A., Wahl-Schott C.

Sodium–calcium exchangers (NCXs) are membrane transporters that play an important role in Ca2+ homeostasis and Ca2+ signaling. The recent crystal structure of NCX_Mj, a member of the NCX family from the archaebacterium Methanococcus jannaschii, provided insight into the atomistic details of sodium–calcium exchange. Here, we extend these findings by providing detailed functional data on purified NCX_Mj using solid supported membrane (SSM)–based electrophysiology, a powerful but unexploited tool for functional studies of electrogenic transporter proteins. We show that NCX_Mj is highly selective for Na+, whereas Ca2+ can be replaced by Mg2+ and Sr2+ and that NCX_Mj can be inhibited by divalent ions, particularly Cd2+. By directly comparing the apparent affinities of Na+ and Ca2+ for NCX_Mj with those for human NCX1, we show excellent agreement, indicating a strong functional similarity between NCX_Mj and its eukaryotic isoforms. We also provide detailed instructions to facilitate the adaption of this method to other electrogenic transporter proteins. Our findings demonstrate that NCX_Mj can serve as a model for the NCX family and highlight several possible applications for SSM-based electrophysiology.

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2020 – Electrophysiological characterization of transport across outer membrane channels from Gram‐negative bacteria in presence of lipopolysaccharides (LPS)
Port-a-Patch, Vesicle Prep Pro and Orbit mini Publication in Angewandte Chemie International Edition (2020) Authors: Wang J., Terrasse R., Bafna J.A., Benier L., Winterhalter M.

Multi‐drug resistance in Gram‐negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics here, we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer, which moreover allows also characterization of membrane protein channels in their native environment. Two major membrane channels from E. coli , OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only a few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution method, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution and significantly lower permeation. We suggest using outer membrane vesicles as a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

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2019 – Structure of the human ClC-1 chloride channel
Port-a-Patch, Vesicle Prep Pro and Orbit mini Publication in PLOS Biology (2019) Authors: Wang K., Preisler, SS, Zhang, L., Cui, Y., Missel, JW., Grønberg C., Gotfryd, K., Lindahl E., Andersson, M., Calloe, K., Egea P.F., Klaerke D.A., Pusch M., Pedersen P.A., Zhou, Z.H., Gourdon, P.

ClC-1 protein channels facilitate rapid passage of chloride ions across cellular membranes, thereby orchestrating skeletal muscle excitability. Malfunction of ClC-1 is associated with myotonia congenita, a disease impairing muscle relaxation. Here, we present the cryo-electron microscopy (cryo-EM) structure of human ClC-1, uncovering an architecture reminiscent of that of bovine ClC-K and CLC transporters. The chloride conducting pathway exhibits distinct features, including a central glutamate residue (“fast gate”) known to confer voltage-dependence (a mechanistic feature not present in ClC-K), linked to a somewhat rearranged central tyrosine and a narrower aperture of the pore toward the extracellular vestibule. These characteristics agree with the lower chloride flux of ClC-1 compared with ClCK and enable us to propose a model for chloride passage in voltage-dependent CLC channels. Comparison of structures derived from protein studied in different experimental conditions supports the notion that pH and adenine nucleotides regulate ClC-1 through interactions between the so-called cystathionine-β-synthase (CBS) domains and the intracellular vestibule (“slow gating”). The structure also provides a framework for analysis of mutations causing myotonia congenita and reveals a striking correlation between mutated residues and the phenotypic effect on voltage gating, opening avenues for rational design of therapies against ClC-1–related diseases. Author summary Chloride transporting CLC proteins are expressed in a wide range of organisms, and the family encompasses several members with numerous roles in human health and disease by allowing movement of chloride ions across the membranes that encapsulate cells and cellular organelles. Structurally, CLCs form dimers possessing a separate ion translocation pathway in each monomer, and they can operate as either channels or transporters that exchange chloride for protons. The CLC channel ClC-1 is critical to skeletal muscle excitability and has been proposed as a target to alleviate neuromuscular disorders. Here, we have analyzed the structure of human ClC-1 and revealed the high similarity of its ion conducting pathway to those observed in other CLC members, including prokaryotic and algal transporters. Our data suggest how ClC-1 is regulated by environmental cues to allow opening and closure, thereby permitting attenuation of muscle function. Our results help with understanding the principal determinants that govern CLC proteins and may guide downstream translational applications to combat muscle pathologies.

Application Note PDF
OmpF – “Lipid Bilayer recordings of OmpF reconstituted in Proteoliposomes “
Port-a-Patch and Vesicle Prep Pro application note:  

Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) positioned by suction on the apertures of our patch clamp chips made from borosilicate glass substrate. Incubation of GUVs with purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation.

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KcsA – “Lipid bilayer recordings of KcsA channels reconstituted in proteoliposomes”
Port-a-Patch and Vesicle Prep Pro application note:  

Solvent-free planar lipid bilayers were formed in an automated manner using suction to attract a giant unilamellar vesicle (GUV) to the patch clamp chip which subsequently bursts across the aperture. Incubation of GUV's with purified KcsA channel protein yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. The rapid formation of protein-containing planar lipid bilayers is of potential use for the efficient electrophysiological characterization of KcsA as shown here and also other ion channel proteins of interest.

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MscL – “Lipid bilayer recordings of a mechanosensitive channel, MscL, using Nanion’s pressure clampchannel, MscL, using Nanion’s pressure clamp”
Port-a-Patch and Vesicle Prep Pro application note:  

Solvent-free planar lipid bilayers were formed in an automated manner using suction to attract a giant unilamellar vesicle (GUV) to the patch clamp chip which subsequently bursts across the aperture. Incubation of GUVs with purified MscL channel protein yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. The rapid formation of protein-containing planar lipid bilayers is of potential use for the efficient electrophysiological characterization of MscL as shown here and also other ion channel proteins of interest.In order to study the effect of pressure, the functional MscL purified was reconstituted in our system. The reconstitution was done in GUVs and then bilayers were formed on a chip (Kreir, Farre et al. 2008). The Port-a-Patch system has a pump controlled by a computer and could apply from +300 to -300 mBar and is controlled via software allowing accurate pressure control. All pressure applications could be visualized and recorded at the same time as the recordings.

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2022 – Temporin B Forms Hetero-Oligomers with Temporin L, Modifies Its Membrane Activity, and Increases the Cooperativity of Its Antibacterial Pharmacodynamic Profile
Port-a-Patch and Vesicle Prep Pro Publication in Biochemistry (2022)  Authors: Ferguson P.M., Clarke M., Manzo G., Hind C.K, Clifford M., Sutton J.M., Lorenz C.D., Phoenix D.A., Mason J.A.

The pharmacodynamic profile of antimicrobial peptides (AMPs) and their in vivo synergy are two factors that are thought to restrict resistance evolution and ensure their conservation. The frog Rana temporaria secretes a family of closely related AMPs, temporins A-L, as an effective chemical dermal defence. The antibacterial potency of temporin L has been shown to increase synergistically in combination with both temporins B and A but this is modest. Here we show that the less potent temporin B enhances the cooperativity of the in vitro antibacterial activity of the more potent temporin L against EMRSA-15 and that this may be associated with an altered interaction with the bacterial plasma membrane, a feature critical for the antibacterial activity of most AMPs. Addition of buforin II, a histone H2A fragment, can further increase the cooperativity. Molecular dynamics simulations indicate temporins B and L readily form hetero-oligomers in models of Gram-positive bacterial plasma membranes. Patch-clamp studies show transmembrane ion conductance is triggered with lower amounts of both peptides and more quickly, when used in combination, but conductance is of a lower amplitude and pores are smaller. Temporin B may therefore act by forming temporin L/B hetero-oligomers that are more effective than temporin L homo-oligomers at bacterial killing and/or by reducing the probability of the latter forming until a threshold concentration is reached. Exploration of the mechanism of synergy between AMPs isolated from the same organism may therefore yield antibiotic combinations with advantageous pharmacodynamic properties.

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2022 – The human TRPA1 intrinsic cold and heat sensitivity involves separate channel structures beyond the N-ARD domain
Vesicle Prep Pro and Port-a-Patch Publication in Nature Communications (2022) Authors: Moparthi L., Sinica V., Filipovic M., Vlachova V., Zygmunt P.M.

The human TRPA1 (hTRPA1) is an intrinsic thermosensitive ion channel responding to both cold and heat, depending on the redox environment. Here, we have studied purified hTRPA1 truncated proteins to gain further insight into the temperature gating of hTRPA1. We found in patch-clamp bilayer recordings that ∆1-688 hTRPA1, without the N-terminal ankyrin repeat domain (N-ARD), was more sensitive to cold and heat, whereas ∆1-854 hTRPA1 that is also lacking the S1-S4 voltage sensing-like domain (VSLD) gained sensitivity to cold but lost its heat sensitivity. The thiol reducing agent TCEP abolished the temperature sensitivity of both ∆1-688 hTRPA1 and ∆1-854 hTRPA1. Cold and heat activity of ∆1-688 hTRPA1 and ∆1-854 hTRPA1 were associated with different structural conformational changes as revealed by intrinsic tryptophan fluorescence measurements. Heat evoked major structural rearrangement of the VSLD as well as the C-terminus domain distal to the transmembrane pore domain S5-S6 (CTD), whereas cold only caused minor conformational changes. As shown for Δ1-854 hTRPA1, a sudden drop in tryptophan fluorescence occurred within 25-20°C indicating a transition between heat and cold conformations of the CTD, and thus it is proposed that the CTD contains a bidirectional temperature switch priming hTRPA1 for either cold or heat. In whole-cell patch clamp electrophysiology experiments, replacement of the cysteines 865, 1021 and 1025 with alanine modulated the cold sensitivity of hTRPA1 when heterologously expressed in HEK293T cells. It is proposed that the hTRPA1 CTD harbors cold and heat sensitive domains allosterically coupled to the S5-S6 pore region and the VSLD, respectively.

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2022 – Cannabinoid non-cannabidiol site modulation of TRPV2 structure and function
Port-a-Patch and Vesicle Prep Pro Publication in Nature Communications (2022) Authors: Zhang L., Simonsen C., Zimova L., Wang K., Moparthi L., Gaudet R., Ekoff M., Nilsson G., Hellmich U., Vlachova V., Gourdon P., Zygmunt P.

TRPV2 is a ligand-operated temperature sensor with poorly defined pharmacology. Here, we combine calcium imaging and patch-clamp electrophysiology with cryo-electron microscopy (cryo-EM) to explore how TRPV2 activity is modulated by the phytocannabinoid Δ9-tetrahydrocannabiorcol (C16) and by probenecid. C16 and probenecid act in concert to stimulate TRPV2 responses including histamine release from rat and human mast cells. Each ligand causes distinct conformational changes in TRPV2 as revealed by cryo-EM. Although the binding for probenecid remains elusive, C16 associates within the vanilloid pocket. As such, the C16 binding location is distinct from that of cannabidiol, partially overlapping with the binding site of the TRPV2 inhibitor piperlongumine. Taken together, we discover a new cannabinoid binding site in TRPV2 that is under the influence of allosteric control by probenecid. This molecular insight into ligand modulation enhances our understanding of TRPV2 in normal and pathophysiology.

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2022 – Recent advances in ion-channel probes for nanopore sensing: Insights into the probe architectures
Port-a-Patch and Vesicle Prep Pro Publication in Analytica Chimica Acta (2022) Authors: Hussein E.A., Rice B., White R.J.

This review introduces the recent advances in the nanopore sensing platform, ion channel probes (ICPs), with a particular focus on the different probe design (2011–2022). The use of ion channel proteins has emerged in different applications to understand the dynamics of many biological processes and characterize or detect biomolecules. The development of utilizing protein channels in nanopore sensing has led to diverse platforms in which the ion channels, or biological nanopores, can be embedded in a lipid membrane. Ion channel probes, where the ion channels are integrated at the tip of a solid probe, enable higher spatially-resolved detection of small molecules and extend the applications of ion channels to map different surfaces and perform chemical imaging. Different probe materials and designs have been exploited throughout the last decade, which opens the door for multiple probe architecture and applications. We provide more insights into the advances of ICP designs that render them well-suited for further applications.

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2022 – Balancing water solubility with membrane permeability in the design of a synthetic ionophore
Port-a-Patch and Vesicle Prep Pro Pre-Print in ChemRxiv (2022) Authors: Yang K., Boles J.E., White L.J., Hilton K.L.F., Lai H.Y., Long Y., Hiscock J.R., Haynes C.J.E.

Synthetic ionophores are promising therapeutic targets, yet current limitations associated with their lipophilicity and poor water solubility prevent the translation of this molecular technology into the clinic. In this work we report investigations into the cation transport ability of a series of antimicrobial supramolecular, self-associating amphiphiles (SSAs). We identify a member of this class of compounds to function as a K+ transporter in cooperative action with a known anionophore. This SSA is soluble in a range of organic solvents and in 100% water, retaining its transport activity when delivered from a purely aqueous solution – therefore overcoming current molecular delivery limitations. These findings shed light on a potential antimicrobial mechanism of action and inform the design of future therapeutic targets that can balance water solubility and membrane penetration

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2022 – Bolaamphiphile Analogues of 12-bis-THA Cl2 Are Potent Antimicrobial Therapeutics with Distinct Mechanisms of Action against Bacterial, Mycobacterial, and Fungal Pathogens
Port-a-Patch and Vesicle Prep Pro Publication in ASM Journals (2022)  Authors: Di Blasio S., Clarke M., Hind C., Asai M., Laurence L., Benvenuti A., Hassan M., Semenya D., Kwun-Wai D., Horrocks V., Manzo G., Van Der Lith S., Lam C., Gentile E., Annette C., Bosse J., Li Y., Panaretou B., Langford P., Robertson B., Lam J., Sutton J., McArthur M., Mason A.

12-Bis-THA Cl2 [12,12′-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride] is a cationic bolalipid adapted from dequalinium chloride (DQC), a bactericidal anti-infective indicated for bacterial vaginosis (BV). Here, we used a structure-activity-relationship study to show that the factors that determine effective killing of bacterial, fungal, and mycobacterial pathogens differ, to generate new analogues with a broader spectrum of activity, and to identify synergistic relationships, most notably with aminoglycosides against Acinetobacter baumannii and Pseudomonas aeruginosa, where the bactericidal killing rate was substantially increased. Like DQC, 12-bis-THA Cl2 and its analogues accumulate within bacteria and fungi. More hydrophobic analogues with larger headgroups show reduced potential for DNA binding but increased and broader spectrum antibacterial activity. In contrast, analogues with less bulky headgroups and stronger DNA binding affinity were more active against Candida spp. Shortening the interconnecting chain, from the most lipophilic twelve-carbon chain to six, improved the selectivity index against Mycobacterium tuberculosis in vitro, but only the longer chain analogue was therapeutic in a Galleria mellonella infection model, with the shorter chain analogue exacerbating the infection. In vivo therapy of Escherichia coli ATCC 25922 and epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) infections in Galleria mellonella was also achieved with longer-chain analogues, as was therapy for an A. baumannii 17978 burn wound infection with a synergistic combination of bolaamphiphile and gentamicin. The present study shows how this class of bolalipids may be adapted further to enable a wider range of potential applications.

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2020 – The structure of the antimicrobial human cathelicidin LL-37 shows oligomerization and channel formation in the presence of membrane mimics
Port-a-Patch and Vesicle Prep Pro Publication in Scientific Reports (2020) Authors: Sancho-Vaello E., Gil-Carton D., François P., Bonetti E.J., Kreir M., Pothula K.R., Kleinekathöfer U., Zeth K.

The human cathelicidin LL-37 serves a critical role in the innate immune system defending bacterial infections. LL-37 can interact with molecules of the cell wall and perforate cytoplasmic membranes resulting in bacterial cell death. To test the interactions of LL-37 and bacterial cell wall components we crystallized LL-37 in the presence of detergents and obtained the structure of a narrow tetrameric channel with a strongly charged core. The formation of a tetramer was further studied by cross-linking in the presence of detergents and lipids. Using planar lipid membranes a small but defined conductivity of this channel could be demonstrated. Molecular dynamic simulations underline the stability of this channel in membranes and demonstrate pathways for the passage of water molecules. Time lapse studies of E. coli cells treated with LL-37 show membrane discontinuities in the outer membrane followed by cell wall damage and cell death. Collectively, our results open a venue to the understanding of a novel AMP killing mechanism and allows the rational design of LL-37 derivatives with enhanced bactericidal activity.

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2021 – Norfluoxetine inhibits TREK-2 K2P channels by multiple mechanisms including state-independent effects on the selectivity filter gate
Port-a-Patch and Vesicle Prep Pro Publication in Journal of General Physiology (2021) Authors: Proks P., Schewe M., Conrad L.J., Rao S., Rathje S., Rödström K.E.J., Carpenter E.P., Baukrowitz T., Tucker S.J.

The TREK subfamily of two-pore domain K+ (K2P) channels are inhibited by fluoxetine and its metabolite, norfluoxetine (NFx). Although not the principal targets of this antidepressant, TREK channel inhibition by NFx has provided important insights into the conformational changes associated with channel gating and highlighted the role of the selectivity filter in this process. However, despite the availability of TREK-2 crystal structures with NFx bound, the precise mechanisms underlying NFx inhibition remain elusive. NFx has previously been proposed to be a state-dependent inhibitor, but its binding site suggests many possible ways in which this positively charged drug might inhibit channel activity. Here we show that NFx exerts multiple effects on single-channel behavior that influence both the open and closed states of the channel and that the channel can become highly activated by 2-APB while remaining in the down conformation. We also show that the inhibitory effects of NFx are unrelated to its positive charge but can be influenced by agonists which alter filter stability, such as ML335, as well as by an intrinsic voltage-dependent gating process within the filter. NFx therefore not only inhibits channel activity by altering the equilibrium between up and down conformations but also can directly influence filter gating. These results provide further insight into the complex allosteric mechanisms that modulate filter gating in TREK K2P channels and highlight the different ways in which filter gating can be regulated to permit polymodal regulation.

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2020 – Ionophore constructed from non-covalent assembly of a G-quadruplex and liponucleoside transports K+-ion across biological membranes
Port-a-Patch and Vesicle Prep Pro Publication in Nature Communications (2020)   Authors: Debnath M., Chakraborty S., Kumar Y.P., Chaudhuri R., Jana B., Dash J.

The selective transport of ions across cell membranes, controlled by membrane proteins, is critical for a living organism. DNA-based systems have emerged as promising artificial ion transporters. However, the development of stable and selective artificial ion transporters remains a formidable task. We herein delineate the construction of an artificial ionophore using a telomeric DNA G-quadruplex (h-TELO) and a lipophilic guanosine (MG). MG stabilizes h-TELO by non-covalent interactions and, along with the lipophilic side chain, promotes the insertion of h-TELO within the hydrophobic lipid membrane. Fluorescence assays, electrophysiology measurements and molecular dynamics simulations reveal that MG/h-TELO preferentially transports K+-ions in a stimuli-responsive manner. The preferential K+-ion transport is presumably due to conformational changes of the ionophore in response to different ions. Moreover, the ionophore transports K+-ions across CHO and K-562 cell membranes. This study may serve as a design principle to generate selective DNA-based artificial transporters for therapeutic applications.

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2020 – Purification and initial characterization of Plasmodium falciparum K+ channels, PfKch1 and PfKch2 produced in Saccharomyces cerevisiae
Vesicle Prep Pro and Port-a-Patch Publication in Microbial Cell Factories (2020) Authors: Molbaek K., Tejada M., Ricke C.H., Scharff-Poulsen P., ElleKVist P., Helix-Nielsen C., Kumar N., Klaerke D.A., Pedersen P.A.

Resistance towards known antimalarial drugs poses a significant problem, urging for novel drugs that target vital proteins in the malaria parasite Plasmodium falciparum. However, recombinant production of malaria proteins is notoriously difficult. To address this, we have investigated two putative K+channels, PfKch1 and PfKch2, identified in the P.falciparum genome. We show that PfKch1 and PfKch2 and a C-terminally truncated version of PfKch1 (PfKch11−1094)could indeed be functionally expressed in vivo, since a K+-uptake deficient Saccharomyces cerevisiae strain was complemented by the P. falciparum cDNAs. PfKch11−1094-GFP and GFP-PfKch2 fusion proteins were overexpressedin yeast, purified and reconstituted in lipid bilayers to determine their electrophysiological activity. Single channel conductance amounted to 16 ± 1 pS for PfKch11−1094-GFP and 28 ± 2 pS for GFP-PfKch2. We predicted regulator of K+-conductance (RCK) domains in the C-terminals of both channels, and we accordingly measured channel activity in the presence of Ca2+.

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2020 – Hepatitis C Virus p7 Induces Membrane Permeabilization by Interacting with Phosphatidylserine
Port-a-Patch and Vesicle Prep Pro Publication in International Journal of Molecular Sciences (2020) Authors: Lee H-R., Lee G.Y., You D-G., Kim H.K., Yoo Y.D.

Hepatitis C virus (HCV) p7 is known to be a nonselective cation channel for HCV maturation. Because the interaction of HCV proteins with host lipids in the endoplasmic reticulum membrane is crucial for the budding process, the identification of p7–lipid interactions could be important for understanding the HCV life cycle. Here, we report that p7 interacts with phosphatidylserine (PS) to induce membrane permeabilization. The interaction of p7 with PS was not inhibited by Gd3+ ions, which have been known to interact with negatively charged lipids, but channel activity and p7-induced mitochondrial depolarization were inhibited by Gd3+ ions. From the present results, we suggest that the p7–PS interaction plays an essential role in regulating its ion channel function and could be a potential molecular target for anti-HCV therapy.

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2020 – Human TRPA1 is an inherently mechanosensitive bilayer-gated ion channel
Port-a-Patch and Vesicle Prep Pro Publication in Cell Calcium (2020) Authors: Moparthi L., Zygmunt P.M.

The role of mammalian Transient Receptor Potential Ankyrin 1 (TRPA1) as a mechanosensor is controversial. Here, we report that purified human TRPA1 (hTRPA1) with and without its N-terminal ankyrin repeat domain responded with pressure-dependent single-channel current activity when reconstituted into artificial lipid bilayers. The hTRPA1 activity was abolished by the thiol reducing agent TCEP. Thus, depending on its redox state, hTRPA1 is an inherent mechanosensitive ion channel gated by force-from-lipids.

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2020 – Calcium activates purified human TRPA1 with and without its N-terminal ankyrin repeat domain in the absence of calmodulin
Port-a-Patch and Vesicle Prep Pro Publication in Cell Calcium (2020) Authors: Moparthi L., Moparthi S.B., Wenger J., Zygmunt P.M.

Extracellular influx of calcium or release of calcium from intracellular stores have been shown to activate mammalian TRPA1 as well as to sensitize and desensitize TRPA1 electrophilic activation. Calcium binding sites on both intracellular N- and C-termini have been proposed. Here, we demonstrate based on Förster resonance energy transfer (FRET) and bilayer patch-clamp studies, a direct calmodulin-independent action of calcium on the purified human TRPA1 (hTRPA1), causing structural changes and activation without immediate subsequent desensitization of hTRPA1 with and without its N-terminal ankyrin repeat domain (N-ARD). Thus, calcium alone activates hTRPA1 by a direct interaction with binding sites outside the N-ARD.

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2020 – Conformational equilibrium shift underlies altered K+ channel gating as revealed by NMR
Port-a-Patch and Vesicle Prep Pro Publication in Nature Communications (2020)   Authors: Iwahasi Y., Toyama Y., Imai S., Itoh H., Osawa M., Inoue M., Shimada I.

The potassium ion (K+) channel plays a fundamental role in controlling K+ permeation across the cell membrane and regulating cellular excitabilities. Mutations in the transmembrane pore reportedly affect the gating transitions of K+ channels, and are associated with the onset of neural disorders. However, due to the lack of structural and dynamic insights into the functions of K+ channels, the structural mechanism by which these mutations cause K+ channel dysfunctions remains elusive. Here, we used nuclear magnetic resonance spectroscopy to investigate the structural mechanism underlying the decreased K+-permeation caused by disease-related mutations, using the prokaryotic K+ channel KcsA. We demonstrated that the conformational equilibrium in the transmembrane region is shifted toward the non-conductive state with the closed intracellular K+-gate in the disease-related mutant. We also demonstrated that this equilibrium shift is attributable to the additional steric contacts in the open-conductive structure, which are evoked by the increased side-chain bulkiness of the residues lining the transmembrane helix. Our results suggest that the alteration in the conformational equilibrium of the intracellular K+-gate is one of the fundamental mechanisms underlying the dysfunctions of K+ channels caused by disease-related mutations.

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2019 – Temporin L and aurein 2.5 have identical conformations but subtly distinct membrane and antibacterial activities
Port-a-Patch and Vesicle Prep Pro Publication in Nature Scientific Reports (2019) Authors: Manzo G., Ferguson P.M., Hind C.H., Clifford M., Gustilo V.B., Ali H., Bansal S.S., Bui T.T., Drake A.F., Atkinson R.A., Sutton J.M., Lorenz C.D., Phoenix D.A., Mason A.J.

Frogs such as Rana temporaria and Litoria aurea secrete numerous closely related antimicrobial peptides (AMPs) as an effective chemical dermal defence. Damage or penetration of the bacterial plasma membrane is considered essential for AMP activity and such properties are commonly ascribed to their ability to form secondary amphipathic, α-helix conformations in membrane mimicking milieu. Nevertheless, despite the high similarity in physical properties and preference for adopting such conformations, the spectrum of activity and potency of AMPs often varies considerably. Hence distinguishing apparently similar AMPs according to their behaviour in, and effects on, model membranes will inform understanding of primary-sequence-specific antimicrobial mechanisms. Here we use a combination of molecular dynamics simulations, circular dichroism and patch-clamp to investigate the basis for differing anti-bacterial activities in representative AMPs from each species; temporin L and aurein 2.5. Despite adopting near identical, α-helix conformations in the steady-state in a variety of membrane models, these two AMPs can be distinguished both in vitro and in silico based on their dynamic interactions with model membranes, notably their differing conformational flexibility at the N-terminus, ability to form higher order aggregates and the characteristics of induced ion conductance. Taken together, these differences provide an explanation of the greater potency and broader antibacterial spectrum of activity of temporin L over aurein 2.5. Consequently, while the secondary amphipathic, α-helix conformation is a key determinant of the ability of a cationic AMP to penetrate and disrupt the bacterial plasma membrane, the exact mechanism, potency and spectrum of activity is determined by precise structural and dynamic contributions from specific residues in each AMP sequence.

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2020 – A pleurocidin analogue with greater conformational flexibility, enhanced antimicrobial potency and in vivo therapeutic efficacy
Vesicle Prep Pro and Port-a-Patch Publication in Communications Biology (2020) Authors: Manzo G., Hind C.K., Ferguson P.M., Amison R.T., Hodgson-Casson A.C., Ciazynska K.A., Weller B.J., Clarke M., Lam C., Man R.C.H., O'Shaughnessy B.G., Clifford M., Bui T.T., Drake A.F., Atkinson R.A., Lam J.K.W., Pitchford S.C., Page C.P., Phoenix D.A., Lorenz C.D., Sutton J.M., Mason A.J.

Antimicrobial peptides (AMPs) are a potential alternative to classical antibiotics that are yet to achieve a therapeutic breakthrough for treatment of systemic infections. The antibacterial potency of pleurocidin, an AMP from Winter Flounder, is linked to its ability to cross bacterial plasma membranes and seek intracellular targets while also causing membrane damage. Here we describe modification strategies that generate pleurocidin analogues with substantially improved, broad spectrum, antibacterial properties, which are effective in murine models of bacterial lung infection. Increasing peptide–lipid intermolecular hydrogen bonding capabilities enhances conformational flexibility, associated with membrane translocation, but also membrane damage and potency, most notably against Gram-positive bacteria. This negates their ability to metabolically adapt to the AMP threat. An analogue comprising D-amino acids was well tolerated at an intravenous dose of 15 mg/kg and similarly effective as vancomycin in reducing EMRSA-15 lung CFU. This highlights the therapeutic potential of systemically delivered, bactericidal AMPs.

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2019 – Minor sequence modifications in temporin B cause drastic changes in antibacterial potency and selectivity by fundamentally altering membrane activity
Port-a-Patch and Vesicle Prep Pro Publication in Nature Scientific Reports (2019)    Authors: Manzo G., Ferguson P.M., Gustilo V.B., Ali H., Bui T.T., Drake A.F., Atkinson R.A., Batoni G., Lorenz C.D., Phoenix D.A., Mason A.J.

Antimicrobial peptides (AMPs) are a potential source of new molecules to counter the increase in antimicrobial resistant infections but a better understanding of their properties is required for effective translation as therapeutics. Details of the mechanism of their interaction with the bacterial plasma membrane are desired since damage or penetration of this structure is considered essential for AMP activity. Relatively modest modifications to AMP primary sequence can induce substantial changes in potency and/or spectrum of activity but, hitherto, have not been predicted to substantially alter the mechanism of interaction with the bacterial plasma membrane. Here we use a combination of molecular dynamics simulations, circular dichroism, liquid- and solid-state NMR and patch clamp to investigate the extent to which temporin B and its analogues can be distinguished both in vitro and in silico on the basis of their interactions with model membranes. Enhancing the hydrophobicity of the N-terminus and cationicity of the C-terminus in temporin B improves its membrane activity and potency against both Gram-negative and Gram-positive bacteria. In contrast, enhancing the cationicity of the N-terminus abrogates its ability to trigger channel conductance and renders it ineffective against Staphylococcus aureus while nevertheless enhancing its potency against Escherichia coli. Our findings suggest even closely related AMPs may target the same bacterium with fundamentally differing mechanisms of action.

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2019 – Molecular understanding of calcium permeation through the open Orai channel
Port-a Patch and Vesicle Prep Pro Publication in PLOS Computational Biology (2019)    Authors: Liu X., Wu G., Yu Y., Chen X., Ji R., Lu J., Li X., Zhang X., Yang X., Shen Y.

The Orai channel is characterized by voltage independence, low conductance, and high Ca2+ selectivity and plays an important role in Ca2+ influx through the plasma membrane (PM). How the channel is activated and promotes Ca2+ permeation is not well understood. Here, we report the crystal structure and cryo-electron microscopy (cryo-EM) reconstruction of a Drosophila melanogaster Orai (dOrai) mutant (P288L) channel that is constitutively active according to electrophysiology. The open state of the Orai channel showed a hexameric assembly in which 6 transmembrane 1 (TM1) helices in the center form the ion-conducting pore, and 6 TM4 helices in the periphery form extended long helices. Orai channel activation requires conformational transduction from TM4 to TM1 and eventually causes the basic section of TM1 to twist outward. The wider pore on the cytosolic side aggregates anions to increase the potential gradient across the membrane and thus facilitate Ca2+ permeation. The open-state structure of the Orai channel offers insights into channel assembly, channel activation, and Ca2+ permeation.

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2018 – Single channel recording of a mitochondrial calcium uniporter
Port-a-Patch and Vesicle Prep Pro Publication in BBRC (2018)   Authors: Wu G., Li S., Zong G., Liu, X., Fei S., Shen L., Guan X., Yanga X., Shen Y.

Mitochondrial calcium uniporter (MCU) is the pore-forming subunit of the entire uniporter complex and plays an important role in mitochondrial calcium uptake. However, the single channel recording of MCU remains controversial. Here, we expressed and purified different MCU proteins and then reconstituted them into planar lipid bilayers for single channel recording. We showed that MCU alone from Pyronema omphalodes (pMCU) is active with prominent single channel Ca2+ currents. In sharp contrast, MCU alone from Homo sapiens (hMCU) is inactive. The essential MCU regulator (EMRE) activates hMCU, and therefore, the complex (hMCU-hEMRE) shows prominent single channel Ca2+ currents. These single channel currents are sensitive to the specific MCU inhibitor Ruthenium Red. Our results clearly demonstrate that active MCU can conduct large amounts of calcium into the mitochondria.

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2019 – Golgi anti-apoptotic proteins are evolutionarily conserved ion channels that regulate cell death in plants
Port-a-Patch and Vesicle Prep Pro Pre-Publication in BioRxiv (2019)   Authors: Sierla M., Prole D.L., Saraiva N., Carrara G., Dinischiotu N., Vaattovaara A., Wrzaczek M., Taylor C.W., Smith F.L., Feys B.

Programmed cell death regulates developmental and stress responses in eukaryotes. Golgi anti-apoptotic proteins (GAAPs) are evolutionarily conserved cell death regulators. Human and viral GAAPs inhibit apoptosis and modulate intracellular Ca2+ fluxes, and viral GAAPs form cation selective channels. Although most mammalian cell death regulators are not conserved at the sequence level in plants, the GAAP gene family shows expansion, with five paralogues (AtGAAP1-5) in the Arabidopsis genome. We pursued molecular and physiological characterization of AtGAAPs making use of the advanced knowledge of their human and viral counterparts. Structural modeling of AtGAAPs predicted the presence of a channel-like pore, and electrophysiological recordings from purified AtGAAP3 reconstituted into lipid bilayers confirmed that plant GAAPs can function as ion channels. AtGAAP1 and AtGAAP4 localized exclusively to the Golgi within the plant cell, while AtGAAP2, AtGAAP3 and AtGAAP5 also showed tonoplastic localization. Gene expression analysis revealed differential spatial expression and abundance of transcript for AtGAAP paralogues in Arabidopsis tissues. We demonstrate that AtGAAP1-5 inhibit Bax-induced cell death in yeast. However, overexpression of AtGAAP1 induces cell death in Nicotiana benthamiana leaves and lesion mimic phenotype in Arabidopsis. We propose that AtGAAPs function as Golgi-localized ion channels that regulate cell death by affecting ionic homeostasis within the cell.

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2018 – Reconstitution and Electrophysiological Characterization of Ion Channels in Lipid Bilayers
Port-a-Patch and Vesicle Prep Pro Publication in Current Protocols in Pharmacology (2018)  Authors: Klaerke D.A., de los Angeles Tejada M., Grøsfjeld Christensen V., Lassen M., Amstrup Pedersen P., Calloe K.

Detergent‐solubilized purified ion channels can be reconstituted into lipid bilayers for electrophysiological analysis. Traditionally, ion channels were inserted into vesicles and subsequently fused with planar “black lipid membranes” formed from lipids dissolved in a hydrophobic solvent such as decane. Provided in this article is a step‐by‐step guide to reconstitute purified ion channel proteins into giant unilamellar vesicles (GUVs). This procedure results in the formation of proteoliposomes that can be used for planar bilayer formation and electrophysiological characterization of single‐channel currents. By using preformed GUVs it is possible to omit the membrane solvent. Compared to traditional preparations, the lipid bilayers formed from GUVs provide an environment that more closely resembles the native cell membrane. Also described is an alternate protocol that entails the production of planar lipid bilayers from GUVs onto which proteins in detergent are added.

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2018 – Romo1 is a mitochondrial nonselective cation channel with viroporin-like characteristics
Port-a-Patch and Vesicle Prep Pro Publication in Journal of Cell Biology (2018)   Authors: Lee G.Y., You D.G., Lee H.R., Hwang S.W., Lee C.J., Do Yoo Y.

Reactive oxygen species (ROS) modulator 1 (Romo1) is a nuclear-encoded mitochondrial inner membrane protein known to regulate mitochondrial ROS production and to act as an essential redox sensor in mitochondrial dynamics. Although its physiological roles have been studied for a decade, the biophysical mechanisms that explain these activities of Romo1 are unclear. In this study, we report that Romo1 is a unique mitochondrial ion channel that differs from currently identified eukaryotic ion channels. Romo1 is a highly conserved protein with structural features of class II viroporins, which are virus-encoded nonselective cation channels. Indeed, Romo1 forms a nonselective cation channel with its amphipathic helical transmembrane domain necessary for pore-forming activity. Notably, channel activity was specifically inhibited by Fe2+ ions, an essential transition metal ion in ROS metabolism. Using structural bioinformatics, we designed an experimental data-guided structural model of Romo1 with a rational hexameric structure. We propose that Romo1 establishes a new category of viroporin-like nonselective cation channel in eukaryotes.

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2018 – Parallel evolution of frog antimicrobial peptides produces identical conformations but subtly distinct membrane and antibacterial activities
Port-a-Patch and Vesicle Prep Pro pre-Publication in bioRxiv (2018)   Authors: Manzo G., Ferguson P.M., Hind C., Clifford M., Gustilo V.B., Ali H., Bansal S.S., Bui T.T., Drake A.F., Atkinson R.A., Sutton M.J., Lorenz C.D., Phoenix D.A., Mason A.J.

Frogs such as Rana temporaria and Litoria aurea secrete numerous closely related antimicrobial peptides (AMPs) as an effective chemical dermal defence. Despite the high similarity in physical properties and preference for adopting secondary amphipathic, α-helix conformations in membrane mimicking milieu, their spectrum of activity and potency often varies considerably. Damage or penetration of the bacterial plasma membrane is considered essential for AMP activity and hence distinguishing apparently similar AMPs according to their behaviour in, and effects on, model membranes will inform understanding of species specific effective antimicrobial mechanisms. Here we use a combination of molecular dynamics simulations, circular dichroism and patch-clamp to investigate the basis for differing anti-bacterial activities in representative AMPs from each species; temporin L and aurein 2.5. Despite adopting near identical, α-helix conformations in the steady-state in a variety of membrane models, these two AMPs can be distinguished both in vitro and in silico based on their dynamic interactions with model membranes; the greater conformational flexibility and the higher amplitude channel conductance induced offers a rationale for the greater potency and broader spectrum of activity of temporin L over aurein 2.5. Specific contributions from individual residues are identified that define the mechanisms of action of each AMP. Our findings suggest AMPs in frogs are examples of parallel evolution whose utility is based on apparently similar but subtly distinct mechanisms of action.

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2018 – Quantifying Permeation of Small Charged Molecules across Channels: Electrophysiology in Small Volumes
Port-a-Patch and Vesicle Prep Pro Publication in ACS Omega (2018)    Authors: Wang J., Benier L., Winterhalter M.

A major bottleneck in the development of small molecule antibiotics is to achieve good permeability across the outer membrane in Gram-negative bacteria. Optimization with respect to permeability surprisingly lacks appropriate methods. Recently we proposed to use the diffusion potential for charged molecules created by their difference in electrophoretic mobility while crossing the outer membrane channel under a concentration gradient. The latter provides semi-quantitative values but the current available setups require large volumes and thus exclude several classes of molecules. Here we propose a simple approach capturing proteoliposomes at aperture of glass surface (planar aperture or conical glass capillary) decreasing the necessary volume below 50 µL. We measured the transport of two charged molecules sulbactam and ceftazidime across the two major porins in E.coli. Both molecules permeate through these porins were observed with sulbactam owes higher permeability.

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2018 – An aromatic cluster in Lysinibacillus sphaericus BinB involved in toxicity and proper in-membrane folding
Port-a-Patch and Vesicle Prep Pro Publication in Archives of Biochemistry and Biophysics (2018)   Authors: Chooduang A., Surya W., Torres J., Boonserm P.

The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. Based on structural alignments with other toxins, an aromatic cluster in the C-terminal domain of BinB (termed here BC) has been proposed to be important for toxicity. We tested this experimentally using BinB mutants bearing single mutations in this aromatic cluster. Consistent with the hypothesis, two of these mutations, F311A and F315A, were not toxic to Culex quinquefasciatus larvae and were unable to permeabilize liposomes or elicit ion channel activity, in contrast to wild-type BinB. Despite these effects, none of these mutations altered significantly the interaction between the activated forms of the two subunits in solution. These results indicate that these aromatic residues on the C-terminal domain of BinB are critical for toxin insertion in membranes. The latter can be by direct contact of these residues with the membrane surface, or by facilitating the formation a membrane-inserting oligomer.

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2018 – Molecular mechanism of the Orai channel activation
Port-a-Patch and Vesicle Prep Pro Pre-publicatin in bioRxiv (2018)   Authors: Liu, X., Wu, G., Yu, Y., Chen, X., Ji, R., Lu, J., Li, X., Zhang, X., Yang, X., Shen, Y.

The Orai channel is characterized by voltage independence, low conductance and high Ca2+ selectivity and plays an important role in Ca2+ influx through the plasma membrane. How the channel is activated and promotes Ca2+ permeation are not well understood. Here, we report the crystal structure and cryo-electron microscopy reconstruction of a Drosophila melanogaster Orai mutant (P288L) channel that is constitutively active according to electrophysiology. The open state of the Orai channel showed a hexameric assembly in which six TM1 helices in the center form the ion-conducting pore, and six TM4 helices in the periphery form extended long helices. Orai channel activation requires conformational transduction from TM4 to TM1 and eventually causes the basic section of TM1 to twist outward. The wider pore on the cytosolic side aggregates anions to increase the potential gradient across the membrane and thus facilitate Ca2+ permeation. The open-state structure of the Orai channel offers insights into channel assembly, channel activation and Ca2+ permeation.

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2017 – Polydim-I antimicrobial activity against MDR bacteria and its model membrane interaction
Port-a-Patch and  Vesicle Prep Pro Publication in PLoS ONE (2017)  Authors: Rangel M., Fernandes dos Santos Castro F., Mota-Lima L.D., Clissa P.B., Martins D.B., Perez dos Santos Cabrera M., Mortari M.R.

The rapid spread of multi-drug resistant pathogens represents a serious threat to public health, considering factors such as high mortality rates, treatment restrictions and high prevalence of multi-drug resistant bacteria in the hospital environment. Antimicrobial peptides (AMPs) may exhibit powerful antimicrobial activity against different and diverse microorganisms, also presenting the advantage of absence or low toxicity towards animal cells. In this study, the evaluation of the antimicrobial activity against multi-drug resistant bacteria of a recently described AMP from wasp, Polydim-I, was performed. Polydim-I presented activity against standard strains (non-carriers of multi-resistant genes) that are susceptible to commercial antimicrobials, and also against multi-drug resistant strains at concentrations bellow 1μg/ml (0.41 μM). This is a rather low concentration among those reported for AMPs. At this concentration we found out that Polydim-I inhibits almost 100% of the tested pathogens growth, while with the ATCC strains the minimum inhibitory concentration (MIC100) is 400 times higher. Also, in relation to in vitro activity of conventional drugs against multi-drug resistant bacteria strains, Polydim-I is almost 10 times more efficient and with broader spectrum. Cationic AMPs are known as multi-target compounds and specially for targeting the phospholipid matrix of bacterial membranes. Exploring the interactions of Polydim-I with lipid bilayers, we have confirmed that this interaction is involved in the mechanism of action. Circular dichroism experiments showed that Polydim-I undergoes a conformational transition from random coil to a mostly helical conformation in the presence of membrane mimetic environments. Zeta potential measurements confirmed the binding and partial charge neutralization of anionic asolectin vesicles, and also suggested a possible aggregation of peptide molecules. FTIR experiments confirmed that some peptide aggregation occurs, which is minimized in the presence of strongly anionic micelles of sodium dodecyl sulfate. Also, Polydim-I induced channel-like structures formation to asolectin lipid bilayers, as demonstrated in the electrophysiology experiments. We suggest that cationic Polydim-I targets the membrane lipids due to electrostatic attraction, partially accumulates, neutralizing the opposite charges and induces pore formation. Similar mechanism of action has already been suggested for other peptides from wasp venoms, especially mastoparans.

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2017 – The Rotavirus NSP4 Viroporin Domain is a Calcium-conducting Ion Channel
Port-a-Patch and Vesicle Prep Pro Publication in Nature Scientific Reports    Authors: Pham T., Perry J.L., Dosey T.L., Delcour L.H.,Hyser J.M.

Viroporins are small virus-encoded ion channel proteins. Most viroporins are monovalent selective cation channels, with few showing the ability to conduct divalent cations, like calcium (Ca2+). Nevertheless, some viroporins are known to disrupt host cell Ca2+ homeostasis, which is critical for virus replication and pathogenesis. Rotavirus nonstructural protein 4 (NSP4) is an endoplasmic reticulum transmembrane glycoprotein that has a viroporin domain (VPD), and NSP4 viroporin activity elevates cytosolic Ca2+ in mammalian cells. The goal of this study was to demonstrate that the NSP4 VPD forms an ion channel and determine whether the channel can conduct Ca2+. Using planar lipid bilayer and liposome patch clamp electrophysiology, we show that a synthetic peptide of the NSP4 VPD has ion channel activity. The NSP4 VPD was selective for cations over anions and channel activity was observed to have both well-defined “square top” openings as well as fast current fluctuations, similar to other viroporins. Importantly, the NSP4 VPD showed similar conductance of divalent cations (Ca2+ and Ba2+) as monovalent cations (K+), but a viroporin defective mutant lacked Ca2+ conductivity. These data demonstrate that the NSP4 VPD is a Ca2+-conducting viroporin and establish the mechanism by which NSP4 disturbs host cell Ca2+ homeostasis.

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2017 – Evaluation of the antimicrobial activity of the mastoparan Polybia-MPII isolated from venom of the social wasp Pseudopolybia vespiceps testacea (Vespidae, Hymenoptera)
Port-a-Patch and Vesicle Prep Pro Publication in International Journal of Antimicrobial Agents (2017)   Authors: Silva J.C., Neto L.M., Neves R.C., Gonçalves J.C., Trentini M.M., Mucury-Filho R., Smidt K.S., Fensterseifer I.C., Silva O.N., Lima L.D., Clissa P.B., Vilela N., Guilhelmelli F., Silva L.P., Rangel M., Kipnis A., Silva-Pereira I., Franco O.L., Junqueira-Kipnis A.P., Bocca A.L., Mortari M.R.

Mastoparans, a class of peptides found in wasp venom, have significant effects following a sting as well as useful applications in clinical practice. Among these is their potential use in the control of micro-organisms that cause infectious diseases with a significant impact on society. Thus, the present study describes the isolation and identification of a mastoparan peptide from the venom of the social wasp Pseudopolybia vespiceps and evaluated its antimicrobial profile against bacteria (Staphylococcus aureus and Mycobacterium abscessus subsp. massiliense), fungi (Candida albicans and Cryptococcus neoformans) and in vivo S. aureus infection. The membrane pore-forming ability was also assessed. The mastoparan reduced in vitro and ex vivo mycobacterial growth by 80% at 12.5 µM in infected peritoneal macrophages but did not affect the shape of bacterial cells at the dose tested (6.25 µM). The peptide also showed potent action against S. aureus in vitro (EC50 and EC90 values of 1.83 µM and 2.90 µM, respectively) and reduced the in vivo bacterial load after 6 days of topical treatment (5 mg/kg). Antifungal activity was significant, with EC50 and EC90 values of 12.9 µM and 15.3 µM, respectively, for C. albicans, and 11 µM and 22.70 µM, respectively, for C. neoformans. Peptides are currently attracting interest for their potential in the design of antimicrobial drugs, particularly due to the difficulty of micro-organisms in developing resistance to them. In this respect, Polybia-MPII proved to be highly effective, with a lower haemolysis rate compared with peptides of the same family.

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2017 – Organelle membrane derived patches: reshaping classical methods for new targets
Port-a-Patch and Vesicle Prep Pro Publication in Nature Scientific Reports (2017)    Authors: Shapovalov G., Ritaine A., Bidaux G., Slomianny, C., Borowiec A-S., Gordienko D., Bultynck G., Skryma R., Prevarskaya N.

Intracellular ion channels are involved in multiple signaling processes, including such crucial ones as regulation of cellular motility and fate. With 95% of the cellular membrane belonging to intracellular organelles, it is hard to overestimate the importance of intracellular ion channels. Multiple studies have been performed on these channels over the years, however, a unified approach allowing not only to characterize their activity but also to study their regulation by partner proteins, analogous to the patch clamp “golden standard”, is lacking. Here, we present a universal approach that combines the extraction of intracellular membrane fractions with the preparation of patchable substrates that allows to characterize these channels in endogenous protein environment and to study their regulation by partner proteins. We validate this method by characterizing activity of multiple intracellular ion channels localized to different organelles and by providing detailed electrophysiological characterization of the regulation of IP3R activity by endogenous Bcl-2. Thus, after synthesis and reshaping of the well-established approaches, organelle membrane derived patch clamp provides the means to assess ion channels from arbitrary cellular membranes at the single channel level.

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2017 – Asymmetric mechanosensitivity in a eukaryotic ion channel
Port-a-Patch and Vesicle Prep Pro Publication in PNAS (2017) Authors: Clausen M.V., Jarerattanachata V., Carpenterc E.P., Mark S. , Sansomb P., Tucker S.J.

Living organisms perceive and respond to a diverse range of mechanical stimuli. A variety of mechanosensitive ion channels have evolved to facilitate these responses, but the molecular mechanisms underlying their exquisite sensitivity to different forces within the membrane remains unclear. TREK-2 is a mammalian two-pore domain (K2P) K+ channel important for mechanosensation, and recent studies have shown how increased membrane tension favors a more expanded conformation of the channel within the membrane. These channels respond to a complex range of mechanical stimuli, however, and it is uncertain how differences in tension between the inner and outer leaflets of the membrane contribute to this process. To examine this, we have combined computational approaches with functional studies of oppositely oriented single channels within the same lipid bilayer. Our results reveal how the asymmetric structure of TREK-2 allows it to distinguish a broad profile of forces within the membrane, and illustrate the mechanisms that eukaryotic mechanosensitive ion channels may use to detect and fine-tune their responses to different mechanical stimuli. Significance: One important way in which living organisms are able to detect and respond to their environment is via the conversion of mechanical forces into electrical signals. However, the molecular mechanisms that enable mammalian “mechanosensitive” ion channels to detect a wide profile of forces within the membrane remain unclear. By studying the functional activity of individual TREK-2 K2P channels inserted in different directions into a lipid bilayer, we are now able to describe how the asymmetric structure of this channel enables it to sense such a broad profile of forces. These results help us understand how eukaryotic ion channels respond to a rich variety of sensory stimuli.

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2017 – Bilayer-Mediated Structural Transitions Control Mechanosensitivity of the TREK-2 K2P Channel
Port-a-Patch and Vesicle Prep Pro Publication in Structure (2017)   Authors: Arya P, Jarerattanachat V., Clausen M.V., Schewe M., McClenaghan C., Argent L., Conrad L.J., Dong Y.Y., Pike A.C.W., Carpenter E.P., Baukrowitz T., Sansom M.S.P., Tucker S.J.

The mechanosensitive two-pore domain (K2P) K+ channels (TREK-1, TREK-2, and TRAAK) are important for mechanical and thermal nociception. However, the mechanisms underlying their gating by membrane stretch remain controversial. Here we use molecular dynamics simulations to examine their behavior in a lipid bilayer. We show that TREK-2 moves from the “down” to “up” conformation in direct response to membrane stretch, and examine the role of the transmembrane pressure profile in this process. Furthermore, we show how state-dependent interactions with lipids affect the movement of TREK-2, and how stretch influences both the inner pore and selectivity filter. Finally, we present functional studies that demonstrate why direct pore block by lipid tails does not represent the principal mechanism of mechanogating. Overall, this study provides a dynamic structural insight into K2P channel mechanosensitivity and illustrates how the structure of a eukaryotic mechanosensitive ion channel responds to changes in forces within the bilayer.

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2016 – Structural and functional characterization of a calcium-activated cation channel from Tsukamurella paurometabola
Port-a-Patch and Vesicle Prep Pro Publication in Nature Communications (2016) Authors: Dhakshnamoorthy B., Rohaim A., Rui H., Blachowicz L., Roux B.

The selectivity filter is an essential functional element of K+ channels that is highly conserved both in terms of its primary sequence and its three-dimensional structure. Here, we investigate the properties of an ion channel from the Gram-positive bacterium Tsukamurella paurometabola with a selectivity filter formed by an uncommon proline-rich sequence. Electrophysiological recordings show that it is a non-selective cation channel and that its activity depends on Ca2+ concentration. In the crystal structure, the selectivity filter adopts a novel conformation with Ca2+ ions bound within the filter near the pore helix where they are coordinated by backbone oxygen atoms, a recurrent motif found in multiple proteins. The binding of Ca2+ ion in the selectivity filter controls the widening of the pore as shown in crystal structures and in molecular dynamics simulations. The structural, functional and computational data provide a characterization of this calcium-gated cationic channel.

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2016 – The N-terminal Ankyrin Repeat Domain Is Not Required for Electrophile and Heat Activation of the Purified Mosquito TRPA1 Receptor
Port-a-Patch and Vesicle Prep Pro Publication in Nature (2016) Authors: Survery S., Moparthi L., Kjellbom P., Högestätt E.D., Zygmunt P.M., Johanson U.

Temperature sensors are crucial for animals to optimize living conditions. The temperature response of the ion channel transient receptor potential A1 (TRPA1) is intriguing, some orthologs have been reported to be activated by cold and others by heat, but the molecular mechanisms responsible for its activation remain elusive. Single-channel electrophysiological recordings of heterologously expressed and purified Anopheles gambiae TRPA1 (AgTRPA1), with and without the N-terminal ankyrin repeat domain, demonstrate that both proteins are functional as they responded to the electrophilic compounds allyl isothiocyanate (AITC) and cinnamaldehyde as well as heat. The proteins similar intrinsic fluorescence properties and corresponding quenching when activated by AITC or heat, suggest lipid bilayer-independent conformational changes outside the N-terminal domain. The results show that AgTRPA1 is an inherent temperature- and chemoreceptor, and analogous to what has been reported for the human TRPA1 ortholog the N-terminal domain may tune the response but is not required for the activation by these stimuli.

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2016 – Photosensitization in Porphyrias and Photodynamic Therapy Involves TRPA1 and TRPV1
Port-a-Patch and Vesicle Prep Pro Publication in The Journal of Neuroscience (2016) Authors: Babes A., Sauer S.K., Moparthi L., Kichko T.I., Neacsu C., Namer B., Filipovic M., Zygmunt P.M., Reeh P.W., Fischer M.J.

Photosensitization, an exaggerated sensitivity to harmless light, occurs genetically in rare diseases, such as porphyrias, and in photodynamic therapy where short-term toxicity is intended. A common feature is the experience of pain from bright light. In human subjects, skin exposure to 405 nm light induced moderate pain, which was intensified by pretreatment with aminolevulinic acid. In heterologous expression systems and cultured sensory neurons, exposure to blue light activated TRPA1 and, to a lesser extent, TRPV1 channels in the absence of additional photosensitization. Pretreatment with aminolevulinic acid or with protoporphyrin IX dramatically increased the light sensitivity of both TRPA1 and TRPV1 via generation of reactive oxygen species. Artificial lipid bilayers equipped with purified human TRPA1 showed substantial single-channel activity only in the presence of protoporphyrin IX and blue light. Photosensitivity and photosensitization could be demonstrated in freshly isolated mouse tissues and led to TRP channel-dependent release of proinflammatory neuropeptides upon illumination. With antagonists in clinical development, these findings may help to alleviate pain during photodynamic therapy and also allow for disease modification in porphyria patients. Significance Statement: Cutaneous porphyria patients suffer from burning pain upon exposure to sunlight and other patients undergoing photodynamic therapy experience similar pain, which can limit the therapeutic efforts. This study elucidates the underlying molecular transduction mechanism and identifies potential targets of therapy. Ultraviolet and blue light generates singlet oxygen, which oxidizes and activates the ion channels TRPA1 and TRPV1. The disease and the therapeutic options could be reproduced in models ranging from isolated ion channels to human subjects, applying protoporphyrin IX or its precursor aminolevulinic acid. There is an unmet medical need, and our results suggest a therapeutic use of the pertinent antagonists in clinical development.

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2016 – Pore architecture of TRIC channels and insights into their gating mechanism
Port-a-Patch and Vesicle Prep Pro Publication in Nature (2016) Authors: Yang H., Hu M., Guo J., Ou X., Cai T., Liu Z.

Intracellular Ca2+ signalling processes are fundamental to muscle contraction, neurotransmitter release, cell growth and apoptosis. Release of Ca2+ from the intracellular stores is supported by a series of ion channels in sarcoplasmic or endoplasmic reticulum (SR/ER). Among them, two isoforms of the trimeric intracellular cation (TRIC) channel family, named TRIC-A and TRIC-B, modulate the release of Ca2+ through the ryanodine receptor or inositol triphosphate receptor, and maintain the homeostasis of ions within SR/ER lumen. Genetic ablations or mutations of TRIC channels are associated with hypertension, heart disease, respiratory defects and brittle bone disease. Despite the pivotal function of TRIC channels in Ca2+ signalling, their pore architectures and gating mechanisms remain unknown. Here we present the structures of TRIC-B1 and TRIC-B2 channels from Caenorhabditis elegans in complex with endogenous phosphatidylinositol-4,5-biphosphate (PtdIns(4,5)P2, also known as PIP2) lipid molecules. The TRIC-B1/B2 proteins and PIP2 assemble into a symmetrical homotrimeric complex. Each monomer contains an hourglass-shaped hydrophilic pore contained within a seven-transmembrane-helix domain. Structural and functional analyses unravel the central role of PIP2 in stabilizing the cytoplasmic gate of the ion permeation pathway and reveal a marked Ca2+-induced conformational change in a cytoplasmic loop above the gate. A mechanistic model has been proposed to account for the complex gating mechanism of TRIC channels.

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2016 – Functional characterization of p7 viroporin from hepatitis C virus produced in a cell-free expression system
Port-a-Patch and Vesicle Prep Pro Publication in Protein Expression and Purification (2016) Authors: Soranzo T., Cortès S., Gilde F., Kreir M., Picart C., Lenormand J.-L.

Using a cell-free expression system we produced the p7 viroporin embedded into a lipid bilayer in a single-step manner. The protein quality was assessed using different methods. We examined the channel forming activity of p7 and verified its inhibition by 5-(N,N-Hexamethylene) amiloride (HMA). Fourier transformed infrared spectroscopy (FTIR) experiments further showed that when p7 was inserted into synthetic liposomes, the protein displayed a native-like conformation similar to p7 obtained from other sources. Photoactivatable amino acid analogs used for p7 protein synthesis enabled oligomerization state analysis in liposomes by cross-linking. Therefore, these findings emphasize the quality of the cell-free produced p7 proteoliposomes which can benefit the field of the hepatitis C virus (HCV) protein production and characterization and also provide tools for the development of new inhibitors to reinforce our therapeutic arsenal against HCV.

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2016 – Human TRPA1 is a heat sensor displaying intrinsic U-shaped thermosensitivity
Port-a-Patch and Vesicle Prep Pro Publication in Scientific Reports (2016) Authors: Moparthi L., Kichko T. I., Eberhardt M., Högestätt E. D., Kjellbom P., Johanson U., Reeh P., W., Leffler A., Filipovic M. R., Zygmunt P. M.

Thermosensitive Transient Receptor Potential (TRP) channels are believed to respond to either cold or heat. In the case of TRP subtype A1 (TRPA1), there seems to be a species-dependent divergence in temperature sensation as non-mammalian TRPA1 is heat-sensitive whereas mammalian TRPA1 is sensitive to cold. It has been speculated but never experimentally proven that TRPA1 and other temperature-sensitive ion channels have the inherent capability of responding to both cold and heat. Here we show that redox modification and ligands affect human TRPA1 (hTRPA1) cold and heat sensing properties in lipid bilayer and whole-cell patch-clamp recordings as well as heat-evoked TRPA1-dependent calcitonin gene-related peptide (CGRP) release from mouse trachea. Studies of purified hTRPA1 intrinsic tryptophan fluorescence, in the absence of lipid bilayer, consolidate hTRPA1 as an intrinsic bidirectional thermosensor that is modified by the redox state and ligands. Thus, the heat sensing property of TRPA1 is conserved in mammalians, in which TRPA1 may contribute to sensing warmth and uncomfortable heat in addition to noxious cold.

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2015 – Quantification of Fluoroquinolone Uptake through the Outer Membrane Channel OmpF of Escherichia coli
Port-a-Patch and Vesicle Prep Pro Publication in Journal of the American Chemical Society (2015) Authors: Cama J., Bajaj H., Pagliara S., Maier T., Braun Y., Winterhalter M., Keyser U.F.

Decreased drug accumulation is a common cause of antibiotic resistance in microorganisms. However, there are few reliable general techniques capable of quantifying drug uptake through bacterial membranes. We present a semiquantitative optofluidic assay for studying the uptake of autofluorescent drug molecules in single liposomes. We studied the effect of the Escherichia coli outer membrane channel OmpF on the accumulation of the fluoroquinolone antibiotic, norfloxacin, in proteoliposomes. Measurements were performed at pH 5 and pH 7, corresponding to two different charge states of norfloxacin that bacteria are likely to encounter in the human gastrointestinal tract. At both pH values, the porins significantly enhance drug permeation across the proteoliposome membranes. At pH 5, where norfloxacin permeability across pure phospholipid membranes is low, the porins increase drug permeability by 50-fold on average. We estimate a flux of about 10 norfloxacin molecules per second per OmpF trimer in the presence of a 1 mM concentration gradient of norfloxacin. We also performed single channel electrophysiology measurements and found that the application of transmembrane voltages causes an electric field driven uptake in addition to concentration driven diffusion. We use our results to propose a physical mechanism for the pH mediated change in bacterial susceptibility to fluoroquinolone antibiotics.

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2016 – Conductance and Capacity of Plain Lipid Membranes under Conditions of Variable Gravity
Port-a-Patch and Vesicle Prep Pro Publication in Journal of Biomedical Science and Engineering (2016) Authors: Sieber M., Kaltenbach S., Hanke W. and Kohn F.P.M

Biological cell membranes are complex structures containing mainly lipids and proteins. Functional aspects of such membranes are usually attributed to membrane integral proteins. However, it is well established that parameters of the lipid matrix are modifying the function of proteins. Additionally, electrical capacity and conductance of the plain lipid matrix of membranes are contributing directly to cellular functions as there is, for example, the propagation of action potentials. Accordingly the dependence of these parameters on changes of gravity might be important in the field of life sciences under space conditions. In this study consequently we have performed experiments in parabolic flight campaigns utilizing the patch-clamp technology to investigate conductance and capacity of plain lipid vesicle membranes under conditions of changing gravity. Both capacity and conductance were found to be gravity dependent. The changes in capacity could be contributed to changes in membrane geometry. Significant permeability in plain lipid membranes could be only observed at high potentials, where spontaneous current fluctuations occurred. The probability of these fluctuations was gravity dependent.

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2015 – Different Ligands of the TRPV3 Cation Channel Cause Distinct Conformational Changes As Revealed by Intrinsic Tryptophan Fluorescence Quenching
Port-a-Patch and Vesicle Prep Pro Publication in Journal of Biological Chemistry (2015) Authors: Billen B., Brams M., Debaveye S., Remeeva A., Alpizar Y.A., Waelkens E., Kreir M., Brüggemann A., Talavera K., Nilius B., Voets T., Ulens C.

TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane protein c are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies.

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2015 – Golgi anti-apoptotic proteins are highly conserved ion channels that affect apoptosis and cell migration
Port-a-Patch and Vesicle Prep Pro Publication in Journal of Biological Chemistry (2015) Authors: Carrara G., Saraiva N., Parsons M., Byrne B., Prole D.L., Taylor C.W., Smith G.L.

Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca(2+) content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently.

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2014 – Temperature-sensitive gating of hCx26: high-resolution Raman spectroscopy sheds light on conformational changes
Port-a-Patch and Vesicle Prep Pro Publication in Biomedical Optics Express (2014) Authors: Kniggendorf A.-K., Meinhardt-Wollweber M., Yuan X., Roth B., Seifert A., Fertig N., Zeilinger C.

The temperature-sensitive gating of human Connexin 26 (hCx26) was analyzed with confocal Raman microscopy. High-resolution Raman spectra covering the spectral range between 400 and 1500 rel. cm−1 with a spectral resolution of 1 cm−1 were fully annotated, revealing notable differences between the spectrum recorded from solubilized hCx26 in Ca2+-buffered POPC at 10°C and any other set of protein conditions (temperature, Ca2+ presence, POPC presence). Spectral components originating from specific amino acids show that the TM1/EL1 parahelix and probably the TM4 trans-membrane helix and the plug domain are involved in the gating process responsible for fully closing the hemichannel.

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2015 – Cell-free expression of a functional pore-only sodium channel
Port-a-Patch and Vesicle Prep Pro Publication in Protein Expression and Purification (2015) Authors: Kovácsová G, Gustavsson E, Wang J, Kreir M, Peuker S, Westenhoff S.

Voltage-gated sodium channels participate in the propagation of action potentials in excitable cells. Eukaryotic NaVs are pseudo homotetrameric polypeptides, comprising four repeats of six transmembrane segments (S1–S6). The first four segments form the voltage-sensing domain and S5 and S6 create the pore domain with the selectivity filter. Prokaryotic NaVs resemble these characteristics, but are truly tetrameric. They can typically be efficiently synthesized in bacteria, but production in vitro with cell-free synthesis has not been demonstrated. Here we report the cell-free expression and purification of a prokaryotic tetrameric pore-only sodium channel. We produced milligram quantities of the functional channel protein as characterized by size-exclusion chromatography, infrared spectroscopy and electrophysiological recordings. Cell-free expression enables advanced site-directed labelling, post-translational modifications, and special solubilization schemes. This enables next-generation biophysical experiments to study the principle of sodium ion selectivity and transport in sodium channels.

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2014 – Structure of a Conserved Golgi Complex-targeting Signal in CoroNaVirus Envelope Proteins
Port-a-Patch and Vesicle Prep Pro Publication in Journal of Biological Chemistry (2014) Authors: Li Y., Surya W., Claudine S., Torres J.

CoroNaVirus envelope (CoV E) proteins are ~100-residue polypeptides with at least one channel-forming α-helical transmembrane (TM) domain. The extramembrane C terminal tail contains a completely conserved proline, at the center of a predicted β coil β motif. This hydrophobic motif has been reported to constitute a Golgi-targeting signal, or a second TM domain. However, no structural data for this, or other extramembrane domains in CoV E proteins, is available. Herein, we show that the E protein in the severe acute respiratory syndrome (SARS) virus has only one TM domain in micelles, whereas the predicted β coil β motif forms a short membrane-bound α helix connected by a disordered loop to the TM domain. However, complementary results suggest that this motif is potentially poised for conformational change, or in dynamic exchange with other conformations.

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2014 – Studying mechanosensitive ion channels with an automated patch clamp
Port-a-Patch and Vesicle Prep Pro Publication in European Biophysics Journal (2014) Authors: Barthmes M.,  Mac Donald F.J., Birkner J.P., Brüggemann A., Wahl-Schott C., Koçer A.

Patch clamp electrophysiology is the main technique to study mechanosensitive ion channels (MSCs), however, conventional patch clamping is laborious and success and output depends on the skills of the operator. Even though automated patch systems solve these problems for other ion channels, they could not be applied to MSCs. Here, we report on activation and single channel analysis of a bacterial mechanosensitive ion channel using an automated patch clamp system. With the automated system, we could patch not only giant unilamellar liposomes but also giant Escherichia coli (E. coli) spheroplasts. The tension sensitivity and channel kinetics data obtained in the automated system were in good agreement with that obtained from the conventional patch clamp. The findings will pave the way to high throughput fundamental and drug screening studies on mechanosensitive ion channels.

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2014 – Human TRPA1 is intrinsically cold- and chemosensitive with and without its N-terminal ankyrin repeat domain
Port-a-Patch and Vesicle Prep Pro Publication in Proceedings of the National Academy of Sciences of the United States of America (2014)   Authors: Moparthi L., Survery S., Kreir M., Simonsen C., Kjellbom P., Högestätt E.D., Johanson U., Zygmunt P.M.

We have purified and reconstituted human transient receptor potential (TRP) subtype A1 (hTRPA1) into lipid bilayers and recorded single-channel currents to understand its inherent thermo- and chemosensory properties as well as the role of the ankyrin repeat domain (ARD) of the N terminus in channel behavior. We report that hTRPA1 with and without its N-terminal ARD (Δ1–688 hTRPA1) is intrinsically cold-sensitive, and thus, cold-sensing properties of hTRPA1 reside outside the N-terminal ARD. We show activation of hTRPA1 by the thiol oxidant 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin) and that electrophilic compounds activate hTRPA1 in the presence and absence of the N-terminal ARD. The nonelectrophilic compounds menthol and the cannabinoid Δ9-tetrahydrocannabiorcol (C16) directly activate hTRPA1 at different sites independent of the N-terminal ARD. The TRPA1 antagonist HC030031 inhibited cold and chemical activation of hTRPA1 and Δ1–688 hTRPA1, supporting a direct interaction with hTRPA1 outside the N-terminal ARD. These findings show that hTRPA1 is an intrinsically cold- and chemosensitive ion channel. Thus, second messengers, including Ca2+, or accessory proteins are not needed for hTRPA1 responses to cold or chemical activators. We suggest that conformational changes outside the N-terminal ARD by cold, electrophiles, and nonelectrophiles are important in hTRPA1 channel gating and that targeting chemical interaction sites outside the N-terminal ARD provides possibilities to fine tune TRPA1-based drug therapies (e.g., for treatment of pain associated with cold hypersensitivity and cardiovascular disease).

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2014 – Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system
Port-a-Patch and Vesicle Prep Pro Publication in Biosensors and Bioelectronics (2014)   Authors: Dondapati S.K., Kreir M., Quast R.B., Wüstenhagen D.A., Brüggemann A., Fertig N., Kubick S.

The potassium channel KcsA was heterologously expressed in a eukaryotic cell-free system. Both, the expression yields and functional analysis of the protein were reported. Qualitative and quantitative analyses of KcsA expression were performed by using 14C-labeled leucine as one of the amino acids supplemented in the cell-free reaction mixture. There was a time dependent increase in the protein yield as well as the intensity of the native tetramer band in insect cell derived microsomes. Electrophysiology measurements demonstrated the functional activity of the microsomes harboring KcsA showing single-channel currents with the typical biophysical characteristics of the ion channel. The channel behavior was asymmetric and showed positive rectification with larger currents towards positive voltages. KcsA channel currents were effectively blocked by potassium selective barium (Ba2+). This functional demonstration of an ion channel in eukaryotic cell-free system has a large potential for future applications including drug screening, diagnostic applications and functional assessment of complex membrane proteins like GPCRs by coupling them to ion channels in cell-free systems. Furthermore, membrane proteins can be expressed directly from linear DNA templates within 90 min, eliminating the need for additional cloning steps, which makes this cell-free system fast and efficient.

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2012 – Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayer
Port-a-Patch and Vesicle Prep Pro Publication in The Journal of Biological Chemistry (2012) Authors: Carnarius C., Kreir M., Krick M., Methfessel C., Moehrle V., Valerius O., Brüggemann A., Steinem C., Fertig N.

In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein.

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2014 – An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity
Port-a-Patch and Vesicle Prep Pro Publication in Proceedings of the National Academy of Sciences of the United States of America (2014) Authors: Danilchanka A., Sun J., Pavlenok M., Maueröder C., Speer A., Siroy A., Marrero J., Trujillo C., Mayhew D.L., Doornbos K.S., Muñoz 'L.E., HerrmannM., EhrtS., Berens C., Niederweisa M.

The ability to control the timing and mode of host cell death plays a pivotal role in microbial infections. Many bacteria use toxins to kill host cells and evade immune responses. Such toxins are unknown in Mycobacterium tuberculosis. Virulent M. tuberculosis strains induce necrotic cell death in macrophages by an obscure molecular mechanism. Here we show that the M. tuberculosis protein Rv3903c (channel protein with necrosis-inducing toxin, CpnT) consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted toxic C-terminal domain. Infection experiments revealed that CpnT is required for survival and cytotoxicity of M. tuberculosis in macrophages. Furthermore, we demonstrate that the C-terminal domain of CpnT causes necrotic cell death in eukaryotic cells. Thus, CpnT has a dual function in uptake of nutrients and induction of host cell death by M. tuberculosis.

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2011 – Voltage-gated sodium channel (NaV) protein dissection creates a set of functional pore-only proteins
Port-a-Patch and Vesicle Prep Pro Publication in Proceedings of the National Academy of Sciences of the United States (2011) Authors: Shaya D., Kreir M., Robbins R.A., Wong S., Hammon J., Brüggemann A., Minor Jr. D.L.

Many voltage-gated ion channel (VGIC) superfamily members contain six-transmembrane segments in which the first four form a voltage-sensing domain (VSD) and the last two form the pore domain (PD). Studies of potassium channels from the VGIC superfamily together with identification of voltage-sensor only proteins have suggested that the VSD and the PD can fold independently. Whether such transmembrane modularity is common to other VGIC superfamily members has remained untested. Here we show, using protein dissection, that the Silicibacter pomeroyi voltage-gated sodium channel (NaVSp1) PD forms a stand-alone, ion selective pore (NaVSp1p) that is tetrameric, α-helical, and that forms functional, sodium-selective channels when reconstituted into lipid bilayers. Mutation of the NaVSp1p selectivity filter from LESWSM to LDDWSD, a change similar to that previously shown to alter ion selectivity of the bacterial sodium channel NaVBh1 (NaChBac), creates a calcium-selective pore-only channel, CaVSp1p. We further show that production of PDs can be generalized by making pore-only proteins from two other extremophile NaVs: one from the hydrocarbon degrader Alcanivorax borkumensis (NaVAb1p), and one from the arsenite oxidizer Alkalilimnicola ehrlichei (NaVAe1p). Together, our data establish a family of active pore-only ion channels that should be excellent model systems for study of the factors that govern both sodium and calcium selectivity and permeability. Further, our findings suggest that similar dissection approaches may be applicable to a wide range of VGICs and, thus, serve as a means to simplify and accelerate biophysical, structural, and drug development efforts.

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2012 – Design, synthesis and functional analysis of dansylated polytheonamide mimic: an artificial Peptide ion channel
Port-a-Patch and Vesicle Prep Pro Publication in Journal of the American Chemical Society (2012) Authors: Itoh H., Matsuoka S., Kreir M., Inoue M.

We report herein the design, total synthesis, and functional analysis of a novel artificial ion channel molecule, designated as dansylated polytheonamide mimic (3). The channel 3 was designed based on an exceptionally potent cytotoxin, polytheonamide B (1). Our strategy for the development of synthetic ion channels, which could be easily derivatized for various functions, involved two key features. First, the structure of 1 was simplified by replacing many of nonproteinogenic amino acid residues which required multistep synthesis by commercially available amino acids while retaining those residues necessary for folding. It significantly reduced the number of synthetic steps and facilitated a practical chemical construction of 3. Second, the introduction of propargyl glycine at residue 44 enabled facile installation of dansyl group as a reporter of the membrane localization of 3. Application of a newly designed protective group strategy provided efficient construction of the 37 amino acid sequence of residues 12–48 through one automatic solid-phase peptide synthesis. After peptide cleavage from the resin, 3 was synthesized via dansyl group introduction and one fragment-coupling reaction with residues 1–11, followed by the global deprotection. The simplified mimic 3 exhibited potent cytotoxicity toward p388 mouse leukemia cells (IC50 = 12 nM), effectively induced ion transport across the lipid bilayers of liposomes, and displayed H+ and Na+ ion channel activities. Because of its simplified yet functional scaffold structure with a potential for diversification, our rationally designed ion channel molecule should be useful as a novel platform for developing various cytotoxic channel molecules with additional desired functions.

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2010 – Permeation of antibiotics through escherichia coli OmpF and OmpC porins: screening for influx on a single-molecule level
Port-a-Patch and Vesicle Prep Pro Publication in Journal of Biomolecular Screening (2010) Authors: Mahendran K.R., Kreir M., Weingart H., Fertig N., Winterhalter M.

A chip-based automated patch-clamp technique provides an attractive biophysical tool to quantify solute permeation through membrane channels. Proteo–giant unilamellar vesicles (proteo-GUVs) were used to form a stable lipid bilayer across a micrometer-sized hole. Because of the small size and hence low capacitance of the bilayer, single-channel recordings were achieved with very low background noise. The latter allowed the characterization of the influx of 2 major classes of antibiotics—cephalosporins and fluoroquinolones—through the major Escherichia coli porins OmpF and OmpC. Analyzing the ion current fluctuations in the presence of antibiotics revealed transport properties that allowed the authors to determine the mode of permeation. The chip-based setup allows rapid solution exchange and efficient quantification of antibiotic permeation through bacterial porins on a single-molecule level.

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2010 – Studying mechanosensitive ion channels using liposomes
Port-a-Patch and Vesicle Prep Pro Publication in Methods in Molecular Biology (2010) Authors: Martinac B., Rohde P.R., Battle A. R., Petrov E., Pal P, Alexander Foo A.FW, Vásquez V., Huynh T. and Kloda A.

Mechanosensitive (MS) ion channels are the primary molecular transducers of mechanical force into electrical and/or chemical intracellular signals in living cells. They have been implicated in innumerable mechanosensory physiological processes including touch and pain sensation, hearing, blood pressure control, micturition, cell volume regulation, tissue growth, or cellular turgor control. Much of what we know about the basic physical principles underlying the conversion of mechanical force acting upon membranes of living cells into conformational changes of MS channels comes from studies of MS channels reconstituted into artificial liposomes. Using bacterial MS channels as a model, we have shown by reconstituting these channels into liposomes that there is a close relationship between the physico-chemical properties of the lipid bilayer and structural dynamics bringing about the function of these channels.

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2009 – Amyloid-beta-Induced Ion Flux in Artificial Lipid Bilayers and Neuronal Cells: Resolving a Controversy
Port-a-Patch and Vesicle Prep Pro Publication in Neurotoxicity Research (2009) Authors: Capone R., Quiroz F.G., Prangkio P., Saluja I., Sauer A.M., Bautista M.R., Turner R.S., Yang J., Mayer M.

Understanding the pathogenicity of amyloid-beta (Aβ) peptides constitutes a major goal in research on Alzheimer’s disease (AD). One hypothesis entails that Aβ peptides induce uncontrolled, neurotoxic ion flux through cellular membranes. The exact biophysical mechanism of this ion flux is, however, a subject of an ongoing controversy which has attenuated progress toward understanding the importance of Aβ-induced ion flux in AD. The work presented here addresses two prevalent controversies regarding the nature of transmembrane ion flux induced by Αβ peptides. First, the results clarify that Αβ can induce stepwise ion flux across planar lipid bilayers as opposed to a gradual increase in transmembrane current; they show that the previously reported gradual thinning of membranes with concomitant increase in transmembrane current arises from residues of the solvent hexafluoroisopropanol, which is commonly used for the preparation of amyloid samples. Second, the results provide additional evidence suggesting that Aβ peptides can induce ion channel-like ion flux in cellular membranes that is independent from the postulated ability of Αβ to modulate intrinsic cellular ion channels or transporter proteins.

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2009 – The M34A mutant of Connexin26 reveals active conductance states in pore-suspending membranes
Port-a-Patch and Vesicle Prep Pro Publication in Journal of Structural Biology (2009) Authors: Gaßmann O., Kreir M., Ambrosi C., Pranskevich J., Oshima A, Röling C., Sosinsky G., Fertig N., Steinem C.

Connexin26 (Cx26) is a member of the connexin family, the building blocks for gap junction intercellular channels. These dodecameric assemblies are involved in gap junction-mediated cell–cell communication allowing the passage of ions and small molecules between two neighboring cells. Mutations in Cx26 lead to the disruption of gap junction-mediated intercellular communication with consequences such as hearing loss and skin disorders. We show here that a mutant of Cx26, M34A, forms an active hemichannel in lipid bilayer experiments. A comparison with the Cx26 wild-type is presented. Two different techniques using micro/nano-structured substrates for the formation of pore-suspending lipid membranes are used. We reconstituted the Cx26 wild-type and Cx26M34A into artificial lipid bilayers and observed single channel activity for each technique, with conductance levels of around 35, 70 and 165 pS for the wild-type. The conductance levels of Cx26M34A were found at around 45 and 70 pS.

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2006 – High-resolution electrophysiology on a chip: Transient dynamics of alamethicin channel formation
Port-a-Patch and Vesicle Prep Pro Publication in Biochimica et Biophysica Acta - Biomembranes (2006) Authors: Sondermann M., George M., Fertig N., Behrends J.C.

Microstructured planar substrates have been shown to be suitable for patch clamp recording from both whole cells and isolated patches of membrane, as well as for measurements from planar lipid bilayers. Here, we further explore this technology with respect to high-resolution, low noise single-channel recording. Using solvent-free lipid bilayers from giant unilamellar vesicles obtained by electro-swelling, we recorded channels formed by the peptaibol alamethicin, a well-studied model system for voltage-dependent channels, focusing on the transient dynamics of single-channel formation upon application of a voltage step. With our setup, we were able to distinctly resolve dwell times well below 100 mus and to perform a thorough statistical analysis of alamethicin gating. Our results show good agreement with models that do not rely on the existence of non-conducting preaggregate states. Microstructured apertures in glass substrates appear promising with respect to future experiments on cellular ion channels reconstituted in suspended lipid membranes.

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2008 – Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes
Port-a-Patch and Vesicle Prep Pro Publication in Lab-on-a-chip (2008) Authors: Kreir M., Farre C., Beckler M., George M., Fertig N.

Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) after gentle suction application through micron-sized apertures in a borosilicate glass substrate. Incubation of GUVs with the purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time-consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation. Bilayer recordings are attractive for investigations of membrane proteins not accessible to patch clamp analysis, like e.g. proteins from organelles. In the presented work, we show the example of the outer membrane protein OmpF from Escherichiacoli. We reconstituted OmpF in proteoliposomes and observed the characteristic trimeric conductance levels and the typical gating induced by pH and transmembrane voltage. Moreover, OmpF is the main entrance for beta-lactam antibiotics and we investigated translocation processes of antibiotics and modulation of OmpF by spermine. We suggest that the rapid formation of porin containing lipid bilayers is of potential for the efficient electrophysiological characterization of the OmpF protein, for studying membrane permeation processes and for the rapid screening of antibiotics.

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2012 – Natural and artificial ion channels for biosensing platforms
Port-a-Patch, Patchliner, SyncroPatch 96 ((a predecessor model of SyncroPatch 384PE) and Vesicle Prep Pro Publication in Analytical and Bioanalytical Chemistry (2012) Authors: Steller L., Kreir M., Salzer R.

The single-molecule selectivity and specificity of the binding process together with the expected intrinsic gain factor obtained when utilizing flow through a channel have attracted the attention of analytical chemists for two decades. Sensitive and selective ion channel biosensors for high-throughput screening are having an increasing impact on modern medical care, drug screening, environmental monitoring, food safety, and biowarefare control. Even virus antigens can be detected by ion channel biosensors. The study of ion channels and other transmembrane proteins is expected to lead to the development of new medications and therapies for a wide range of illnesses. From the first attempts to use membrane proteins as the receptive part of a sensor, ion channels have been engineered as chemical sensors. Several other types of peptidic or nonpeptidic channels have been investigated. Various gating mechanisms have been implemented in their pores. Three technical problems had to be solved to achieve practical biosensors based on ion channels: the fabrication of stable lipid bilayer membranes, the incorporation of a receptor into such a structure, and the marriage of the modified membrane to a transducer. The current status of these three areas of research, together with typical applications of ion-channel biosensors, are discussed in this review.

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2020 – Structure and mechanism of bactericidal mammalian perforin-2, an ancient agent of innate immunity
Orbit mini and Vesicle Prep Pro Publication in Science Advances (2020)   Authors: Ni T., Jiao F., Yu X., Aden S., Ginger L., Williams S.I., Bai F., Pražák V., Karia D., Stansfeld P., Zhang P., Munson G., Anderluh G.,Scheuring S., Gilbert R.J.C.

Perforin-2 (MPEG1) is thought to enable the killing of invading microbes engulfed by macrophages and other phagocytes, forming pores in their membranes. Loss of perforin-2 renders individual phagocytes and whole organisms significantly more susceptible to bacterial pathogens. Here, we reveal the mechanism of perforin-2 activation and activity using atomic structures of pre-pore and pore assemblies, high-speed atomic force microscopy, and functional assays. Perforin-2 forms a pre-pore assembly in which its pore-forming domain points in the opposite direction to its membrane-targeting domain. Acidification then triggers pore formation, via a 180° conformational change. This novel and unexpected mechanism prevents premature bactericidal attack and may have played a key role in the evolution of all perforin family proteins.

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2022 – An oomycete NLP cytolysin forms transient small pores in lipid membranes
Orbit mini and Vesicle Prep Pro Publication in Science Advances (2022)  Authors: Pirc K., Clifton L.A., Yilmaz N., Saltalmacchiamo A., Mally M., Snoj T., Žnidaršič N., Srnko M., Borišek J., Parkkila P., Albert I., Podobnik M., Numata K., Nürnberger T., Viitala T., Derganac J., Magistrato A., Lakey J.H., Anderluh G.

Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1–like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.

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2017 – Engineering a pH responsive pore forming protein
Orbit mini and Vesicle Prep Pro Publication in Scientific Reports (2017)    Authors: Kisovec M., Rezelj S., Knap P., Cajnko M.M., Caserman S., Flašker A., Žnidaršič N., Repič M., Mavri J., Ruan Y., Scheuring S., Podobnik M., Anderluh G.

Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH.

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2015 – Bilayer-Spanning DNA Nanopores with Voltage- Switching between Open and Closed State
Orbit 16 and Vesicle Prep Pro Publication in American Chemical Society Nano (2015) Authors: Seifert A., Göpfrich K., Burns J.R., Fertig N., Keyser U.F., Howorka S.

Membrane-spanning nanopores from folded DNA are a recent example of biomimetic man-made nanostructures that can open up applications in biosensing, drug delivery, and nanofluidics. In this report, we generate a DNA nanopore based on the archetypal six-helix-bundle architecture and systematically characterize it via single-channel current recordings to address several fundamental scientific questions in this emerging field. We establish that the DNA pores exhibit two voltage-dependent conductance states. Low transmembrane voltages favor a stable high-conductance level, which corresponds to an unobstructed DNA pore. The expected inner width of the open channel is confirmed by measuring the conductance change as a function of poly(ethylene glycol) (PEG) size, whereby smaller PEGs are assumed to enter the pore. PEG sizing also clarifies that the main ion-conducting path runs through the membrane-spanning channel lumen as opposed to any proposed gap between the outer pore wall and the lipid bilayer. At higher voltages, the channel shows a main low-conductance state probably caused by electric-field-induced changes of the DNA pore in its conformation or orientation. This voltage-dependent switching between the open and closed states is observed with planar lipid bilayers as well as bilayers mounted on glass nanopipettes. These findings settle a discrepancy between two previously published conductances. By systematically exploring a large space of parameters and answering key questions, our report supports the development of DNA nanopores for nanobiotechnology.

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2015 – High-Resolution Size-Discrimination of Single Nonionic Synthetic Polymers with a Highly Charged Biological Nanopore
Orbit 16 and Vesicle Prep Pro Publication in American Chemical Society Nano (2015) Authors: Baaken G., Halimeh I., Bacri, Pelta J., Oukhaled A., Behrends J.C.

Electrophysiological studies of the interaction of polymers with pores formed by bacterial toxins provide a window on single molecule interaction with proteins in real time, report on the behavior of macromolecules in confinement, and enable label-free single molecule sensing. Using pores formed by the staphylococcal toxin α-hemolysin (aHL), a particularly pertinent observation was that, under high salt conditions (3–4 M KCl), the current through the pore is blocked for periods of hundreds of microseconds to milliseconds by poly(ethylene glycol) (PEG) oligomers (degree of polymerization approximately 10–60). Notably, this block showed monomeric sensitivity on the degree of polymerization of individual oligomers, allowing the construction of size or mass spectra from the residual current values. Here, we show that the current through the pore formed by aerolysin (AeL) from Aeromonas hydrophila is also blocked by PEG but with drastic differences in the voltage-dependence of the interaction. In contrast to aHL, AeL strongly binds PEG at high transmembrane voltages. This fact, which is likely related to AeL’s highly charged pore wall, allows discrimination of polymer sizes with particularly high resolution. Multiple applications are now conceivable with this pore to screen various nonionic or charged polymers.

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2015 – Antibiotic translocation through porins studied in planar lipid bilayers using parallel platforms
Orbit 16, Port-a-Patch and Vesicle Prep Pro Publication in Analyst (2015) Authors: Weichbrodt C., Bajaj H., Baaken G., Wang J., Guinot S., Kreir M, Behrends J.C., Winterhalter M., Fertig N.

In general, the method of choice to characterize the conductance properties of channel-forming bacterial porins is electrophysiology. Here, the classical method is to reconstitute single porins into planar lipid bilayers to derive functional information from the observed channel conductance. In addition to an estimated pore size, ion selectivity or transport properties in general are of importance. For the latter, measuring the ion current fluctuation can provide some information about the mode of transport of charged molecules penetrating the proteins. For instance, increasing the external voltage modifies the residence time in the channel: charged molecules with the ability to permeate through channels will travel faster whereas non-permeating molecules get pushed to the constriction zone with enhanced residence time. Here, we are interested in the ability of antibiotics to permeate channels and compare different techniques to reveal fast events.

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2022 – Highly shape- and size-tunable membrane nanopores made with DNA
Orbit 16, Orbit mini and Vesicle Prep Pro Publication in Nature Nanotechnology (2022) Authors: Xing Y., Dorey A., Jayasinghe L., Howorka S.

Membrane nanopores are key for molecular transport in biology, portable DNA sequencing, label-free single-molecule analysis and nanomedicine. Transport traditionally relies on barrel-like channels of a few nanometres width, but there is considerable scientific and technological interest for much wider structures of tunable shape. Yet, these nanopores do not exist in nature and are challenging to build using existing de novo routes for proteins. Here, we show that rational design with DNA can drastically expand the structural and functional range of membrane nanopores. Our design strategy bundles DNA duplexes into pore subunits that modularly arrange to form tunable pore shapes and lumen widths of up to tens of nanometres. Functional units for recognition or signalling can be optionally attached. By dialling in essential parameters, we demonstrate the utility and potential of the custom-engineered nanopores by electrical direct single-molecule sensing of 10-nm-sized proteins using widely used research and hand-held analysis devices. The designer nanopores illustrate how DNA nanotechnology can deliver functional biomolecular structures to be used in synthetic biology, single-molecule enzymology and biophysical analysis, as well as portable diagnostics and environmental screening.

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2023 – The Unusual Aggregation and Fusion Activity of the Antimicrobial Peptide W-BP100 in Anionic Vesicles
Vesicle Prep Pro Publication in Membranes (2023) Authors: Ferreira A.R., Ferreira M., Nunes C. Reis S., Teixeira C., Gomes P., Gameiro P.

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

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