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Antibody engagement with the membrane-proximal external region (MPER) of the envelope glycoprotein (Env) of HIV-1 constitutes a distinctive molecular recognition phenomenon, the full appreciation of which is crucial for understanding the mechanisms that underlie the broad neutralization of the virus. Recognition of the HIV-1 Env antigen seems to depend on two specific features developed by antibodies with MPER specificity: (i) a large cavity at the antigen-binding site that holds the epitope amphipathic helix; and (ii) a membrane-accommodating Fab surface that engages with viral phospholipids. Thus, besides the main Fab–peptide interaction, molecular recognition of MPER depends on semi-specific (electrostatic and hydrophobic) interactions with membranes and, reportedly, on specific binding to the phospholipid head groups. Here, based on available cryo-EM structures of Fab–Env complexes of the anti-MPER antibody 10E8, we sought to delineate the functional antibody–membrane interface using as the defining criterion the neutralization potency and binding affinity improvements induced by Arg substitutions. This rational, Arg-based mutagenesis strategy revealed the position-dependent contribution of electrostatic interactions upon inclusion of Arg-s at the CDR1, CDR2 or FR3 of the Fab light chain. Moreover, the contribution of the most effective Arg-s increased the potency enhancement induced by inclusion of a hydrophobic-at-interface Phe at position 100c of the heavy chain CDR3. In combination, the potency and affinity improvements by Arg residues delineated a protein–membrane interaction site, whose surface and position support a possible mechanism of action for 10E8-induced neutralization. Functional delineation of membrane-interacting patches could open new lines of research to optimize antibodies of therapeutic interest that target integral membrane epitopes.

Introduction: Plasma membranes are not the homogeneous bilayers of uniformly distributed lipids but the lipid complex with laterally separated lipid raft membrane domains, which provide receptor, ion channel and enzyme proteins with a platform. The aim of this article is to review the mechanistic interaction of drugs with membrane lipid rafts and address the question whether drugs induce physicochemical changes in raft-constituting and raft-surrounding membranes. Methods: Literature searches of PubMed/MEDLINE and Google Scholar databases from 2000 to 2020 were conducted to include articles published in English in internationally recognized journals. Collected articles were independently reviewed by title, abstract and text for relevance. Results: The literature search indicated that pharmacologically diverse drugs interact with raft model membranes and cellular membrane lipid rafts. They could physicochemically modify functional protein-localizing membrane lipid rafts and the membranes surrounding such domains, affecting the raft organizational integrity with the resultant exhibition of pharmacological activity. Raft-acting drugs were characterized as ones to decrease membrane fluidity, induce liquid-ordered phase or order plasma membranes, leading to lipid raft formation; and ones to increase membrane fluidity, induce liquid-disordered phase or reduce phase transition temperature, leading to lipid raft disruption. Conclusion: Targeting lipid raft membrane domains would open a new way for drug design and development. Since angiotensin-converting enzyme 2 receptors which are a cell-specific target of and responsible for the cellular entry of novel coronavirus are localized in lipid rafts, agents that specifically disrupt the relevant rafts may be a drug against coronavirus disease 2019.

Cell membranes are very complex biological systems including a large variety of lipids and proteins. Therefore, they are difficult to extract and directly investigate with biophysical methods. For many decades, the characterization of simpler biomimetic lipid membranes, which contain only a few lipid species, provided important physico-chemical information on the most abundant lipid species in cell membranes. These studies described physical and chemical properties that are most likely similar to those of real cell membranes. Indeed, biomimetic lipid membranes can be easily prepared in the lab and are compatible with multiple biophysical techniques. Lipid phase transitions, the bilayer structure, the impact of cholesterol on the structure and dynamics of lipid bilayers, and the selective recognition of target lipids by proteins, peptides, and drugs are all examples of the detailed information about cell membranes obtained by the investigation of biomimetic lipid membranes. This review focuses specifically on the advances that were achieved during the last decade in the field of biomimetic lipid membranes mimicking the mammalian plasma membrane. In particular, we provide a description of the most common types of lipid membrane models used for biophysical characterization, i.e., lipid membranes in solution and on surfaces, as well as recent examples of their applications for the investigation of protein-lipid and drug-lipid interactions. Altogether, promising directions for future developments of biomimetic lipid membranes are the further implementation of natural lipid mixtures for the development of more biologically relevant lipid membranes, as well as the development of sample preparation protocols that enable the incorporation of membrane proteins in the biomimetic lipid membranes.

Giant vesicles are widely used as a model membrane system, both for basic biological systems and for their promising applications in the development of smart materials and cell mimetics, as well as in driving new technologies in synthetic biology and for the cosmetics and pharmaceutical industry. The reader is guided to use giant vesicles, from the formation of simple membrane platforms to advanced membrane and cell system models. It also includes fundamentals for understanding lipid or polymer membrane structure, properties and behavior. Every chapter includes ideas for further applications and discussions on the implications of the observed phenomena towards understanding membrane-related processes. The Giant Vesicle Book is meant to be a road companion, a trusted guide for those making their first steps in this field as well as a source of information required by experts. Key Features • A complete summary of the field, covering fundamental concepts, practical methods, core theory, and the most promising applications • A start-up package of theoretical and experimental information for newcomers in the field • Extensive protocols for establishing the required preparations and assays • Tips and instructions for carefully performing and interpreting measurements with giant vesicles or for observing them, including pitfalls • Approaches developed for investigating giant vesicles as well as brief overviews of previous studies implementing the described techniques • Handy tables with data and structures for ready reference Part I: The making of Chapter 1 Preparation methods for giant unilamellar vesicles - Rumiana Dimova, Pasquale Stano, Carlos M. Marques and Peter Walde Chapter 2 Preparation and properties of giant plasma membrane vesicles and giant unilamellar vesicles from natural membranes - Joseph H. Lorent and Ilya Levental Chapter 3 Protein reconstitution in giant vesicles - Matthias Garten, Daniel Lévy and Patricia Bassereau　 Chapter 4 GUVs with cytoskeleton - Tobias Härtel and Petra Schwille Part II: Giant vesicles theoretically and in silico Chapter 5 Understanding giant vesicles – a theoretical perspective - Reinhard Lipowsky Chapter 6 Simulating membranes, vesicles, and cells - Thorsten Auth, Dmitry A. Fedosov and Gerhard Gompper Chapter 7 Theory of vesicle dynamics in flow and electric fields - Petia M. Vlahovska and Chaouqi Misbah Chapter 8 Particle-membrane interactions - Jaime Agudo-Canalejo, Reinhard Lipowsky Chapter 9 Theory of polymer-membrane interactions - Fabrice Thalmann and Carlos M. Marques Part III: GUV-based techniques and what one can learn from them Chapter 10 Application of optical microscopy techniques on giant unilamellar vesicles - Luis A. Bagatolli Chapter 11 Mechanics assays of synthetic lipid membranes based on micropipette aspiration - Elisa Parra and David Needham Chapter 12 Atomic force microscopy of giant unilamellar vesicles - Andreas Janshoff Chapter 13 Manipulation and biophysical characterization of GUVs with an optical stretcher - Gheorghe Cojoc, Antoine Girot, Ulysse Delabre and Jochen Guck Chapter 14 Vesicle fluctuation analysis - John Hjort Ipsen, Allan Grønhøj Hansen and Tripta Bhatia Chapter 15 Using electric fields to assess membrane material properties in GUVs - Rumiana Dimova and　　Karin A. Riske Chapter 16 Creating membrane nanotubes from GUVs - Coline Prévost, Mijo Simunovic and Patricia Bassereau Chapter 17 Measuring GUV adhesion - Kheya Sengupta and Ana Smith Chapter 18 Phase diagrams and tie lines in GUVs - Matthew C. Blosser, Caitlin Cornell, Scott P. Rayermann and Sarah L. Keller Chapter 19 Vesicle dynamics in flow: an experimental approach - Victor Steinberg and Michael Levant Chapter 20 Membrane permeability measurements - Begoña Ugarte-Uribe, Ana J. García-Sáez and Mireille M. A. E. Claessens Part IV: GUVs as membrane interaction platforms Chapter 21 - Lipid and protein mobility in GUVs - Begoña Ugarte-Uribe, Kushal Kumar Das and Ana J. García-Sáez Chapter 22 Shining light on membranes - Rosangela Itri, Carlos M. Marques and Mauricio S. Baptista Chapter 23 Protein-membrane interactions - Eva M Schmid and Daniel A Fletcher Chapter 24 Effects of antimicrobial peptides and detergents on GUVs - Karin A. Riske Chapter 25 Lipid-polymer interactions: effect on GUVs shapes and behavior - Brigitte Pépin-Donat, François Quemeneur and Clément Campillo Part V: GUVs as complex membrane containers Chapter 26 Polymersomes - Praful Nair, David Christian and Dennis E. Discher Chapter 27 Giant hybrid polymer/lipid vesicles - Thi Phuong Tuyen Dao, Khalid Ferji, Fabio Fernandes, Manuel Prieto, Sébastien Lecommandoux, Emmanuel Ibarboure, Olivier Sandre and Jean-François Le Meins Chapter 28 Giant unilamellar vesicles: from protocell models to the construction of minimal cells - Masayuki Imai and Peter Walde Chapter 29 Encapsulation of aqueous two-phase systems and gels within giant lipid vesicles - Allyson M. Marianelli and Christine D. Keating Chapter 30 Droplet-supported giant lipid vesicles as compartments for synthetic biology - Johannes P. Frohnmayer, Marian Weiss, Lucia T. Benk, Jan-Willi Janiesch, Barbara Haller, Rafael B. Lira, Rumiana Dimova, Ilia Plazman and Joachim P. Spatz Appendices Appendix 1 List of lipids and physical constants of lipid bilayers Appendix 2 List of membrane dyes and fluorescent groups conjugated to lipids Appendix 3 List of detergents Appendix 4 List of water-soluble dyes or their fluorescent groups and their structures Rumiana Dimova leads an experimental lab in biophysics at the Max Planck Institute of Colloids and Interfaces in Potsdam, Germany. She has been working with giant vesicles already from the beginning of her scientific career. After being introduced into the magic of their preparation during her studies as a student in Bulgaria, she remained fascinated by their application and over the years pursued a variety of projects employing giant vesicles as a platform to develop new methods for the biophysical characterization of membranes and processes involving them. Until now, these studies have resulted in more than hundred peer-reviewed publications. Recently, she was also awarded the Emmy Noether distinction for women in physics of the European Physical Society. Carlos Marques , a CNRS senior scientist, founded the MCube group at the Charles Sadron Institute in Strasbourg, France, where he gears experimental and theoretical research towards the understanding of the physical properties of self-assembled lipid bilayers. Trained as a polymer theoretician, Carlos first got interested in membranes because they interact with polymers and published the first prediction for the membrane changes expected when polymers adsorb on lipid bilayers. He then expanded the scope of his group to include experiments and numerical simulations, and has now published many papers based on research with giant unilamellar vesicles, including the first study of lipid oxidation in GUVs and the discovery of the so-called PVA method for vesicle growth.

Myelin ensheathes selected axonal segments within the nervous system, resulting primarily in nerve impulse acceleration, as well as mechanical and trophic support for neurons. In the central and peripheral nervous systems, various proteins that contribute to the formation and stability of myelin are present, which also harbor pathophysiological roles in myelin disease. Many myelin proteins have common attributes, including small size, hydrophobic segments, multifunctionality, longevity, and regions of intrinsic disorder. With recent advances in protein biophysical characterization and bioinformatics, it has become evident that intrinsically disordered proteins (IDPs) are abundant in myelin, and their flexible nature enables multifunctionality. Here, we review known myelin IDPs, their conservation, molecular characteristics and functions, and their disease relevance, along with open questions and speculations. We place emphasis on classifying the molecular details of IDPs in myelin, and we correlate these with their various functions, including susceptibility to post-translational modifications, function in protein–protein and protein–membrane interactions, as well as their role as extended entropic chains. We discuss how myelin pathology can relate to IDPs and which molecular factors are potentially involved.

The interaction of the neuronal protein α-synuclein with lipid membranes appears crucial in the context of Parkinson’s disease, but the underlying mechanistic details, including the roles of different lipids in pathogenic protein aggregation and membrane disruption, remain elusive. Here, we used single-vesicle resolution fluorescence and label-free scattering microscopy to investigate the interaction kinetics of monomeric α-synuclein with surface-tethered vesicles composed of different negatively charged lipids. Supported by a theoretical model to account for structural changes in scattering properties of surface-tethered lipid vesicles, the data demonstrate stepwise vesicle disruption and asymmetric membrane deformation upon α-synuclein binding to phosphatidylglycerol vesicles at protein concentrations down to 10 nM (∼100 proteins per vesicle). In contrast, phosphatidylserine vesicles were only marginally affected. These insights into structural consequences of α-synuclein interaction with lipid vesicles highlight the contrasting roles of different anionic lipids, which may be of mechanistic relevance for both normal protein function (e.g., synaptic vesicle binding) and dysfunction (e.g., mitochondrial membrane interaction).

This study aimed to evaluate the antibacterial activities of α-terpineol against common foodborne pathogenic bacteria by agar well diffusion, broth microdilution, and colony counting assay. Propulsive research was conducted to reveal the antibacterial mechanisms, including morphology, infrared spectroscopy, membrane fluidity, membrane permeability, proton motive force, and oxidative phosphorylation. Results indicated that the antibacterial activity of α-terpineol decreased in the following order: Escherichia coli O157:H7, Salmonella typhimurium, Listeria monocytogenes, and Staphylococcus aureus. With an initial cell count of 8 log CFU/mL, α-terpineol at 0.8% (v/v) reduced E. coli O157:H7 and S. aureus by approximately 5.6 and 3.9 log CFU/mL within 1 h, respectively. Remarkable destruction in cell envelopes and intracellular organizations was observed. The hydroxyl of α-terpineol might form glycosidic bonds with carbohydrates and hydrogen bonds with PO2− and COO− via infrared spectroscopy analysis. Generalized polarization of Laurdan revealed that the polar head groups of phospholipids transformed into close packed. The anisotropy variations of trimethyl amino-diphenylhexatriene (TMA-DPH) and DPH suggested membrane fluidity decreased. The N-phenyl-1-naphthylamine intake assay indicated that α-terpineol impaired the cell wall. Propidium iodide staining was indicative of damaged plasma membranes. Electron transport in the cytoplasmic membrane was impaired, inducing reactive oxygen species accumulation. Both membrane electrical potential and membrane pH gradient collapsed. The disruption of proton motive force and the leakage of ATP resulted in a deficit of intracellular ATP. Our research revealed the interaction between the hydroxyl group of α-terpineol and bacteria affects membrane function contributing to the bacteria’s death.Key points• α-Terpineol hydroxy formed glycosidic bonds and hydrogen bonds with bacteria• α-Terpineol increased the membrane gelation and reduced the membrane fluidity• Proton motive force and oxidative phosphorylation were impaired

Deciphering cellular interactions is essential to both understand the mechanisms underlying a broad range of human diseases, but also to manipulate therapies targeting these diseases. Here, the formation of cell doublets resulting from specific membrane ligand‐receptor interactions is discovered. Based on this phenomenon, the study developed DoubletSeeker, a novel high‐throughput method for the reliable identification of ligand‐receptor interactions. The study shows that DoubletSeeker can accurately identify T cell receptor (TCR)‐antigen interactions with high sensitivity and specificity. Notably, DoubletSeeker effectively captured paired TCR‐peptide major histocompatibility complex (pMHC) information during a highly complex library‐on‐library screening and successfully identified three mutant TCRs that specifically recognize the MART‐1 epitope. In turn, DoubletSeeker can act as an antigen discovery platform that allows for the development of novel immunotherapy targets, making it valuable for investigating fundamental tumor immunology. This study introduces DoubletSeeker, an innovative method to screen ligand‐receptor interactions by examining the formation of cell doublets from membrane protein interactions. DoubletSeeker demonstrates efficiency in identifying various ligand‐receptor interactions, such as TCRs and pMHCs, with high sensitivity and specificity. Notably, when coupled with single‐doublet sequencing technology, DoubletSeeker enables the decoding of paired TCR‐pMHC information in a library‐on‐library screening approach.

The calcium release-activated calcium (CRAC) channel consists of STIM1, a Ca2+ sensor in the endoplasmic reticulum (ER), and Orai1, the Ca2+ ion channel in the plasma membrane. Ca2+ store depletion triggers conformational changes and oligomerization of STIM1 proteins and their direct interaction with Orai1. Structural alterations include the transition of STIM1 C-terminus from a folded to an extended conformation thereby exposing CAD (CRAC activation domain)/SOAR (STIM1-Orai1 activation region) for coupling to Orai1. In this study, we discovered that different point mutations of F394 in the small alpha helical segment (STIM1 α2) within the CAD/SOAR apex entail a rich plethora of effects on diverse STIM1 activation steps. An alanine substitution (STIM1 F394A) destabilized the STIM1 quiescent state, as evident from its constitutive activity. Single point mutation to hydrophilic, charged amino acids (STIM1 F394D, STIM1 F394K) impaired STIM1 homomerization and subsequent Orai1 activation. MD simulations suggest that their loss of homomerization may arise from altered formation of the CC1α1-SOAR/CAD interface and potential electrostatic interactions with lipid headgroups in the ER membrane. Consistent with these findings, we provide experimental evidence that the perturbing effects of F394D depend on the distance of the apex from the ER membrane. Taken together, our results suggest that the CAD/SOAR apex is in the immediate vicinity of the ER membrane in the STIM1 quiescent state and that different mutations therein can impact the STIM1/Orai1 activation cascade in various manners. Graphic abstractLegend: Upon intracellular Ca2+ store depletion of the endoplasmic reticulum (ER), Ca2+ dissociates from STIM1. As a result, STIM1 adopts an elongated conformation and elicits Ca2+ influx from the extracellular matrix (EM) into the cell due to binding to and activation of Ca2+-selective Orai1 channels (left). The effects of three point mutations within the SOARα2 domain highlight the manifold roles of this region in the STIM1/Orai1 activation cascade: STIM1 F394A is active irrespective of the intracellular ER Ca2+ store level, but activates Orai1 channels to a reduced extent (middle). On the other hand, STIM1 F394D/K cannot adopt an elongated conformation upon Ca2+ store-depletion due to altered formation of the CC1α1-SOAR/CAD interface and/or electrostatic interaction of the respective side-chain charge with corresponding opposite charges on lipid headgroups in the ER membrane (right).

Polymer nanoparticles offer significant benefits for improving delivery of biological therapeutics such as DNA and proteins, as they allow the cargo to be protected until it is delivered to a target cell. However, there are still challenges with achieving efficient delivery to the optimal cellular region. One significant roadblock is escape of nanoparticles from within the endosomal/lysosomal compartments into the cytosol. Here, we review the recent advances in understanding endosomal escape of polymer nanoparticles. We also discuss the current progress on investigating how nanoparticle structure can control endosomal escape. It is important to understand the fundamental biological processes that govern endosomal escape in order to design more effective therapeutic delivery systems.