DPPA as a Potential Cell Membrane Component Responsible for Binding Amyloidogenic Protein Human Cystatin C.

A phospholipid bilayer is a typical structure that serves crucial functions in various cells and organelles. However, it is not unusual for it to take part in pathological processes. The cell membrane may be a binding target for amyloid-forming proteins, becoming a factor modulating the oligomerization process leading to amyloid deposition-a hallmark of amyloidogenic diseases-e.g., Alzheimer's disease. The information on the mechanisms governing the oligomerization influenced by the protein-membrane interactions is scarce. Therefore, our study aims to describe the interactions between DPPA, a cell membrane mimetic, and amyloidogenic protein human cystatin C. Circular dichroism spectroscopy and differential scanning calorimetry were used to monitor (i) the secondary structure of the human cystatin C and (ii) the phase transition temperature of the DPPA, during the protein-membrane interactions. NMR techniques were used to determine the protein fragments responsible for the interactions, and molecular dynamics simulations were applied to provide a molecular structure representing the interaction. The obtained data indicate that the protein interacts with DPPA, submerging itself into the bilayer via the AS region. Additionally, the interaction increases the content of α-helix within the protein's secondary structure and stabilizes the whole molecule against denaturation.

Methods for the Characterization of the Colloidal Properties of Bacterial Membrane Vesicles.

Bacterial membrane vesicles (BMVs) are extracellular vesicles secreted by either Gram-positive or Gram-negative bacteria. These BMVs typically possess a diameter between 20 and 250 nm. Due to their size, when these BMVs are suspended in another medium, they could be constituents of a colloidal system. It has been hypothesized that investigating BMVs as colloidal particles could help characterize BMV interactions with other environmentally relevant surfaces. Developing a more thorough understanding of BMV interactions with other surfaces would be critical for developing predictive models of their environmental fate. However, this bio-colloidal perspective has been largely overlooked for BMVs, despite the wealth of methods and expertise available to characterize colloidal particles. A particular strength of taking a more colloid-centric approach to BMV characterization is the potential to quantify a particle's attachment efficiency (α). These values describe the likelihood of attachment during particle-particle or particle-surface interactions, especially those interactions which are governed by physicochemical interactions (such as those described by DLVO and xDLVO theory). Elucidating the influence of physical and electrochemical properties on these attachment efficiency values could give insights into the primary factors driving interactions between BMVs and other surfaces. This chapter details methods for the characterization of BMVs as colloids, beginning with size and surface charge (i.e., electrophoretic mobility/zeta potential) measurements. Afterward, this chapter will address experimental design, especially column experiments, targeted for BMV investigation and the determination of α values.

Spray Drying of Bacterial Membrane Vesicles for Vaccine Delivery.

Extracellular vesicles are nanosized lipid-bilayered spheres secreted from every living cell and they serve physiological and pathophysiological functions. Bacterial membrane vesicles are shed from both Gram-negative and Gram-positive bacteria and harbor many virulence factors, nuclear material, polysaccharides, proteins, and antigenic determinants, which are essential for immune recognition and evasion. Hence, bacterial membrane vesicles are very promising vaccine candidates. Spray drying is a well-established pharmaceutical technique to produce inhalable dry powders with enhanced stability for formulations of vaccines. In this chapter, we illustrate general guidelines for spray drying of bacterial extracellular vesicles to improve their stability without compromising their immunogenic protective effect. We discuss some of the most important experiments to characterize the generated spray-dried bacterial membrane vesicle powder vaccine.

Superposition of Substrate Deformation Fields Induced by Molecular Clutches Explains Cell Spatial Sensing of Ligands.

Cells can sense the physical properties of the extracellular matrices (ECMs), such as stiffness and ligand density, through cell adhesions to actively regulate their behaviors. Recent studies have shown that varying ligand spacing of ECMs can influence adhesion size, cell spreading, and even stem cell differentiation, indicating that cells have the spatial sensing ability of ECM ligands. However, the mechanism of the cells' spatial sensing remains unclear. In this study, we have developed a lattice-spring motor-clutch model by integrating cell membrane deformation, the talin unfolding mechanism, and the lattice spring for substrate ligand distribution to explore how the spatial distribution of integrin ligands and substrate stiffness influence cell spreading and adhesion dynamics. By applying the Gillespie algorithm, we found that large ligand spacing reduces the superposition effect of the substrate's displacement fields generated by pulling force from motor-clutch units, increasing the effective stiffness probed by the force-sensitive receptors; this finding explains a series of previous experiments. Furthermore, using the mean-field theory, we obtain the effective stiffness sensed by bound clutches analytically; our analysis shows that the bound clutch number and ligand spacing are the two key factors that affect the superposition effects of deformation fields and, hence, the effective stiffness. Overall, our study reveals the mechanism of cells' spatial sensing, i.e., ligand spacing changes the effective stiffness sensed by cells due to the superposition effect of deformation fields, which provides a physical clue for designing and developing biological materials that effectively control cell behavior and function.

Influence of phospholipid head and tail molecular structures on cell membrane mechanical response under tension.

Biological cell membranes are primarily comprised of a diverse lipid bilayer with multiple phospholipid (lipid) types, each of which is comprised of a hydrophilic headgroup and two hydrophobic hydrocarbon tails. The lipid type determines the molecular structure of head and tail groups, which can affect membrane mechanics at nanoscale and subsequently cell viability under mechanical loading. Hence, using molecular dynamics simulations, the current study investigated seven membrane phospholipids and the effect of their structural differences on physical deformation, mechanoporation damage, and mechanical failure of the membranes under tension. The inspected phospholipids showed similar yield stresses and strains, as well as pore evolution and damage, but significantly different failure strains. In general, failure occurred at a lower strain for lipids with a larger equilibrium area per lipid. The obtained results suggest that larger headgroup structure, greater degree of unsaturation, and tail-length asymmetry influenced the phospholipids' ability to pack against each other, increased the fluidity and equilibrium area per lipid of the membrane, and resulted in lower failure strain. Overall, this study provides insights on how different phospholipid structures affect membrane physical responses at the molecular level and serves as a reference for future studies of more complex membrane systems with intricate biophysical properties.

Intracellular delivery strategies using membrane-interacting peptides and proteins.

While the cellular cytosol and organelles contain attractive targets for disease treatments, it remains a challenge to deliver therapeutic biomacromolecules to these sites. This is due to the selective permeability of the plasma and endosomal membranes, especially for large and hydrophilic therapeutic cargos such as proteins and nucleic acids. In response, many different delivery systems and molecules have been devised to help therapeutics cross these barriers to reach cytosolic targets. Among them are peptide and protein-based systems, which have several advantages over other natural and synthetic materials including their ability to interact with cell membranes. In this review, we will describe recent advances and current challenges of peptide and protein strategies that leverage cell membrane association and modulation to enable cytosolic delivery of biomacromolecule cargo. The approaches covered here include peptides and proteins derived from or inspired by natural sequences as well as those designed de novo for delivery function.

Analysis of protein-protein and protein-membrane interactions by isotope-edited infrared spectroscopy.

The objective of this work is to highlight the power of isotope-edited Fourier transform infrared (FTIR) spectroscopy in resolving important problems encountered in biochemistry, biophysics, and biomedical research, focusing on protein-protein and protein membrane interactions that play key roles in practically all life processes. An overview of the effects of isotope substitutions in (bio)molecules on spectral frequencies and intensities is given. Data are presented demonstrating how isotope-labeled proteins and/or lipids can be used to elucidate enzymatic mechanisms, the mode of membrane binding of peripheral proteins, regulation of membrane protein function, protein aggregation, and local and global structural changes in proteins during functional transitions. The use of polarized attenuated total reflection FTIR spectroscopy to identify the spatial orientation and the secondary structure of a membrane-bound interfacial enzyme and the mode of lipid hydrolysis is described. Methods of production of site-directed, segmental, and domain-specific labeling of proteins by the synthetic, semisynthetic, and recombinant strategies, including advanced protein engineering technologies such as nonsense suppression and frameshift quadruplet codons are overviewed.

Using Nanoscale Passports To Understand and Unlock Ion Channels as Gatekeepers of the Cell.

Natural ion channels are proteins embedded in the cell membrane that control many aspects of cell and human physiology by acting as gatekeepers, regulating the flow of ions in and out of cells. Advances in nanotechnology have influenced the methods for studying ion channels in vitro, as well as ways to unlock the delivery of therapeutics by modulating them in vivo. This review provides an overview of nanotechnology-enabled approaches for ion channel research with a focus on the synthesis and applications of synthetic ion channels. Further, the uses of nanotechnology for therapeutic applications are critically analyzed. Finally, we provide an outlook on the opportunities and challenges at the intersection of nanotechnology and ion channels. This work highlights the key role of nanoscale interactions in the operation and modulation of ion channels, which may prompt insights into nanotechnology-enabled mechanisms to study and exploit these systems in the near future.

A nondestructive membrane engineering method using an amphiphilic polymer.

The cellular signaling process or ion transport is mediated by membrane proteins (MPs) located on the cell surface, and functional studies of MPs have mainly been conducted using cells endogenously or transiently expressing target proteins. Reconstitution of purified MPs in the surface of live cells would have advantages of short manipulation time and ability to target cells in which gene transfection is difficult. However, direct reconstitution of MPs in live cells has not been established. The traditional detergent-mediated reconstitution method of MPs into a lipid bilayer cannot be applied to live cells because this disrupts and reforms the lipid bilayer structure, which is detrimental to cell viability. In this study, we demonstrated that GPCRs (prostaglandin E2 receptor 4 [EP4] and glucagon-like peptide-1 receptor [GLP1R]) or serotonin receptor 3A (5HT3A), a ligand-gated ion channel, stabilized with amphiphilic poly-γ-glutamate (APG), can be reconstituted into mammalian cell plasma membranes without affecting cell viability. Furthermore, 5HT3A reconstituted in mammalian cells showed ligand-dependent Ca2+ ion transport activity. APG-mediated reconstitution of GPCR in synthetic liposomes showed that electrostatic interaction between APG and membrane surface charge contributed to the reconstitution process. This APG-mediated membrane engineering method could be applied to the functional modification of cell membranes with MPs in live cells.

Membrane prewetting by condensates promotes tight-junction belt formation.

Biomolecular condensates enable cell compartmentalization by acting as membraneless organelles1. How cells control the interactions of condensates with other cellular structures such as membranes to drive morphological transitions remains poorly understood. We discovered that formation of a tight-junction belt, which is essential for sealing epithelial tissues, is driven by a wetting phenomenon that promotes the growth of a condensed ZO-1 layer2 around the apical membrane interface. Using temporal proximity proteomics in combination with imaging and thermodynamic theory, we found that the polarity protein PATJ mediates a transition of ZO-1 into a condensed surface layer that elongates around the apical interface. In line with the experimental observations, our theory of condensate growth shows that the speed of elongation depends on the binding affinity of ZO-1 to the apical interface and is constant. Here, using PATJ mutations, we show that ZO-1 interface binding is necessary and sufficient for tight-junction belt formation. Our results demonstrate how cells exploit the collective biophysical properties of protein condensates at membrane interfaces to shape mesoscale structures.

Nonequilibrium Dynamic Phase Diagram for Transmembrane Transport of Active Particles.

In recent years, there has been considerable push toward the biomedical applications with active particles, which have great potential to revolutionize disease diagnostics and therapy. The direct penetration of active particles through the cell membrane leads to more efficient intracellular delivery than previously considered endocytosis processes but may cause membrane disruption. Understanding fundamental behaviors of cell membranes in response to such extreme impacts by active particles is crucial to develop active particle-based biomedical technologies and manage health and safety issues in this emerging field. Unfortunately, the physical principles underlying the nonequilibrium behaviors from endocytosis to direct penetration remain elusive, and experiments are challenging. Here, we present a computed dynamic phase diagram for transmembrane transport of active particles and identify four characteristic dynamic phases in endocytosis and direct penetration according to the particle activity and membrane tension. The boundaries dividing these phases are analytically obtained with theoretical models, elucidating the nonequilibrium physics and criteria for the transition between different phases. Furthermore, we numerically and experimentally show three distinct dynamic regimes related to the interplay between necking and wrapping during the endocytosis process of active particles, which strikingly contrast the regimes for passive particles. Overall, these findings could be useful for sharpening the understanding of basic principles underlying biological issues related to the safe and efficient biomedical applications of such emerging matters.

Quantifying biomolecular organisation in membranes with brightness-transit statistics.

Cells crucially rely on the interactions of biomolecules at their plasma membrane to maintain homeostasis. Yet, a methodology to systematically quantify biomolecular organisation, measuring diffusion dynamics and oligomerisation, represents an unmet need. Here, we introduce the brightness-transit statistics (BTS) method based on fluorescence fluctuation spectroscopy and combine information from brightness and transit times to elucidate biomolecular diffusion and oligomerisation in both cell-free in vitro and in vitro systems incorporating living cells. We validate our approach in silico with computer simulations and experimentally using oligomerisation of EGFP tethered to supported lipid bilayers. We apply our pipeline to study the oligomerisation of CD40 ectodomain in vitro and endogenous CD40 on primary B cells. While we find a potential for CD40 to oligomerize in a concentration or ligand depended manner, we do not observe mobile oligomers on B cells. The BTS method combines sensitive analysis, quantification, and intuitive visualisation of dynamic biomolecular organisation.

Tensing Flipper: Photosensitized Manipulation of Membrane Tension, Lipid Phase Separation, and Raft Protein Sorting in Biological Membranes.

The lateral organization of proteins and lipids in the plasma membrane is fundamental to regulating a wide range of cellular processes. Compartmentalized ordered membrane domains enriched with specific lipids, often termed lipid rafts, have been shown to modulate the physicochemical and mechanical properties of membranes and to drive protein sorting. Novel methods and tools enabling the visualization, characterization, and/or manipulation of membrane compartmentalization are crucial to link the properties of the membrane with cell functions. Flipper, a commercially available fluorescent membrane tension probe, has become a reference tool for quantitative membrane tension studies in living cells. Here, we report on a so far unidentified property of Flipper, namely, its ability to photosensitize singlet oxygen (1O2) under blue light when embedded into lipid membranes. This in turn results in the production of lipid hydroperoxides that increase membrane tension and trigger phase separation. In biological membranes, the photoinduced segregated domains retain the sorting ability of intact phase-separated membranes, directing raft and nonraft proteins into ordered and disordered regions, respectively, in contrast to radical-based photo-oxidation reactions that disrupt raft protein partitioning. The dual tension reporting and photosensitizing abilities of Flipper enable simultaneous visualization and manipulation of the mechanical properties and lateral organization of membranes, providing a powerful tool to optically control lipid raft formation and to explore the interplay between membrane biophysics and cell function.

Preparation and Structural Analysis of Lipoteichoic Acid on Cell Membranes Derived from Lactic Acid Bacteria.

Gram-positive bacteria, including lactic acid bacteria (LAB), possess lipoteichoic acid (LTA) on the cell surface. LTA is an amphiphilic molecule typically composed of hydrophilic glycerolphosphate polymer and hydrophobic anchor glycolipid moieties. It is involved in physiological properties of the cell surface and also plays roles in interactions with the host. Appropriate preparation procedures, such as extraction and purification, are required to clarify the structure-activity relationship. Structural diversity of LTA has been reported at the bacterial species and strain levels, and structural differences might affect interactions with the host. This chapter introduces techniques for preparation and structural analysis of LTA derived from LAB. It consists of four sections, covering butanol extraction, hydrophobic interaction chromatography, immunoblotting, and structural analysis. Technical notes containing supplemental information about the individual steps are also provided.

Precise control of transmembrane current via regulating bionic lipid membrane composition.

The transport of ions through biological ion channels is regulated not only by their structural characteristics but also by the composition of the phospholipid membrane, which serves as a carrier for nanochannels. Inspired by the modulation of ion currents by lipid membrane composition, exemplified by the activation of the K+ channel of Streptomyces A by anionic lipids, we present a biomimetic nanochannel system based on combining DNA nanotechnology with two-dimensional graphene oxide (GO) nanosheets. By designing multibranched DNA nanowires, we assemble programmable DNA scaffold networks (DSNs) on the GO surface to precisely control membrane composition. Modulating the DSN layers from one to five enhances DNA composition, yielding a maximum 12-fold enhancement in ion current, primarily due to charge effects. Incorporating DNAzymes facilitates reversible modulation of membrane composition, enabling cyclic conversion of ion current. This approach offers a pathway for creating devices with highly efficient, tunable ion transport, applicable in diverse fields like mass transport, environmental protection, biomimetic channels, and biosensors.

PCA, PC-CVA, and Random Forest of GCIB-SIMS Data for the Elucidation of Bacterial Envelope Differences in Antibiotic Resistance Research.

Antibiotic resistance can rapidly spread through bacterial populations via bacterial conjugation. The bacterial membrane has an important role in facilitating conjugation, thus investigating the effects on the bacterial membrane caused by conjugative plasmids, antibiotic resistance, and genes involved in conjugation is of interest. Analysis of bacterial membranes was conducted using gas cluster ion beam-secondary ion mass spectrometry (GCIB-SIMS). The complexity of the data means that data analysis is important for the identification of changes in the membrane composition. Preprocessing of data and several analytical methods for identification of changes in bacterial membranes have been investigated. GCIB-SIMS data from Escherichia coli samples were subjected to principal components analysis (PCA), principal components-canonical variate analysis (PC-CVA), and Random Forests (RF) data analysis with the aim of extracting the maximum biological information. The influence of increasing replicate data was assessed, and the effect of diminishing biological variation was studied. Optimized m/z region-specific scaling provided improved clustering, with an increase in biologically significant peaks contributing to the loadings. PC-CVA improved clustering, provided clearer loadings, and benefited from larger data sets collected over several months. RF required larger sample numbers and while showing overlap with the PC-CVA, produced additional peaks of interest. The combination of PC-CVA and RF allowed very subtle differences between bacterial strains and growth conditions to be elucidated for the first time. Specifically, comparative analysis of an E. coli strain with and without the F-plasmid revealed changes in cyclopropanation of fatty acids, where the addition of the F-plasmid led to a reduction in cyclopropanation.

Materials evaluation using cell-sized liposomes.

Cell membranes play a vital role in delineating the internal cellular environment from the external surroundings, going beyond mere compartmentalization. Researchers have delved into the structural organization, properties, and functional roles of biological membranes, paving the way for their application in substance identification, detection, and quantification. This review introduces various studies and their implications for future research. It underscores the advantages of employing cell-sized liposomes, which enable real-time observation for rapid detection and analysis of diverse materials. The utility of cell-sized liposomes extends to their size, dynamic shape changes, and phase-separation, offering valuable insights into the evaluation of targeted materials.

Pyrazolone-Protein Interaction Enables Long-Term Retention Staining and Facile Artificial Biorecognition on Cell Membranes.

Cell membrane genetic engineering has been utilized to confer cell membranes with functionalities for diagnostic and therapeutic purposes but concerns over cost and variable modification results. Although nongenetic chemical modification and phospholipid insertion strategies are more convenient, they still face bottlenecks in either biosafety or stability of the modifications. Herein, we show that pyrazolone-bearing molecules can bind to proteins with high stability, which is mainly contributed to by the multiple interactions between pyrazolone and basic amino acids. This new binding model offers a simple and versatile noncovalent approach for cell membrane functionalization. By binding to cell membrane proteins, pyrazolone-bearing dyes enabled precise cell tracking in vitro (>96 h) and in vivo (>21 days) without interfering with the protein function or causing cell death. Furthermore, the convenient anchor of pyrazolone-bearing biotin on cell membranes rendered the biorecognition to avidin, showing the potential for artificially creating cell targetability.

Activity of Membrane-Permeabilizing Lpt Peptides.

Herein, we investigated the toxicity and membrane-permeabilizing capabilities of Lpt and Lpt-like peptides, belonging to type I toxin-antitoxin systems carried by plasmid DNA of Lacticaseibacillus strains. These 29 amino acid peptides are predicted to form α-helical structures with a conserved central hydrophobic sequence and differently charged hydrophilic termini. Like Lpt, the expression of Lpt-like in E. coli induced growth arrest, nucleoid condensation, and cell membrane damage, suggesting membrane interaction as the mode of action. The membrane permeabilization activity of both peptides was evaluated by using liposome leakage assays, dynamic light scattering, and CD spectroscopy. Lpt and Lpt-like showed liposome leakage activity, which did not lead to liposome disruption but depended on peptide concentration. Lpt was generally more effective than Lpt-like, probably due to different physical chemical properties. Leakage was significantly reduced in larger liposomes and increased with negatively charged PCPS liposomes, indicating that electrostatic interactions and membrane curvature influence peptide activity. Contrary to most membrane-active peptides, Lpt an Lpt-like progressively lost their α-helical structure upon interaction with liposomes. Our data are inconsistent with the formation of membrane-spanning peptide pores but support a mechanism relying on the transient failure of the membrane permeability barrier possibly through the formation of "lipid pores".