Fungal membrane determinants affecting sensitivity to antifungal cyclic lipopeptides from Bacillus spp.

Bacillus spp. produce numerous antimicrobial metabolites. Among these metabolites, cyclic lipopeptides (CLP) including fengycins, iturins, and surfactins are known to have varying antifungal activity against phytopathogenic fungi. The differential activities of CLP have been attributed to diverse mechanisms of action on fungal membranes. However, the precise biochemical determinants driving their antifungal modes of action have not been conclusively identified. In this study, three plant pathogenic fungi of varying lipopeptide sensitivities, Alternaria solani, Cladosporium cucumerinum, and Fusarium sambucinum, were studied to determine how their cell membrane lipid compositions may confer sensitivity and/or tolerance to fengycin, iturin, and surfactin. Results indicated that sensitivity to all three lipopeptides correlated with lower ergosterol content and elevated phospholipid fatty acid unsaturation. Fungal sensitivity to surfactin was also notably different than fengycin and iturin, as surfactin was influenced more by lower phosphatidylethanolamine amounts, higher levels of phosphatidylinositol, and less by phospholipid fatty acyl chain length. Results from this study provide insight into the fungal membrane composition of A. solani, F. sambucinum, and C. cucumerinum and the specific membrane characteristics influencing the antifungal effectiveness of fengycin, iturin, and surfactin. Understanding of these determinants should enable more accurate prediction of sensitivity-tolerance outcomes for other fungal species exposed to these important CLP.

Cadmium and calcium ions' effects on the growth of Pleurotus ostreatus mycelia are related to phosphatidylethanolamine content.

Heavy metal Cd2+ can easily be accumulated by fungi, causing significant stress, with the fungal cell membrane being one of the primary targets. However, the understanding of the mechanisms behind this stress remains limited. This study investigated the changes in membrane lipid molecules of Pleurotus ostreatus mycelia under Cd2+ stress and the antagonistic effect of Ca2+ on this stress. Cd2+ in the growth media significantly inhibited mycelial growth, with increasing intensity at higher concentrations. The addition of Ca2+ mitigated this Cd2+-induced growth inhibition. Lipidomic analysis showed that Cd2+ reduced membrane lipid content and altered lipid composition, while Ca2+ counteracted these changes. The effects of both Cd2+ and Ca2+ on lipids are dose dependent and phosphatidylethanolamine appeared most affected. Cd2+ also caused a phosphatidylcholine/phosphatidylethanolamine ratio increase at high concentrations, but Ca2+ helped maintain normal levels. The acyl chain length and unsaturation of lipids remained unaffected, suggesting Cd2+ doesn't alter acyl chain structure of lipids. These findings suggest that Cd2+ may affect the growth of mycelia by inhibiting the synthesis of membrane lipids, particular the synthesis of phosphatidylethanolamine, providing novel insights into the mechanisms of Cd2+ stress in fungi and the role of Ca2+ in mitigating the stress.

Clathrin-Mediated Endocytosis of Multiple Nanoparticles Tends to Be Less Cooperative: A Computational Study.

The internalization of nanoparticles is of great significance for their biological applications. Clathrin-mediated endocytosis (CME) is one of the main endocytic pathways. However, there is still a lack of a fundamental understanding regarding the internalization of multiple nanoparticles via CME. Therefore, in this study, we conducted computational investigations to uncover detailed molecular mechanisms and kinetic pathways for differently shaped nanoparticles in the presence of clathrin. Particular focus is given to understanding the CME of multiple-nanoparticle systems. We found that unlike receptor-mediated endocytosis, multiple nanoparticles did not get cooperatively wrapped by the membrane but tended to undergo independent endocytosis in the presence of clathrin. To further investigate the endocytosis mechanism, we studied the effects of clathrins, nanoparticle shape, nanoparticle size, nanoparticle arrangement, and membrane surface tension. The self-assembly of clathrin prefers independent endocytosis for multiple nanoparticles. Besides, the cooperative behavior is weak with increasing nanoparticle-shape anisotropy. However, when the membrane tension is reduced, the endocytosis pathway for multiple nanoparticles is cooperative endocytosis. Moreover, we found that the self-assembly of clathrins reduces the critical size of nanoparticles to undergo cooperative wrapping by the cell membrane. Our results provide valuable insights into the molecular mechanisms of multiple nanoparticles through CME and offer useful guidance for the design of nanoparticles as drug/gene delivery carriers.

Cell Membrane-Anchored AND Logic Gate Aptasensor for Tumor Cell-Specific Imaging with Improved Accuracy.

Accurate and rapid imaging of tumor cells is of vital importance for early cancer diagnosis and intervention. Aptamer-based fluorescence sensors have become a potent instrument for bioimaging, while false positives and on-target off-tumors linked to single-biomarker aptasensors compromise the specificity and sensitivity of cancer imaging. In this paper, we describe a sequential response aptasensor for precise cancer cell identification that is based on a DNA "AND" logic gate. Specifically, the sensor consists of three single-stranded DNA, including the P-strand that can sensitively respond to an acid environment, the L-strand containing the ATP aptamer sequence, and the R-strand for target cell anchoring. These DNA strands hybridize with one another to create a Y-shaped structure (named Y-ALGN). The aptamer in the R-strand is utilized to anchor the sensor to the target cell membrane primarily. Responding to the extracellular acidic environment of the tumor (input 1), the I-motif sequence forms a tetramer structure so that the P-strand is released from the Y-shaped structure and exposes the ATP binding sites in the L-strand. Extracellular ATP, as input 2, continuously operates the DNA aptasensor to complete the logic computation. Upon the sequential response of both protons and ATP molecules, the aptasensor is activated with restored fluorescence on a particular cancer cell membrane. Benefiting from the precise computation capacity of the "AND" logic gate, the Y-ALGN aptasensor can distinguish between MCF-7 cells and normal cells with high accuracy. As a simple and dual-stimuli-responsive strategy, this nanodevice would offer a fresh approach for accurately diagnosing tumor cells.

Aggression to Biomembranes by Hydrophobic Tail Chains under the Stimulus of Reductant.

Stimulus-responsive materials hold significant promise for antitumor applications due to their variable structures and physical properties. In this paper, a series of peptides with a responsive viologen derivative, Pep-CnV (n = 1, 2, 3) were designed and synthesized. The process and mechanism of the interaction were studied and discussed. An ultraviolet-visible (UV) spectrophotometer and fluorescence spectrophotometer were used to study their redox responsiveness. Additionally, their secondary structures were measured by Circular Dichroism (CD) in the presence or absence of the reductant, Na2SO3. DPPC and DPPG liposomes were prepared to mimic normal and tumor cell membranes. The interaction between Pep-CnV and biomembranes was investigated by the measurements of surface tension and cargo leakage. Results proved Pep-CnV was more likely to interact with the DPPG liposome and destroy its biomembrane under the stimulus of the reductant. And the destruction increased with the length of the hydrophobic tail chain. Pep-CnV showed its potential as an intelligent antitumor agent.

MeCP2 is a naturally supercharged protein with cell membrane transduction capabilities.

The intrinsically disordered protein MeCP2 is a global transcriptional regulator encoded by the MECP2 gene. Although the structured domains of MeCP2 have been the subject of multiple studies, its unstructured regions have not been that extensively characterized. In this work, we show that MeCP2 possesses properties akin to those of supercharged proteins. By utilizing its unstructured portions, MeCP2 can successfully transduce across cell membranes and localize to heterochromatic foci in the nuclei, displaying uptake levels a third lower than a MeCP2 construct fused to the cell-penetrating peptide TAT. MeCP2 uptake can further be enhanced by the addition of compounds that promote endosomal escape following cellular trafficking by means of macropinocytosis. Using a combination of in silico prediction algorithms and live-cell imaging experiments, we mapped the sequence in MeCP2 responsible for its cellular incorporation, which bears a striking resemblance to TAT itself. Transduced MeCP2 was shown to interact with HDAC3. These findings provide valuable insight into the properties of MeCP2 and may be beneficial for devising future protein-based treatment strategies.

Structural Rearrangement of the AT1 Receptor Modulated by Membrane Thickness and Tension.

Membrane-embedded mechanosensitive (MS) proteins, including ion channels and G-protein coupled receptors (GPCRs), are essential for the transduction of external mechanical stimuli into biological signals. The angiotensin II type 1 (AT1) receptor plays many important roles in cardiovascular regulation and is associated with diseases such as hypertension and congestive heart failure. The membrane-mediated activation of the AT1 receptor is not well understood, despite this being one of the most widely studied GPCRs within the context of biased agonism. Here, we use extensive molecular dynamics (MD) simulations to characterize the effect of the local membrane environment on the activation of the AT1 receptor. We show that membrane thickness plays an important role in the stability of active and inactive states of the receptor, as well as the dynamic interchange between states. Furthermore, our simulation results show that membrane tension is effective in driving large-scale structural changes in the inactive state such as the outward movement of transmembrane helix 6 to stabilize intermediate active-like conformations. We conclude by comparing our simulation observations with AlphaFold 2 predictions, as a proxy to experimental structures, to provide a framework for how membrane mediated stimuli can facilitate activation of the AT1 receptor through the ß-arrestin signaling pathway.

Membrane Activity of Melittin and Magainin-I at Low Peptide-to-Lipid Ratio: Different Types of Pores and Translocation Mechanisms.

Antimicrobial peptides (AMPs) are believed to be a prominent alternative to the common antibiotics. However, despite decades of research, there are still no good clinical examples of peptide-based antimicrobial drugs for system application. The main reasons are loss of activity in the human body, cytotoxicity, and low selectivity. To overcome these challenges, a well-established structure-function relationship for AMPs is critical. In the present study, we focused on the well-known examples of melittin and magainin to investigate in detail the initial stages of AMP interaction with lipid membranes at low peptide-to-lipid ratio. By combining the patch-clamp technique with the bioelectrochemical method of intramembrane field compensation, we showed that these peptides interact with the membrane in different ways: melittin inserts deeper into the lipid bilayer than magainin. This difference led to diversity in pore formation. While magainin, after a threshold concentration, formed the well-known toroidal pores, allowing the translocation of the peptide through the membrane, melittin probably induced predominantly pure lipidic pores with a very low rate of peptide translocation. Thus, our results shed light on the early stages of peptide-membrane interactions and suggest new insights into the structure-function relationship of AMPs based on the depth of their membrane insertion.

Thermodynamic Study on Biomimetic Legionella gormanii Bacterial Membranes.

The presented studies were aimed at determining the interactions in model membranes (Langmuir monolayers) created of phospholipids (PL) isolated from Legionella gormanii bacteria cultured with (PL + choline) or without (PL - choline) choline and to describe the impact of an antimicrobial peptide, human cathelicidin LL-37, on PL's monolayer behavior. The addition of choline to the growth medium influenced the mutual proportions of phospholipids extracted from L. gormanii. Four classes of phospholipids-phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL), and their mixtures-were used to register compression isotherms with or without the LL-37 peptide in the subphase. Based on them the excess area (Ae), excess (ΔGe), and total (ΔGm) Gibbs energy of mixing were determined. The thermodynamic analyses revealed that the PL - choline monolayer showed greater repulsive forces between molecules in comparison to the ideal system, while the PL + choline monolayer was characterized by greater attraction. The LL-37 peptide affected the strength of interactions between phospholipids' molecules and reduced the monolayers stability. Accordingly, the changes in interactions in the model membranes allowed us to determine the difference in their susceptibility to the LL-37 peptide depending on the choline supplementation of bacterial culture.

A solution NMR methodology enabling the elucidation of small molecule phospholipid membrane adhesion and passive permeation parameters.

Quantifying small molecule uptake across a biological membrane of a target cell is crucial for the development of efficacious and selective drugs. However, current methods to obtaining such data are not trivial. Herein, we present an accessible, higher-throughput (20 minutes), 1H NMR spectroscopy assay, which enables the quantification of small molecule phospholipid passive membrane permeation and membrane adhesion parameters.

Endocytosis efficiency and targeting ability by the cooperation of nanoparticles.

Cooperative wrapping of nanoparticles (NPs) with small sizes is an important pathway for the uptake of NPs by cell membranes. However, the cooperative wrapping efficiency and the effects of NPs' rigidity remain ambiguous. With the aid of computer simulations, we show that the complete wrapping mechanism of cooperative endocytosis is that the aggregation of NPs leads to greater wrapping forces than the single NP case, which triggers the increase of the wrapping degree and in turn further increases the wrapping forces until they are finally fully taken up. The effects of the NP size, initial distance, interaction strength, arrangement and stiffness on cooperative endocytosis were systematically studied. The cooperative wrapping efficiency increases as the NP radius increases. Hexagonal close packed NPs have the highest internalization efficiency. When the interactions are strong, softer NPs exhibit higher endocytosis efficiency. We further propose two strategies by combining NPs with different wrapping properties for targeting applications. By combining two NPs decorated with different types of ligands, the combination NPs can only be fully endocytosed by the cell membrane with two cognate types of receptors and adhere to the normal cell membrane with only one type of receptor. We also design composite NPs using a large NP non-covalently decorated with several small NPs. By harnessing the competition between the ligand-receptor interactions and the excluded volume interactions between the small NPs and the lipid membrane, the composite NPs have targeting ability towards the cancer cell membrane. The design concept of combining NPs with different wrapping properties for drug targeting applications may be very promising in biomedicine.

Engineering Coatings Inspired by Cell Membrane Thermal Dynamics to Enhance Photothermal Therapy and Osteogenesis for Implant Infections.

Photothermal therapy (PTT) encounters challenges of rapid thermal loss and potential tissue damage. In response, we propose a Heat-Boost and Lock implant coating strategy inspired by the thermal adaptation of biological membranes, enabling precise local photothermal utilization. This coating incorporates a poly(tannic acid) (pTA) bridging layer on implants, facilitating stable layer-by-layer integration of a black phosphorus (BP) photothermal layer and a top cell membrane Heat-Boost and Lock layer. The cell membrane layer significantly curtails photothermal loss (extending the heat retention by 17.62%) and stores energy within its phospholipid bilayer, boosting photothermal effects near implants (achieving a temperature increasement of 275%). Theoretical analysis indicates that these local heat preservation properties of the cell membrane arise from its low thermal conductivity and phase-change properties. In a Staphylococcus aureus-infected bone implant model, our coating demonstrates precise antibacterial action around implants (reach an antibacterial ratio of 99.52%). The synergetic locking function of cell membrane and pTA delays BP biodegradation, ensuring favorable photothermal stability and long-term osteo-inductive performance (increasing the bone volume fraction by 53.45%). Beyond providing an endogenic biointerface, this strategy extends the application of cell membrane in local thermal management, offering possibilities for effective and safe PTT modalities.

Lipid lateral diffusion: mechanisms and modulators.

The lateral diffusion of lipids within a membrane is of paramount importance, serving as a central mechanism in numerous physiological processes including cell signaling, membrane trafficking, protein activity regulation, and energy transduction pathways. This review offers a comprehensive overview of lateral lipid diffusion in model biomembrane systems explored through the lens of neutron scattering techniques. We examine diverse models of lateral diffusion and explore the various factors influencing this fundamental process in membrane dynamics. Additionally, we offer a thorough summary of how different membrane-active compounds, including drugs, antioxidants, stimulants, and membrane proteins, affect lipid lateral diffusion. Our analysis unveils the intricate interplay between these additives and membranes, shedding light on their dynamic interactions. We elucidate that this interaction is governed by a complex combination of multiple factors including the physical state and charge of the membrane, the concentration of additives, the molecular architecture of the compounds, and their spatial distribution within the membrane. In conclusion, we briefly discuss the future directions and areas requiring further investigation in the realm of lateral lipid diffusion, highlighting the need to study more realistic membrane systems.

In Situ Synthesis and Visualization of Membrane SNAP25 Nano-Organization.

Cryo-electron tomography (cryo-ET) can provide insights into the structure and states of natural membrane environments to explore the role of SNARE proteins at membrane fusion and understand the relationship between their subcellular localization/formation and action mechanism. Nevertheless, the identification of individual molecules in crowded and low signal-to-noise ratio membrane environments remains a significant challenge. In this study, cryo-ET is employed to image near-physiological state 293T cell membranes, specifically utilizing in situ synthesized gold nanoparticles (AuNPs) bound with cysteine-rich protein tags to single-molecularly labeled synaptosomal-associated protein 25 (SNAP25) on the membrane surface. The high-resolution images reveal that SNAP25 is predominantly located in regions of high molecular density within the cell membrane and aggregates into smaller clusters, which may increase the fusion efficiency. Remarkably, a zigzag arrangement of SNAP25 is observed on the cell membrane. These findings provide valuable insights into the functional mechanisms of SNARE proteins.

Biomimetic Lipid Raft: Domain Stability and Interaction with Physiologically Active Molecules.

The cell membrane, also called the plasma membrane, is the membrane on the cytoplasmic surface that separates the extracellular from the intracellular. It is thin, about 10 nm thick when viewed with an electron microscope, and is composed of two monolayers of phospholipid membranes (lipid bilayers) containing many types of proteins. It is now known that this cell membrane not only separates the extracellular from the intracellular, but is also involved in sensory stimuli such as pain, itching, sedation, and excitement. Since the "Fluid mosaic model" was proposed for cell membranes, molecules have been thought to be homogeneously distributed on the membrane surface. Later, at the end of the twentieth century, the existence of "Phase-separated microdomain structures" consisting of ordered phases rich in saturated lipids and cholesterol was suggested, and these were termed "Lipid rafts." A model in which lipid rafts regulate cell signaling has been proposed and is the subject of active research.This chapter first outlines the physicochemical properties and thermodynamic models of membrane phase separation (lipid rafts), which play an important role in cell signaling. Next, how physiologically active molecules such as local anesthetics, cooling agents (menthol), and warming agents (capsaicin) interact with artificial cell membranes will be presented.It is undeniable that the plasma membrane contains many channels and receptors that are involved in the propagation of sensory stimuli. At the same time, however, it is important to understand that the membrane exerts a significant influence on the intensity and propagation of these stimuli.

Effect of molecular structure on membrane diffusion: Triphenylmethanes across Escherichia coli studied by second harmonic light scattering.

Understanding how the structure of molecules affects their permeability across cell membranes is crucial for many topics in biomedical research, including the development of drugs. In this work, we examine the transport rates of structurally similar triphenylmethane dyes, malachite green (MG) and brilliant green (BG), across the membranes of living Escherichia coli (E. coli) cells and biomimetic liposomes. Using the time-resolved second harmonic light scattering technique, we found that BG passively diffuses across the E. coli cytoplasmic membrane (CM) 3.8 times faster than MG. In addition, BG exhibits a diffusion rate 3.1 times higher than MG across the membranes of liposomes made from E. coli polar lipid extracts. Measurements on these two molecules, alongside previously studied crystal violet (CV), another triphenylmethane molecule, are compared against the set of propensity rules developed by Lipinski and co-workers for assessing the permeability of hydrophobic ion-like drug molecules through biomembranes. It indicates that BG's increased diffusion rate is due to its higher lipophilicity, with a distribution coefficient 25 times greater than MG. In contrast, CV, despite having similar lipophilicity to MG, shows negligible permeation through the E. coli CM on the observation scale, attributed to its more hydrogen bonding sites and larger polar surface area. Importantly, cell viability tests revealed that BG's antimicrobial efficacy is ∼2.4 times greater than that of MG, which aligns well with its enhanced diffusion into the E. coli cytosol. These findings offer valuable insights for drug design and development, especially for improving the permeability of poorly permeable drug molecules.

Molecular Dynamics Simulations Show That Short Peptides Can Drive Synthetic Cell Division by Binding to the Inner Membrane Leaflet.

An important functionality of lifelike "synthetic cells" is to mimic cell division. Currently, specialized proteins that induce membrane fission in living cells are the primary candidates for dividing synthetic cells. However, interactions between lipid membranes and proteins that are not found in living cells may also be suitable. Here, we discuss the potential of short membrane-anchored peptides to induce cell division. Specifically, we used the coarse-grained MARTINI model to investigate the interaction between short membrane-anchored peptides and a lipid bilayer patch. The simulation revealed that the anchored peptide induces significant spontaneous curvature and suggests that the lipid-peptide complex can be considered as a conically shaped "bulky headgroup" lipid. By systematically increasing the electrostatic charge of the peptide, we find that membrane-anchored peptides may generate sufficiently large constriction forces even at dilute coverages. Finally, we show that when the peptide has an opposite charge to the membrane, the peptide may induce division by binding the inner membrane leaflet of a synthetic cell, that is, cell division from within.

Employing Metadynamics to Predict the Membrane Partitioning of Carboxy-2H-Azirine Natural Products.

Natural products containing the carboxy-2H-azirine moiety are an exciting target for investigation due to their broad-spectrum antimicrobial activity and new chemical space they afford for novel therapeutic development. The carboxy-2H-azirine moiety, including those appended to well-characterized chemical scaffolds, is understudied, which creates a challenge for understanding potential modes of inhibition. In particular, some known natural product carboxy-2H-azirines have long hydrophobic tails, which could implicate them in membrane-associated processes. In this study, we examined a small set of carboxy-2H-azirine natural products with varied structural features that could alter membrane partitioning. We compared the predicted membrane partitioning and alignment of these compounds to those of established membrane embedders with similar chemical scaffolds. To accomplish this, we developed parameters within the framework of the CHARMM36 force field for the 2H-azirine functional group and performed metadynamics simulations of the partitioning into a model bacterial membrane from aqueous solution. We determined that the carboxy-2H-azirine functional group is strongly hydrophilic, imbuing the long-chain natural products with amphipathicity similar to the known membrane-embedding molecules to which they were compared. For the long-chain analogs, the carboxy-2H-azirine head group stays within 1 nm of the phosphate layer, while the hydrophobic tail sits within the membrane. The carboxy-2H-azirine lacking the long alkyl chain instead partitions completely into aqueous solution.

Infrared Spectroscopy of Live Cells Using High-Aspect-Ratio Metal-on-Dielectric Metasurfaces.

Fourier transform infrared (FTIR) spectroscopy is widely used for molecular analysis. However, for the materials situated in an aqueous environment, a precondition for live biological objects such as cells, transmission-based FTIR is prevented by strong water absorption of mid-infrared (MIR) light. Reflection-based cellular assays using internal reflection elements (IREs) such as high-index prisms or flat plasmonic metasurfaces mitigate these issues but suffer from a shallow probing volume localized near the plasma membrane. Inspired by the recent introduction of high-aspect-ratio nanostructures as a novel platform for manipulating cellular behavior, we demonstrate that the integration of plasmonic metasurfaces with tall dielectric nanostructures dramatically enhances the sensing capabilities of FTIR spectroscopy. We also demonstrate the ability of a metal-on-dielectric metasurface to transduce intracellular processes, such as protein translocation to high-curvature membrane regions during cell adhesion, into interpretable spectral signatures of the reflected light.

Cellular Output and Physicochemical Properties of the Membrane-Derived Vesicles Depend on Chemical Stimulants.

Synthetic liposomes are widely used as drug delivery vehicles in biomedical treatments, such as for mRNA-based antiviral vaccines like those recently developed against SARS-CoV-2. Extracellular vesicles (EVs), which are naturally produced by cells, have emerged as a next-generation delivery system. However, key questions regarding their origin within cells remain unresolved. In this regard, plasma membrane vesicles (PMVs), which are essentially produced from the cellular plasma membrane (PM), present a promising alternative. Unfortunately, their properties relevant to biomedical applications have not be extensively studied. Therefore, we conducted a thorough investigation of the methods used in the production of PMVs. By leveraging advanced fluorescence techniques in microscopy and flow cytometry, we demonstrated a strong dependence of the physicochemical attributes of PMVs on the chemicals used during their production. Following established protocols employing chemicals such as paraformaldehyde (PFA), N-ethylmaleimide (NEM) or dl-dithiothreitol (DTT) and by developing a modified NEM-based method that involved a hypotonic shock step, we generated PMVs from THP-1 CD1d cells. We systematically compared key parameters such as vesicle output, their size distribution, vesicular content analysis, vesicular membrane lipid organization and the mobility of a transmembrane protein. Our results revealed distinct trends: PMVs isolated using NEM-based protocols closely resembled natural vesicles, whereas PFA induced significant molecular cross-linking, leading to notable changes in the biophysical properties of the vesicles. Furthermore, our novel NEM protocol enhanced the efficiency of PMV production. In conclusion, our study highlights the unique characteristics of chemically produced PMVs and offers insights into their potentially diverse yet valuable biological functions.