Biological Membranes Research Articles

Fluorescence lifetime imaging microscopy of flexible and rigid dyes probes the biophysical properties of synthetic and biological membranes

Sensing of the biophysical properties of membranes using molecular reporters has recently regained wide-spread attention. This was elicited by the development of new probes of exquisite optical properties and increased performance, combined with developments in fluorescence detection. Here, we report on fluorescence lifetime imaging (FLIM) of various rigid and flexible fluorescent dyes to probe the biophysical properties of synthetic and biological membranes at steady state as well as upon the action of external membrane-modifying agents. We tested the solvatochromic dyes Nile Red and NBD, the viscosity sensor Bodipy C12, the flipper dye FliptR as well as the dyes DiO, Bodipy C16, lissamine-rhodamine, Atto647 which are dyes with no previous reported environmental sensitivity. The performance of the fluorescent probes, many of which are commercially available, was benchmarked with the well-known environmental reporters, with Nile Red and Bodipy C12 being specific reporters of medium hydration and viscosity, respectively. We show that some widely used ordinary dyes with no previous report of sensing capabilities can exhibit competing performance compared to highly sensitive commercially available or custom-based solvatochromic, molecular rotors or flipper in a wide range of biophysics experiments. Compared to other methods, FLIM is a minimally invasive and non-destructive method with optical resolution. It enables biophysical mapping at steady state or assessment of the changes induced by membrane-active molecules at subcellular level in both synthetic and biological membranes when intensity measurements fail to do so. The results have important consequences for the specific choice of the sensor and take into consideration factors such as probe sensitivity, response to environmental changes, ease and speed of data analysis and the probe's intracellular distribution, as well as potential side effects induced by labelling and imaging.

Lipid metabolism in crocodilians: a field with promising applications in the field of ecotoxicology.

In the last years, lipid physiology has become an important research target for systems biology applied to the field of ecotoxicology. Lipids are not only essential components of biological membranes, but also participate in extra and intracellular signaling processes and as signal transducers and amplifiers of regulatory cascades. Particularly in sauropsids, lipids are the main source of energy for reproduction, growth, and embryonic development. In nature, organisms are exposed to different stressors, such as parasites, diseases and environmental contaminants, which interact with lipid signaling and metabolic pathways, disrupting lipid homeostasis. The system biology approach applied to ecotoxicological studies is crucial to evaluate metabolic regulation under environmental stress produced by xenobiotics. In this review, we cover information of molecular mechanisms that contribute to lipid metabolism homeostasis in sauropsids, specifically in crocodilian species. We focus on the role of lipid metabolism as a powerful source of energy and its importance during oocyte maturation, which has been increasingly recognized in many species, but information is still scarce in crocodiles. Finally, we highlight priorities for future research on the influence of environmental stressors on lipid metabolism, their potential effect on the reproductive system and thus on the offspring, and their implications on crocodilians conservation.

Altered Plasma Membrane Lipid Composition in Hypertensive Neutrophils Impacts Epithelial Sodium Channel (ENaC) Endocytosis

Biological membranes are composed of a lipid bilayer with embedded proteins, including ion channels like the epithelial sodium channel (ENaC), which are critical for sodium homeostasis and implicated in arterial hypertension (HTN). Changes in the lipid composition of the plasma membrane can significantly impact cellular processes related to physiological functions. We hypothesized that the observed overexpression of ENaC in neutrophils from HTN patients might result from alterations in the structuring domains within the plasma membrane, disrupting the endocytic processes responsible for ENaC retrieval. This study assessed the structural lipid composition of neutrophil plasma membranes from HTN patients along with the expression patterns of key elements regulating ENaC at the plasma membrane. Our findings suggest alterations in microdomain structure and SGK1 kinase activity, which could prolong ENaC presence on the plasma membrane. Additionally, we propose that the proteasomal and lysosomal degradation pathways are insufficient to diminish ENaC presence at the plasma membrane in HTN. These results highlight the importance of understanding ENaC retrieval mechanisms and suggest that targeting these mechanisms could provide insights for developing drugs to prevent and treat HTN.

Applications of Electrochemistry at the ITIES in Drug Discovery and Development – A Review

AbstractThe field of electrochemistry at the interface between two immiscible electrolyte solutions (ITIES) has been continuously expanding over the years due to their vast number of applications, including to investigate the partitioning of ionizable drugs at liquid‐liquid systems. The aim of this Review is to highlight the great potential of ITIES as simple model of biological membranes to gather information on drug partition, lipophilicity, and pharmacokinetics that can be very useful for researchers in the field of drug discovery for development of new drugs with enhanced permeability. Relevant contributions and perspectives to improve the applicability of ITIES in partition studies were highlighted and discussed. The second part of this Review pretends to highlight the application of electrochemistry at the ITIES as experimental technique to investigate interactions between small ligands, including drugs, and DNA, a topic of high research interest in pharmaceutical and biological sciences, which remains with lots of opportunities to explore.

Cholesterol Changes Interfacial Water Alignment in Model Cell Membranes.

The nanoscopic layer of water that directly hydrates biological membranes plays a critical role in maintaining the cell structure, regulating biochemical processes, and managing intermolecular interactions at the membrane interface. Therefore, comprehending the membrane structure, including its hydration, is essential for understanding the chemistry of life. While cholesterol is a fundamental lipid molecule in mammalian cells, influencing both the structure and dynamics of cell membranes, its impact on the structure of interfacial water has remained unknown. We used surface-specific vibrational sum-frequency generation spectroscopy to study the effect of cholesterol on the structure and hydration of monolayers of the lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and egg sphingomyelin (SM). We found that for the unsaturated lipid DOPC, cholesterol intercalates in the membrane without significantly changing the orientation of the lipid tails and the orientation of the water molecules hydrating the headgroups of DOPC. In contrast, for the saturated lipids DPPC and SM, the addition of cholesterol leads to clearly enhanced packing and ordering of the hydrophobic tails. It is also observed that the orientation of the water hydrating the lipid headgroups is enhanced upon the addition of cholesterol. These results are important because the orientation of interfacial water molecules influences the cell membranes' dipole potential and the strength and specificity of interactions between cell membranes and peripheral proteins and other biomolecules. The lipid nature-dependent role of cholesterol in altering the arrangement of interfacial water molecules offers a fresh perspective on domain-selective cellular processes, such as protein binding.

Polymeric Molecular Envelope Technology for Improved Therapeutics Efficacy: A Review.

The review explores the enhancement of therapeutic efficacy through the innovative use of polymeric molecular envelope technology (MET). It delves into the diverse methods employed to achieve superior therapeutic outcomes, shedding light on strategies for improving drug delivery and bioavailability. MET is a promising approach to improve the solubility and bioavailability of poorly water-soluble drugs. This technology involves the use of a molecular envelope of cyclic oligosaccharides called cyclodextrins, which is a supramolecular assembly of amphiphilic molecules that encapsulate and solubilize hydrophobic drug molecules. This can further improve the solubility of the drug by increasing its surface area and reducing its crystallinity. Moreover, MET also protects the drug from degradation and enhances its permeability across biological membranes. Furthermore, the review thoroughly examines the MET, including its methods of preparation, applications in drug encapsulation, and the evaluation of its potential to optimize therapeutic outcomes. By adopting current research and key findings, this review provides valuable insights into the transformative potential of polymeric molecular envelope technology for advancing the field of therapeutics.

Liposomal Copermeation Assay Reveals Unexpected Membrane Interactions of Commonly Prescribed Drugs.

The permeation of small molecules across biological membranes is a crucial process that lies at the heart of life. Permeation is involved not only in the maintenance of homeostasis at the cell level but also in the absorption and biodistribution of pharmacologically active substances throughout the human body. Membranes are formed by phospholipid bilayers that represent an energy barrier for permeating molecules. Crossing this energy barrier is assumed to be a singular event, and permeation has traditionally been described as a first-order kinetic process, proportional only to the concentration gradient of the permeating substance. For a given membrane composition, permeability was believed to be a unique property dependent only on the permeating molecule itself. We provide experimental evidence that this long-held view might not be entirely correct. Liposomes were used in copermeation experiments with a fluorescent probe, where simultaneous permeation of two substances occurred over a single phospholipid bilayer. Using an assay of six commonly prescribed drugs, we have found that the presence of a copermeant can either enhance or suppress the permeation rate of the probe molecule, often more than 2-fold in each direction. This can have significant consequences for the pharmacokinetics and bioavailability of commonly prescribed drugs when used in combination and provide new insight into so-far unexplained drug-drug interactions as well as changing the perspective on how new drug candidates are evaluated and tested.

Nile tilapia (Oreochromis niloticus) show high tolerance to acute ammonia exposure but lose metabolic scope during prolonged exposure at low concentration

Ammonia is a respiratory gas that is produced during the process of protein deamination. In the unionised form (NH3), it readily crosses biological membranes and is highly toxic to fish. In the present study we examined the effects of unionized ammonia (UIA), on the resting oxygen consumption (MO2), ventilation frequency (fV), heart rate (HR) and heart rate variability (HRV) in Nile tilapia (Oreochromis niloticus). Fish were either exposed to progressively increasing UIA concentrations, up to 97 µM over a 5 h period, or to a constant UIA level of 7 µM over a 24 h period. For both treatment groups resting MO2, HR and fV were recorded as physiological variables. Relative to the control group, the fish groups exposed to the incremental UIA levels did not exhibit significant changes in their MO2, HR and fV at UIA concentrations of 4, 10, 35, or 61 µM compared to control fish. Exposure to 97 µM UIA, however, elicited abrupt and significant downregulations (p < 0.05) in all three responses, as MO2, HR and fv decreased by 25, 54 and 76 % respectively, compared to control measurements. Heart rate became increasingly irregular with increasing UIA concentrations, and heart rate variability was significantly increased at 61 and 97 µM UIA. Prolonged exposure elicited significant changes at exposure 7 µM UIA. Standard (SMR) and maximum metabolic rate (MMR) were significantly reduced, as was the corresponding fV and HR. It is evident from this study that Nile tilapia is tolerant to short term exposure to UIA up to 61 µM but experience a significant metabolic change under conditions of prolonged UIA exposures even at low concentrations.

Plant Protease Inhibitors as Emerging Antimicrobial Peptide Agents: A Comprehensive Review

Antimicrobial peptides (AMPs) are important mediator molecules of the innate defense mechanisms in a wide range of living organisms, including bacteria, mammals, and plants. Among them, peptide protease inhibitors (PPIs) from plants play a central role in their defense mechanisms by directly attacking pathogens or by modulating the plant’s defense response. The growing prevalence of microbial resistance to currently available antibiotics has intensified the interest concerning these molecules as novel antimicrobial agents. In this scenario, PPIs isolated from a variety of plants have shown potential in inhibiting the growth of pathogenic bacteria, protozoans, and fungal strains, either by interfering with essential biochemical or physiological processes or by altering the permeability of biological membranes of invading organisms. Moreover, these molecules are active inhibitors of a range of proteases, including aspartic, serine, and cysteine types, with some showing particular efficacy as trypsin and chymotrypsin inhibitors. In this review, we provide a comprehensive analysis of the potential of plant-derived PPIs as novel antimicrobial molecules, highlighting their broad-spectrum antimicrobial efficacy, specificity, and minimal toxicity. These natural compounds exhibit diverse mechanisms of action and often multifunctionality, positioning them as promising molecular scaffolds for developing new therapeutic antibacterial agents.

Electric field-induced clustering in nanocomposite films of highly polarizable inclusions

A nanocomposite film containing highly polarizable inclusions in a fluid background is explored when an external electric field is applied perpendicular to the planar film. For small electric fields, the induced dipole moments of the inclusions are all polarized in field direction, resulting in a mutual repulsion between the inclusions. Here we show that this becomes qualitatively different for high fields: the total system self-organizes into a state which contains both polarizations, parallel and antiparallel to the external field such that a fraction of the inclusions is counter-polarized to the electric field direction. We attribute this unexpected counter-polarization to the presence of neighboring dipoles which are highly polarized and locally revert the direction of the total electric field. Since dipoles with opposite moments are attractive, the system shows a wealth of novel equilibrium structures for varied inclusion density and electric field strength. These include fluids and solids with homogeneous polarizations as well as equilibrium clusters and demixed states with two different polarization signatures. Based on computer simulations of an linearized polarization model, our results can guide the control of nanocomposites for various applications, including sensing external fields, directing light within plasmonic materials, and controlling the functionality of biological membranes.

Characterization of SIPs-type aquaporins and their roles in response to environmental cues in rice (Oryza sativa L.)

BackgroundAquaporins (AQPs) facilitate water diffusion across biological membranes and are involved in all phases of growth and development. Small and basic intrinsic proteins (SIPs) belong to the fourth subfamily of the plant AQPs. Although SIPs are widely present in higher plants, reports on SIPs are limited. Rice is one of the major food crops in the world, and water use is an important factor affecting rice growth and development; therefore, this study aimed to provide information relevant to the function and environmental response of the rice SIP gene family.ResultsThe rice (Oryza sativa L. japonica) genome encodes two SIP-like genes, OsSIP1 and OsSIP2, whose products are predominantly located in the endoplasmic reticulum (ER) membrane but transient localization to the plasma membrane is not excluded. Heterologous expression in a yeast aquaglyceroporin-mutant fps1Δ showed that both OsSIP1 and OsSIP2 made the cell more sensitive to KCl, sorbitol and H2O2, indicating facilitated permeation of water and hydrogen peroxide. In addition, the yeast cells expressing OsSIP2 were unable to efflux the toxic methylamine taken up by the endogenous MEP permeases, but OsSIP1 showed subtle permeability to methylamine, suggesting that OsSIP1 may have a wider conducting pore than OsSIP2. Expression profiling in different rice tissues or organs revealed that OsSIP1 was expressed in all tissues tested, whereas OsSIP2 was preferentially expressed in anthers and weakly expressed in other tissues. Consistent with this, histochemical staining of tissues expressing the promoter-β-glucuronidase fusion genes revealed their tissue-specific expression profile. In rice seedlings, both OsSIPs were upregulated to varied levels under different stress conditions, including osmotic shock, high salinity, unfavorable temperature, redox challenge and pathogen attack, as well as by hormonal treatments such as GA, ABA, MeJA, SA. However, a reduced expression of both OsSIPs was observed under dehydration treatment.ConclusionsOur results suggest that SIP-like aquaporins are not restricted to the ER membrane and are likely to be involved in unique membrane functions in substrate transport, growth and development, and environmental response.

Shear-Induced Nonequilibrium Patterns in Lipid Bilayer Membranes Exhibiting Phase Separation.

The nonequilibrium dynamics of a fluid lipid membrane under external stimuli is an important issue that spans disciplines such as soft matter, biophysical chemistry, and interface science. This study investigated the dynamic response of lipid vesicles with order-disorder phase separation, which mimics a plasma membrane heterogeneity, to shear flow. Lipid vesicles were immobilized in a microfluidic chamber, and shear-induced nonequilibrium patterns on the membrane surface were observed by an optical microscope. We found that phase-separated membranes exhibit a dissipative structure of stripe patterns along the vortex flow on the membrane surface, and the number of stripes increased with the flow rate. At a high flow rate, the membrane exhibited a stripe-to-wave transition, where striped domains often migrated and the replacement of two different phases happened at vortex centers with time. We obtained a dynamic phase diagram of the shear-induced wave pattern by changing the flow rate, membrane components, and temperature. These findings could provide insight into the dissipative structures of lipid membranes out of equilibrium and flow-mediated mechanotransduction of biological membranes.

Dehydration of Lipid Membranes Drives Redistribution of Cholesterol Between Lateral Domains.

Cholesterol-rich lipid rafts are found to facilitate membrane fusion, central to processes like viral entry, fertilization, and neurotransmitter release. While the fusion process involves local, transient membrane dehydration, the impact of reduced hydration on cholesterol's structural organization in biological membranes remains unclear. Here, we employ confocal fluorescence microscopy and atomistic molecular dynamics simulations to investigate cholesterol behavior in phase-separated lipid bilayers under controlled hydration. We unveiled that dehydration prompts cholesterol release from raft-like domains into the surrounding fluid phase. Unsaturated phospholipids undergo more significant dehydration-induced structural changes and lose more hydrogen bonds with water than sphingomyelin. The results suggest that cholesterol redistribution is driven by the equalization of biophysical properties between phases and the need to satisfy lipid hydrogen bonds. This underscores the role of cholesterol-phospholipid-water interplay in governing cholesterol affinity for a specific lipid type, providing a new perspective on the regulatory role of cell membrane heterogeneity during membrane fusion.

Inorganic polyphosphate and ion transport across biological membranes.

Inorganic polyphosphate (polyP) is widely recognized for playing important roles and processes involved in energy and phosphate storage, regulation of gene expression, and calcium signaling. The less well-known role of polyP is as a direct mediator of ion transport across biological membranes. Here, we will briefly summarize current knowledge of the molecular mechanisms of how polyP can be involved in membrane ion transport. We discuss three types of mechanisms that might involve polyP: (1) formation of non-protein channel complex that includes calcium, polyP, and polyhydroxybutyrate (PHB); (2) modulation of the channel activity of PHBlated protein channels; and (3) direct effects of polyP on the function of the voltage-gated ion channels in the process that do not involve PHB.

Transport Dynamics of Water Molecules Confined between Lipid Membranes.

Water molecules confined between biological membranes exhibit a distinctive non-Gaussian displacement distribution, far different from that of bulk water. Here, we introduce a new transport equation for water molecules in the intermembrane space, quantitatively explaining molecular dynamics simulation results. We find that the unique transport dynamics of water molecules stems from the lateral diffusion coefficient fluctuation caused by their longitudinal motion in the direction perpendicular to the membranes. We also identify an interfacial region where water possesses distinct physical properties, which is unaffected by changes in the intermembrane separation.

Graphical models for identifying pore-forming proteins.

Pore-forming toxins (PFTs) are proteins that form lesions in biological membranes. Better understanding of the structure and function of these proteins will be beneficial in a number of biotechnological applications, including the development of new pest control methods in agriculture. When searching for new pore formers, existing sequence homology-based methods fail to discover truly novel proteins with low sequence identity to known proteins. Search methodologies based on protein structures would help us move beyond this limitation. As the number of known structures for PFTs is very limited, it's quite challenging to identify new proteins having similar structures using computational approaches like deep learning. In this article, we therefore propose a sample-efficient graphical model, where a protein structure graph is first constructed according to consensus secondary structures. A semi-Markov conditional random fields model is then developed to perform protein sequence segmentation. We demonstrate that our method is able to distinguish structurally similar proteins even in the absence of sequence similarity (pairwise sequence identity < 0.4)-a feat not achievable by traditional approaches like HMMs. To extract proteins of interest from a genome-wide protein database for further study, we also develop an efficient framework for UniRef50 with 43 million proteins.

Low voltage electric field mediating gravity-driven membrane bioreactor in treating saline wastewater: removal improvement and membrane fouling control

Gravity-driven membrane bioreactor (GDMBR) endowed with low maintenance and energy consumption owing to its combinations between biological degradation and membrane rejection, however, has not been reported in treating the saline wastewater due to the potential biological inhibition. This study investigated the effects of salt on GDMBR performance, and introduced a novel low voltage electric field (eGDMBR) with hopes to mediate the biological activity and improve the filtration performance. The influence of different low voltage electric fields (0.25 V, 0.50 V, and 0.75 V) on the flux variations and contaminants removal of eGDMBR were investigated. The results demonstrated that integrating low voltage electric field into GDMBR would not impact the appearance of flux stabilization, but delivered a significant influence on its stabilized flux level, and 0.50 V was the optimized voltage, contributing to a substantial flux improvement of 47.10 % in eGDMBR-0.50. Moreover, with the assistance of low voltage electric fields, eGDMBR process obtained an effective removal of chemical oxygen demand (COD), ammonia nitrogen (NH4+-N) and total phosphorus (TP), accounting for average removal efficiency of 54.01 %, 73.08 % and 25.74 %, respectively. Compared to the control, coupling 0.50 V voltage electric field to GDMBR was beneficial to engineer a loose, porous and heterogeneous structure of membrane biofilm and also reduce the accumulation of extracellular polymeric substance (EPS), significantly contributing to alleviating the membrane fouling. Furthermore, with the assistance of low voltage electric fields, the Proteobacteria was enriched by a relative abundance of 58.58 % in the membrane biofilm of eGDMBR-0.50, which was in favor of the degradation of COD and NH4+-N. Therefore, these findings indicated that eGDMBR conferred significant superiorities in treating the saline wastewater and mitigating membrane fouling.

Unlocking the specificity of antimicrobial peptide interactions for membrane-targeted therapies

Antimicrobial peptides (AMPs) are increasingly recognized as potent therapeutic agents, with their selective affinity for pathological membranes, low toxicity profile, and minimal resistance development making them particularly attractive in the pharmaceutical landscape. This study offers a comprehensive analysis of the interaction between specific AMPs, including magainin-2, pleurocidin, CM15, LL37, and clavanin, with lipid bilayer models of very different compositions that have been ordinarily used as biological membrane models of healthy mammal, cancerous, and bacterial cells. Employing unbiased molecular dynamics simulations and metadynamics techniques, we have deciphered the intricate mechanisms by which these peptides recognize pathogenic and pathologic lipid patterns and integrate into lipid assemblies. Our findings reveal that the transverse component of the peptide's hydrophobic dipole moment is critical for membrane interaction, decisively influencing the molecule's orientation and expected therapeutic efficacy. Our approach also provides insight on the kinetic and dynamic dependence on the peptide orientation in the axial and azimuthal angles when coming close to the membrane. The aim is to establish a robust framework for the rational design of peptide-based, membrane-targeted therapies, as well as effective quantitative descriptors that can facilitate the automated design of novel AMPs for these therapies using machine learning methods.

Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes

The organization of membrane proteins between and within membrane-bound compartments is critical to cellular function. Yet we lack approaches to regulate this organization in a range of membrane-based materials, such as engineered cells, exosomes, and liposomes. Uncovering and leveraging biophysical drivers of membrane protein organization to design membrane systems could greatly enhance the functionality of these materials. Towards this goal, we use de novo protein design, molecular dynamic simulations, and cell-free systems to explore how membrane-protein hydrophobic mismatch could be used to tune protein cotranslational integration and organization in synthetic lipid membranes. We find that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the expression and co-translational insertion of membrane proteins into synthetic membranes. We use this principle to sort proteins both between and within membranes, thereby achieving one-pot assembly of vesicles with distinct functions and controlled split-protein assembly, respectively. Our results shed light on protein organization in biological membranes and provide a framework to design self-organizing membrane-based materials with applications such as artificial cells, biosensors, and therapeutic nanoparticles.

Morphology Observation of Two-Dimensional Monolayers of Model Proteins on Water Surface as Revealed by Dropping Method.

We have investigated the morphology of two-dimensional monolayers of gramicidin-D (GD) and alamethicin (Al) formed on the water surface by the dropping method (DM) using surface tension measurement (STm), Brewster angle microscopy (BAM), and atomic force microscopy (AFM). Dynamic light scattering (DLS) revealed that GD in alcoholic solutions formed a dimeric helical structure. According to the CD and NMR spectroscopies, GD molecules existed in dimer form in methanol and lipid membrane environments. The STm results and BAM images revealed that the GD dimer monolayer was in a liquid expanded (LE) state, whereas the Al monolayer was in a liquid condensed (LC) state. The limiting molecular area (A0) was 6.2 ± 0.5 nm2 for the GD-dimer and 3.6 ± 0.5 nm2 for the Al molecule. The AFM images also showed that the molecular long axes of both the GD-dimer and Al were horizontal to the water surface. The stability of each monolayer was confirmed by the time dependence of the surface pressure (π) observed using the STm method. The DM monolayer preparation method for GD-dimer and Al peptide molecules is a useful technique for revealing how the model biological membrane's components assemble in two dimensions on the water surface.