Membrane Reorganization Research Articles

Thermodynamic Investigation of the Mechanism of Heat Production During Membrane Depolarization.

When an action potential passes through a neuron, heat is first produced and then reabsorbed by the neuronal membrane, resulting in a small measurable temperature spike. Here, we describe the thermodynamics and molecular features of the heat production using a coarse-grained molecular dynamics approach. We study a simple unicomponent lipid bilayer membrane surrounded by physiological salt solution with and without an external electric field, which represents an imbalanced charge across the membrane. We show that the temperature increases significantly upon removal of the electric field under constant pressure conditions. The potential energy converted to heat is initially stored mainly in the imbalanced ion distribution across the membrane and the elastic energy of the membrane has only a minor role to play. We demonstrate that the mechanism of heat production involves interaction between ions as well as lipid headgroup dipoles while the interactions between polar water molecules and lipid headgroup dipoles absorbs a considerable portion of such produced heat upon removal of the electric field. Our data provide novel thermodynamic insights into the molecular processes governing membrane reorganization upon discharging of lipid membranes and insight into energy metabolism in nerves.

PsbS contributes to photoprotection in Chlamydomonas reinhardtii independently of energy dissipation

Photosynthetic organisms are frequently exposed to excess light conditions and hence to photo-oxidative stress. To counteract photo-oxidative damage, land plants and most algae make use of non- photochemical quenching (NPQ) of excess light energy, in particular the rapidly inducible and relaxing qE-mechanism. In vascular plants, the constitutively active PsbS protein is the key regulator of qE. In the green algae C. reinhardtii, however, qE activation is only possible after initial high-light (HL) acclimation for several hours and requires the synthesis of LHCSR proteins which act as qE regulators. The precise function of PsbS, which is transiently expressed during HL acclimation in C. reinhardtii, is still unclear. Here, we investigated the impact of different PsbS amounts on HL acclimation characteristics of C. reinhardtii cells. We demonstrate that lower PsbS amounts negatively affect HL acclimation at different levels, including NPQ capacity, electron transport characteristics, antenna organization and morphological changes, resulting in an overall increased HL sensitivity and lower vitality of cells. Contrarily, higher PsbS amounts do not result in a higher NPQ capacity, but nevertheless provide higher fitness and tolerance towards HL stress. Strikingly, constitutively expressed PsbS protein was found to be degraded during HL acclimation. We propose that PsbS is transiently required during HL acclimation for the reorganization of thylakoid membranes and/or antenna proteins along with the activation of NPQ and adjustment of electron transfer characteristics, and that degradation of PsbS is essential in the fully HL acclimated state.

Membrane binding and rearrangement by chikungunya virus capping enzyme nsP1

Alphavirus genome replication is carried out by the viral replication complex inside modified membrane structures called spherules. The viral nonstructural protein 1 (nsP1) is the only membrane-associated protein that anchors the replication complex to the cellular membranes. Although an internal amphipathic helix of nsP1 is critical for membrane association, the mechanism of nsP1 interaction with membranes and subsequent membrane reorganization is not well understood. We studied the membrane interaction of chikungunya virus (CHIKV) nsP1 and show that both the CHIKV nsP1 protein and the amphipathic peptide specifically bind to negatively charged phospholipid vesicles. Using cryo-electron microscopy, we further show that nsP1 forms a contiguous coat on lipid vesicles and induces structural reorganization, while the amphipathic peptide alone failed to deform the membrane bilayer. This suggests that although amphipathic helix of nsP1 is required for initial membrane binding, the remaining cytoplasmic domain of nsP1 is involved in the subsequent membrane reorganization.

Complex cytotoxicity mechanism of bundles formed from self-organised 1-D anodic TiO2 nanotubes layers

The present study reports on a comprehensive investigation of mechanisms of in vitro cytotoxicity of high aspect ratio (HAR) bundles formed from anodic TiO2 nanotube (TNT) layers. Comparative cytotoxicity studies were performed using two types of HAR TNTs (diameter of ∼110 nm), differing in initial thickness of the nanotubular layer (∼35 μm for TNTs-1 vs. ∼10 μm for TNTs-2). Using two types of epithelial cell lines (MDA-MB-231, HEK-293), it was found that nanotoxicity is highly cell-type dependent and plausibly associates with higher membrane fluidity and decreased rigidity of cancer cells enabling penetration of TNTs to the cell membrane towards disruption of membrane integrity and reorganization of cytoskeletal network. Upon penetration, TNTs dysregulated redox homeostasis followed by DNA fragmentation and apoptotic/necrotic cell death. Both TNTs exhibited haemolytic activity and rapidly activated polarization of RAW 264.7 macrophages. Throughout the whole study, TNTs-2 possessing a lower aspect ratio manifested significantly higher cytotoxic effects. Taken together, this is the first report comprehensively investigating the mechanisms underlying the nanotoxicity of bundles formed from self-organised 1-D anodic TNT layers. Except for description of nanotoxicity of industrially-interesting nanomaterials, the delineation of the nanotoxicity paradigm in cancer cells could serve as solid basis for future efforts in rational engineering of TNTs towards selective anticancer nanomedicine.

EJP18 peptide derived from the juxtamembrane domain of epidermal growth factor receptor represents a novel membrane-active cell-penetrating peptide.

Membrane-active peptides have been extensively studied to probe protein-membrane interactions, to act as antimicrobial agents and cell-penetrating peptides (CPPs) for the delivery of therapeutic agents to cells. Hundreds of membrane-active sequences acting as CPPs have now been described including bioportides that serve as single entity modifiers of cell physiology at the intracellular level. Translation of promising CPPs in pre-clinical studies have, however, been disappointing as only few identified delivery systems have progressed to clinical trials. To search for novel membrane-active peptides a sequence from the EGFR juxtamembrane region was identified (named EJP18), synthesised, and examined in its L- and D-form for its ability to mediate the delivery of a small fluorophore and whole proteins to cancer cell lines. Initial studies identified the peptide as being highly membrane-active causing extensive and rapid plasma membrane reorganisation, blebbing, and toxicity. At lower, non-toxic concentrations the peptides outperformed the well-characterised CPP octaarginine in cellular delivery capacity for a fluorophore or proteins that were associated with the peptide covalently or via ionic interactions. EJP18 thus represents a novel membrane-active peptide that may be used as a naturally derived model for biophysical protein-membrane interactions or for delivery of cargo into cells for therapeutic or diagnostic applications.

A Profound Membrane Reorganization Defines Susceptibility of <i>Plasmodium falciparum</i> Infected Red Blood Cells to Lysis by Granulysin and Perforin

Malaria remains one of the most serious health problems in developing countries. The causative agent of malaria, Plasmodium spp., have a complex life cycle involving multiple developmental stages as well as different morphological, biochemical and metabolic requirements. We recently found that gd T cells control parasite growth using pore-forming proteins to deliver their cytotoxic proteases, the granzymes, into blood residing parasites.Here, we demonstrate that Plasmodium falciparum infection depletes the red blood cells of cholesterol, which renders the parasite surrounding membranes susceptible to lysis by prokaryotic membrane disrupting proteins, such as lymphocytic granulysin or the human cathelicidin LL-37. Interestingly, not the cholesterol depletion but rather the simultaneous exposure of phosphatidylserine, a negatively charged phospholipid, triggers resistance of late stage parasitized red blood cells towards the eukaryotic pore forming protein perforin. Overall, we establish here a pathogen-host interaction that involves host cell membrane remodeling that defines the susceptibility towards cytolytic molecules.

Role of neuroendocrine modulation and biochemistry in the sepsis in Piaractus mesopotamicus

Sepsis is a systemic process with multifactorial pathophysiology that affects most animal species. It is responsible for high rates of morbidity and mortality. This work aimed to study the biochemical and neuroendocrine changes of the sepsis process in Piaractus mesopotamicus after Aeromonas hydrophila inoculation analyzing changes in blood leukocyte and differences in neuroendocrine-biochemical modulation using RNA-seq. Fish showed hypercortisolemia, inhibition of glucose absorption, followed by hypocortisolemia and then hyperglycemia. Thyroid hormones (T3 and T4) showed immediate decrease in serum and T4 increased 6 h post-inoculation (HPI). Sepsis-induced hormonal alterations triggered changes in the metabolic pathways increasing protein and lipid catabolism, use of transient anaerobic glycolysis and liver injury. A reference transcriptome was constructed based on blood leukocytes from P. mesopotamicus. The assembly resulted in total 266,272 contigs with a N50 of 2786 bp. There was a reorganization of plasma membrane of leukocytes at the beginning of the septic process with increased expression of neuroendocrine receptors and with continuous flow of neurotransmitters, hormones and solutes with compensatory regulation at 6 HPI. Three and nine HPI seemed to be critical, the expression of a number of transcription factors was increased, including the modulatory DEGs related to glucocorticoid and thyroid hormones induced and suppressed (FDR < 0.05). Neuroendocrine modulation can regulate leukocytes and biochemical parameters of peripheral blood, being important sources for the study of the pathophysiology of sepsis. These finding highlights the importance of further studies focusing on biochemical-neuroendocrine changes in blood leukocytes and systemic sepsis.

The role of endothelial MERTK during the inflammatory response in lungs.

As a key homeostasis regulator in mammals, the MERTK receptor tyrosine kinase is crucial for efferocytosis, a process that requires remodeling of the cell membrane and adjacent actin cytoskeleton. Membrane and cytoskeletal reorganization also occur in endothelial cells during inflammation, particularly during neutrophil transendothelial migration (TEM) and during changes in permeability. However, MERTK’s function in endothelial cells remains unclear. This study evaluated the contribution of endothelial MERTK to neutrophil TEM and endothelial barrier function. In vitro experiments using primary human pulmonary microvascular endothelial cells found that neutrophil TEM across the endothelial monolayers was enhanced when MERTK expression in endothelial cells was reduced by siRNA knockdown. Examination of endothelial barrier function revealed increased passage of dextran across the MERTK-depleted monolayers, suggesting that MERTK helps maintain endothelial barrier function. MERTK knockdown also altered adherens junction structure, decreased junction protein levels, and reduced basal Rac1 activity in endothelial cells, providing potential mechanisms of how MERTK regulates endothelial barrier function. To study MERTK’s function in vivo, inflammation in the lungs of global Mertk-/- mice was examined during acute pneumonia. In response to P. aeruginosa, more neutrophils were recruited to the lungs of Mertk-/- than wildtype mice. Vascular leakage of Evans blue dye into the lung tissue was also greater in Mertk-/- mice. To analyze endothelial MERTK’s involvement in these processes, we generated inducible endothelial cell-specific (iEC) Mertk-/- mice. When similarly challenged with P. aeruginosa, iEC Mertk-/- mice demonstrated no difference in neutrophil TEM into the inflamed lungs or in vascular permeability compared to control mice. These results suggest that deletion of MERTK in human pulmonary microvascular endothelial cells in vitro and in all cells in vivo aggravates the inflammatory response. However, selective MERTK deletion in endothelial cells in vivo failed to replicate this response.

How Does the Seed Pre-Germinative Metabolism Fight Against Imbibition Damage? Emerging Roles of Fatty Acid Cohort and Antioxidant Defence.

During seed imbibition, lipids are engaged in membrane reorganization while facing free radical-mediated oxidative injury. In the present work, we explored changes in lipid components at different timepoints of imbibition (0.5, 2, 4, 6, and 8 h) in the legume Medicago truncatula, by combining biochemical approaches with targeted lipidomics and untargeted metabolomics. ROS and RNS (reactive oxygen and nitrogen species) accumulation was observed throughout the tested timepoints whereas lipid peroxidation increased at 4 h of imbibition. The seed response to oxidative damage was evidenced by a significant increase in tocopherols starting from 0.5 h of imbibition as well as by the reduction in total thiol content occurring at 2 h of imbibition. Since under physiological conditions, the proper functions of the cell membranes are strongly dependent on the qualitative and quantitative balance of fatty acid residues in phospholipids, the investigation was expanded to the fatty acid cohort of M. truncatula seeds. Total saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), omega(ω)-3 and omega(ω)-6 fatty acids showed fluctuations during seed imbibition. The most remarkable finding was the profile of the ω-3 PUFA docosopentaenoic acid (DPA, 7 cis, 10 cis, 13 cis, 16 cis, and 19 cis-22:5) that showed a peak (up to 1.0% of the total fatty acid content) at 0.5 and 8 h of imbibition, concomitant with the peaks observed in tocopherol levels. It is possible that the observed changes in DPA alter the physical properties of membranes, as reported in animal cells, triggering signaling pathways relevant for the cell defense against oxidative injury. Furthermore, the content and balance between tocopherols and PUFAs is regarded as a determinant of storage stability. No enhancement in trans-fatty acids occurred throughout imbibition, suggesting for a proper antioxidant response carried by the seed. Fatty acids profiles were integrated with data from untargeted metabolomics showing changes in lipid sub-pathways, among which fatty acid amide, lyso-phospholipids, and phospholipid metabolism. The emerging lipid profiles and dynamics are discussed in view of the overall imbibition damage generated during M. truncatula seed imbibition.

Visualization and Quantification of Phagocytosis by Neutrophils.

Phagocytosis by phagocytes such as neutrophils is a crucial part of the host innate immune response against invading pathogens. Phagocytosis is a complex process that initiates with the binding of the particles on the cell surface of the phagocytes through the interaction of pattern recognition receptors with ligands on the surface of the pathogens. During this process, phagocytes undergo extensive membrane reorganization and cytoskeleton rearrangement at their cell surface. To gain better insight about the molecular mechanisms of this dynamic cellular process, visualization and quantification in a high-throughput manner is essential. Here, we describe a microscope-based method to visualize and quantify phagocytic uptake of pathogens (such as bacteria and fungi) and model particulates that are larger than 0.5μm (such as Zymosan A and IgG-coated beads).

Weathering the Cold: Modifying Membrane and Storage Fatty Acid Composition of Seeds to Improve Cold Germination Ability in Upland Cotton (Gossypium hirsutum L.)

Cotton is widely cultivated in temperate regions across the world and is often constrained by a short planting window that is bookended by low, suboptimal temperatures. With the growing interest in early season planting, improvements in the cold germination ability of cotton will be necessary to ensure the production stability of early planted crops. The importance of saturation levels of membrane and storage lipids in enhancing cold tolerance in plants, as well as improving cold germination ability in seeds have been widely researched in a range of plant species. While studies have shown that higher levels of unsaturated lipids can enhance cold germination ability and reduce seedling injury in other crops, similar efforts have been fairly limited in cotton. This review looks at the functional properties of membrane and storage lipids, and their role in membrane stability and reorganization during the early stages of germination. Additionally, the importance of storage lipid composition as an energy source to the growing embryo is described in the context of cellular energetics (i.e., fatty acid catabolism). Finally, perspectives in improving the cold germination of upland cotton by manipulating the fatty acid composition of both membrane and storage lipid content of seeds are presented.

Sphingolipids as Potential Therapeutic Targets against Enveloped Human RNA Viruses.

Several notable human diseases are caused by enveloped RNA viruses: Influenza, AIDS, hepatitis C, dengue hemorrhagic fever, microcephaly, and Guillain–Barré Syndrome. Being enveloped, the life cycle of this group of viruses is critically dependent on host lipid biosynthesis. Viral binding and entry involve interactions between viral envelope glycoproteins and cellular receptors localized to lipid-rich regions of the plasma membrane. Subsequent infection by these viruses leads to reorganization of cellular membranes and lipid metabolism to support the production of new viral particles. Recent work has focused on defining the involvement of specific lipid classes in the entry, genome replication assembly, and viral particle formation of these viruses in hopes of identifying potential therapeutic targets for the treatment or prevention of disease. In this review, we will highlight the role of host sphingolipids in the lifecycle of several medically important enveloped RNA viruses.

TORC2-Gad8-dependent myosin phosphorylation modulates regulation by calcium.

Cells respond to changes in their environment through signaling networks that modulate cytoskeleton and membrane organization to coordinate cell-cycle progression, polarized cell growth and multicellular development. Here, we define a novel regulatory mechanism by which the motor activity and function of the fission yeast type one myosin, Myo1, is modulated by TORC2-signalling-dependent phosphorylation. Phosphorylation of the conserved serine at position 742 (S742) within the neck region changes both the conformation of the neck region and the interactions between Myo1 and its associating calmodulin light chains. S742 phosphorylation thereby couples the calcium and TOR signaling networks that are involved in the modulation of myosin-1 dynamics to co-ordinate actin polymerization and membrane reorganization at sites of endocytosis and polarised cell growth in response to environmental and cell-cycle cues.

Phospholipid Scramblases Remodel the Shape of Asymmetric Membranes.

The cell membrane and many organellar membranes are asymmetric and highly curved. In experiments, it is challenging to reconstitute and characterize membranes that differ in the lipid composition of their leaflets. Here we use molecular dynamics simulations to study the large-scale membrane shape changes associated with lipid shuttling between asymmetric leaflets. We exploit leaflet asymmetry to create a stable, near-spherical vesicle bud connected to a flat bilayer under periodic boundary conditions. Then we demonstrate how the lipid scramblase nhTMEM16 relaxes the lipid-number asymmetry. By mediating the flipping of lipids, this transmembrane protein dissipates the mechanochemical gradient between the leaflets and drives a large-scale membrane reorganization, converting the vesicle bud into a flat membrane. Our procedure to exploit bilayer asymmetry for simulations of highly curved membranes can be used to study the function of other lipid transporters and membrane-shaping proteins.

Virus Impact on Lipids and Membranes.

Viruses manipulate cellular lipids and membranes at each stage of their life cycle. This includes lipid-receptor interactions, the fusion of viral envelopes with cellular membranes during endocytosis, the reorganization of cellular membranes to form replication compartments, and the envelopment and egress of virions. In addition to the physical interactions with cellular membranes, viruses have evolved to manipulate lipid signaling and metabolism to benefit their replication. This review summarizes the strategies that viruses use to manipulate lipids and membranes at each stage in the viral life cycle.

Shiga Toxin Induces Lipid Compression: A Mechanism for Generating Membrane Curvature.

Biomembranes are hard to compress laterally, and membrane area compressibility has not been associated with biological processes. Using X-ray surface scattering, we observed that bacterial Shiga toxin compresses lipid packing in a gel phase monolayer upon binding to its cellular receptor, the glycolipid Gb3. This toxin-induced reorganization of lipid packing reached beyond the immediate membrane patch that the protein was bound to, and linkers separating the Gb3 carbohydrate and ceramide moieties modulated the toxin's capacity to compress the membrane. Within a natural membrane, asymmetric compression of the toxin-bound leaflet could provide a mechanism to initiate narrow membrane bending, as observed upon toxin entry into cells. Such lipid compression and long-range membrane reorganization by glycolipid-binding proteins represent novel concepts in membrane biology that have direct implications for the construction of endocytic pits in clathrin-independent endocytosis.

Nanomechanical insights: Amyloid beta oligomer-induced senescent brain endothelial cells

Senescent cells accumulate in various peripheral tissues during aging and have been shown to exacerbate age-related inflammatory responses. We recently showed that exposure to neurotoxic amyloid β (Aβ1–42) oligomers can readily induce a senescence phenotype in human brain microvascular endothelial cells (HBMECs). In the present work, we used atomic force microscopy (AFM) to further characterize the morphological properties such as cell membrane roughness and cell height and nanomechanical properties such as Young's modulus of the membrane (membrane stiffness) and adhesion resulting from the interaction between AFM tip and cell membrane in Aβ1–42 oligomer-induced senescent human brain microvascular endothelial cells. Morphological imaging studies showed a flatter and spread-out nucleus in the senescent HBMECs, both characteristic features of a senescent phenotype. Furthermore, the mean cell body roughness and mean cell height were lower in senescent HBMECs compared to untreated normal HBMECs. We also observed increased stiffness and alterations in the adhesion properties in Aβ1–42 oligomer-induced senescent endothelial cells compared to the untreated normal HBMECs suggesting dynamic reorganization of cell membrane. We then show that vascular endothelial growth factor receptor 1 (VEGFR-1) knockdown or overexpression of Rho GTPase Rac 1 in the endothelial cells inhibited senescence and reversed these nanomechanical alterations, confirming a direct role of these pathways in the senescent brain endothelial cells. These results illustrate that nanoindentation and topographic analysis of live senescent brain endothelial cells can provide insights into cerebrovascular dysfunction in neurodegenerative diseases such as Alzheimer's disease.

Compartmentalization of the proteasome-interacting proteins during sperm capacitation

Ubiquitination is a stable, reversible posttranslational modification of target proteins by covalent ligation of the small chaperone protein ubiquitin. Most commonly ubiquitination targets proteins for degradation/recycling by the 26S proteasome in a well-characterized enzymatic cascade. Studies using human and non-human mammalian spermatozoa revealed the role of the ubiquitin-proteasome system (UPS) in the regulation of fertilization, including sperm-zona pellucida (ZP) interactions as well as the early events of sperm capacitation, the remodeling of the sperm plasma membrane and acrosome, and for the acquisition of sperm fertilizing ability. The present study investigated the activity of UPS during in vitro capacitation of fresh boar spermatozoa in relation to changes in sperm proteome. Parallel and sequential treatments of ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions were used to isolate putative sperm proteasome-associated sperm proteins in a compartment-specific manner. A differential proteomic approach employing 1D PAGE revealed differences in accumulated proteins at the molecular weights of 60, 58, 49, and 35 kDa, and MS analysis revealed the accumulation of proteins previously reported as proteasome co-purifying proteins, as well as some novel proteins. Among others, P47/lactadherin, ACRBP, ADAM5, and SPINK2 (alias SAAI) were processed by the proteasome in a capacitation dependent manner. Furthermore, the capacitation-induced reorganization of the outer acrosomal membrane was slowed down in the presence of proteasomal inhibitors. These novel results support the proposed role of UPS in sperm capacitation and open several new lines of inquiry into sperm capacitation mechanism.

Analysis of lipoprotein transport depletion in Vibrio cholerae using CRISPRi

Genes necessary for the survival or reproduction of a cell are an attractive class of antibiotic targets. Studying essential genes by classical genetics, however, is inherently problematic because it is impossible to knock them out. Here, we screened a set of predicted essential genes for growth inhibition using CRISPR-interference (CRISPRi) knockdown in the human pathogen Vibrio cholerae We demonstrate that CRISPRi knockdown of 37 predicted essential genes inhibits V. cholerae viability, thus validating the products of these genes as potential drug target candidates. V. cholerae was particularly vulnerable to lethal inhibition of the system for lipoprotein transport (Lol), a central hub for directing lipoproteins from the inner to the outer membrane (OM), with many of these lipoproteins coordinating their own essential processes. Lol depletion makes cells prone to plasmolysis and elaborate membrane reorganization, during which the periplasm extrudes into a mega outer membrane vesicle or "MOMV" encased by OM which dynamically emerges specifically at plasmolysis sites. Our work identifies the Lol system as an ideal drug target, whose inhibition could deplete gram-negative bacteria of numerous proteins that reside in the periplasm.

TNFR1 membrane reorganization promotes distinct modes of TNFα signaling.

Signaling by the ubiquitously expressed tumor necrosis factor receptor 1 (TNFR1) after ligand binding plays an essential role in determining whether cells exhibit survival or death. TNFR1 forms distinct signaling complexes that initiate gene expression programs downstream of the transcriptional regulators NFκB and AP-1 and promote different functional outcomes, such as inflammation, apoptosis, and necroptosis. Here, we investigated the ways in which TNFR1 was organized at the plasma membrane at the nanoscale level to elicit different signaling outcomes. We confirmed that TNFR1 forms preassembled clusters at the plasma membrane of adherent cells in the absence of ligand. After trimeric TNFα binding, TNFR1 clusters underwent a conformational change, which promoted lateral mobility, their association with the kinase MEKK1, and activation of the JNK/p38/NFκB pathway. These phenotypes required a minimum of two TNFR1-TNFα contact sites; fewer binding sites resulted in activation of NFκB but not JNK and p38. These data suggest that distinct modes of TNFR1 signaling depend on nanoscale changes in receptor organization.