Plasma Membrane Of Eukaryotic Cells Research Articles

Membrane cholesterol is essential for triterpenoid saponin augmentation of a saporin-based immunotoxin directed against CD19 on human lymphoma cells

Triterpenoid saponins from Saponinum Album (SA) exert potent lytic effects on eukaryotic cell plasma membranes and, when used at sub-lytic concentrations, significantly augment the cytotoxicity of saporin-based immunotoxins (IT). To help elucidate the mechanism(s) behind these two phenomena we investigated the role of cholesterol to both.Human Daudi lymphoma cells were lipid deprived using a combination of three different approaches. Following treatment, the total cellular lipid content was analyzed by electrospray ionization mass spectrometry (ESI-MS) and plasma membrane (PM) cholesterol content measured using the lipophilic fluorescent probe NR12S. Maximal lipid deprivation of cells resulted in a complete loss of sensitivity to lysis by SA. Similarly augmentation of the anti-CD19 immunotoxin (IT) BU12-SAPORIN by SA was lost but without a concomitant loss of intrinsic IT cytotoxicity. The lytic activity of SA was restored following incubation of lipid deprived Daudi cells with Synthecol or LDL. The augmentative effect of SA on IT cytotoxicity for Daudi cells was restored following repletion of PM cholesterol levels with LDL. NR12S fluorescence and ESI-MS analysis of cellular lipids demonstrated that restoration of SA lytic activity by Synthecol was entirely due to increased PM cholesterol levels. Restoration of cellular and PM cholesterol levels by LDL also restored the augmentative effect of SA for IT, an effect associated with repletion of PM cholesterol with minor changes in some phospholipid species. These results indicate that the lytic and IT augmentative properties of SA are cholesterol-dependent in contrast to intrinsic IT cytotoxicity that is at least partially cholesterol independent.

Mechanoregulation of Clathrin-Mediated Endocytosis in Isolated Cells and Developing Tissues

Clathrin-coated assemblies bear the largest fraction of the endocytic load from the plasma membrane of eukaryotic cells. However, dynamics of clathrin-mediated endocytosis (CME) have not been established within tissues of multicellular organisms due to experimental and analytical bottlenecks in determining the lifespan of clathrin-coated structures. We found that clathrin coat growth rates obtained from fluorescence microscopy acquisitions can be utilized as reporters of CME dynamics. Growth rates can be assembled within time windows shorter than the average clathrin coat lifetime and, thereby, allow probing the changes in CME dynamics in real time. Furthermore, this novel approach is applicable to tissues as it is not prone to particle detection and tracking errors, which result in underestimation of the clathrin coat lifetimes. Exploiting these advantages, we detected spatial and temporal changes in CME dynamics within Drosophila amnioserosa tissues at different stages of embryo development. We also found that increased membrane tension impedes CME through inhibition of formation and dissolution of clathrin-coated structures. Therefore, the parameters defining clathrin coat dynamics (i.e., lifetime, formation density and growth rates) can be utilized to monitor the spatiotemporal gradients of the plasma membrane tension during cell migration and spreading.

Molecular Views of a Eukaryotic Plasma Membrane Model

Plasma membranes are composed of hundreds of lipid species and different types of membrane proteins. Lipid-lipid and lipid-protein interactions play a crucial role in regulating cell membranes functions and are involved in many diseases when altered. Despite advances in experimental methods, the interplay between lipids and proteins is not completely understood. In this context, computer simulations have become a powerful tool to investigate the dynamic behavior of plasma membrane components and their interactions at a near atomic resolution. The goal of our research is to work towards large-scale modeling of plasma membranes using computer simulations. In this study, we placed ten different types of eukaryotic membrane proteins in a membrane model containing various lipid species found in the plasma membrane of eukaryotic cells. In our simulation setup, membrane proteins are present in different ratios, for a total of ca. 150 protein molecules, embedded in more than 60,600 lipids. The protein-membrane system was prepared using the Martini force field, with in house software. Using molecular dynamic simulations, we investigate the local lipid environment around proteins, and provide a molecular view of a realistic membrane model.

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It.

Sphingolipids are structural components in the plasma membranes of eukaryotic cells. Their metabolism produces bioactive signaling molecules that modulate fundamental cellular processes. The segregation of sphingolipids into distinct membrane domains is likely essential for cellular function. This review presents the early studies of sphingolipid distribution in the plasma membranes of mammalian cells that shaped the most popular current model of plasma membrane organization. The results of traditional imaging studies of sphingolipid distribution in stimulated and resting cells are described. These data are compared with recent results obtained with advanced imaging techniques, including super-resolution fluorescence detection and high-resolution secondary ion mass spectrometry (SIMS). Emphasis is placed on the new insight into the sphingolipid organization within the plasma membrane that has resulted from the direct imaging of stable isotope-labeled lipids in actual cell membranes with high-resolution SIMS. Super-resolution fluorescence techniques have recently revealed the biophysical behaviors of sphingolipids and the unhindered diffusion of cholesterol analogs in the membranes of living cells are ultimately in contrast to the prevailing hypothetical model of plasma membrane organization. High-resolution SIMS studies also conflicted with the prevailing hypothesis, showing sphingolipids are concentrated in micrometer-scale membrane domains, but cholesterol is evenly distributed within the plasma membrane. Reductions in cellular cholesterol decreased the number of sphingolipid domains in the plasma membrane, whereas disruption of the cytoskeleton eliminated them. In addition, hemagglutinin, a transmembrane protein that is thought to be a putative raft marker, did not cluster within sphingolipid-enriched regions in the plasma membrane. Thus, sphingolipid distribution in the plasma membrane is dependent on the cytoskeleton, but not on favorable interactions with cholesterol or hemagglutinin. The alternate views of plasma membrane organization suggested by these findings are discussed.

A Ligand-Binding Assay to Measure the Affinity and Specificity of Sterol-Binding Proteins In Vitro.

Sterols are major constituents of the plasma membrane of eukaryotic cells and serve as a precursor for several classes of signaling molecules, including steroids and hydroxy sterols. They maintain the functionality and permeability barrier of the plasma membrane through lipid-lipid and lipid-protein interactions. The S. cerevisiae pathogen-related yeast proteins 1, 2, and 3 (Pry) belong to a large protein superfamily known as CAP/SCP/TAPS. Members of this superfamily have been implicated in a wide variety of processes, including immune defense in mammals and plants, pathogen virulence, sperm maturation and fertilization, venom toxicity, and prostate and brain cancer. Pry proteins bind and export sterols in vivo and the purified Pry1 protein binds sterols and related small hydrophobic compounds in vitro. Here we describe a method to determine lipid binding of a purified protein in vitro.

The Ins and Outs of Lipid Flip-Flop.

Our current view of cellular membranes centers on the fluid-mosaic model, which envisions the cellular membrane as a "liquidlike" bilayer of lipids, cholesterol, and proteins that freely diffuse in two dimensions. In stark contrast, the exchange of materials between the leaflets of a bilayer was presumed to be prohibited by the large enthalpic barrier associated with translocating hydrophilic materials, such as a charged lipid headgroup, through the hydrophobic membrane core. This static picture with regard to lipid translocation (or "flip-flop" as it is affectionately known) has been a long-held belief in the study of membrane dynamics. The current accepted membrane model invokes specific protein flippase (inward moving), floppase (outward moving), and scramblase (bidirectional) enzymes that assist in the movement of lipids between the leaflets of cellular membranes. The low rate of protein-free lipid flip-flop has also been a cornerstone of our understanding of the bilateral organization of cellular membrane components, specifically the asymmetric distribution of lipid species found in the luminal and extracellular leaflets of the plasma membrane of eukaryotic cells. Much of the previous work contributing to our current understanding of lipid flip-flop has utilized fluorescent- or spin-labeled lipids. However, there is growing evidence that these lipid probes do not accurately convey the dynamics and thermodynamics of native (unlabeled) lipid motion. This Account summarizes our research efforts directed toward developing a deep physical and chemical understanding of protein-free lipid flip-flop in phospholipid membrane models using sum-frequency vibrational spectroscopy (SFVS). Our use of SFVS enables the direct measurement of native lipid flip-flop in model membranes. In particular, we have explored the kinetic rates and activation thermodynamics of lipid translocation as a means of deciphering the underlying chemical and physical directors governing this process. By means of transition state theory, the contributions from enthalpy and entropy on the activation energy barrier to lipid flip-flop have been explored in detail for a variety of lipid species and membrane compositions. Specifically, the effect of lipid structure and packing and the inclusion of cholesterol and transmembrane peptides on the rates and thermodynamics of lipid translocation have been investigated in detail. It is our hope that these studies will provide a new perspective on lipid translocation in biological membranes and the role of lipid flip-flop in generating and maintaining cell membrane lipid asymmetry.

Staphylococcus aureus α-toxin-mediated cation entry depolarizes membrane potential and activates p38 MAP kinase in airway epithelial cells.

Membrane potential (Vm)-, Na(+)-, or Ca(2+)-sensitive fluorescent dyes were used to analyze changes in Vm or intracellular ion concentrations in airway epithelial cells treated with Staphylococcus aureus α-toxin (Hla), a major virulence factor of pathogenic strains of these bacteria. Gramicidin, a channel-forming peptide causing membrane permeability to monovalent cations, a mutated form of Hla, rHla-H35L, which forms oligomers in the plasma membranes of eukaryotic cells but fails to form functional transmembrane pores, or the cyclodextrin-derivative IB201, a blocker of the Hla pore, were used to investigate the permeability of the pore. Na(+) as well as Ca(2+) ions were able to pass the Hla pore and accumulated in the cytosol. The pore-mediated influx of calcium ions was blocked by IB201. Treatment of cells with recombinant Hla resulted in plasma membrane depolarization as well as in increases in the phosphorylation levels of paxillin (signaling pathway mediating disruption of the actin cytoskeleton) and p38 MAP kinase (signaling pathway resulting in defensive actions). p38 MAP kinase phosphorylation, but not paxillin phosphorylation, was elicited by treatment of cells with gramicidin. Although treatment of cells with rHla-H35L resulted in the formation of membrane-associated heptamers, none of these cellular effects were observed in our experiments. This indicates that formation of functional Hla-transmembrane pores is required to induce the cell physiological changes mediated by α-toxin. Specifically, the changes in ion equilibria and plasma membrane potential are important activators of p38 MAP kinase, a signal transduction module involved in host cell defense.

Uncovering homo-and hetero-interactions on the cell membrane using single particle tracking approaches

The plasma membrane of eukaryotic cells is responsible for a myriad of functions that regulate cell physiology and plays a crucial role in a multitude of processes that include adhesion, migration, signaling recognition and cell–cell communication. This is accomplished by specific interactions between different membrane components such as lipids and proteins on the lipid bilayer but also through interactions with the underlying cortical actin cytoskeleton on the intracellular side and the glycocalyx matrix in close proximity to the extracellular side. Advanced biophysical techniques, including single particle tracking (SPT) have revealed that the lateral diffusion of molecular components on the plasma membrane represents a landmark manifestation of such interactions. Indeed, by studying changes in the diffusivity of individual membrane molecules, including sub-diffusion, confined diffusion and/or transient arrest of molecules in membrane compartments, it has been possible to gain insight on the nature of molecular interactions and to infer on its functional role for cell response. In this review, we will revise some exciting results where SPT has been crucial to reveal homo- and hetero-interactions on the cell membrane.

Investigating Lipid-Protein Interactions in a Complex Biological Membrane Model

Biological membranes are crucial as they define essential compartments for cells. Cell membranes are heterogeneous mixtures of membrane proteins and lipids. Their structure and function are fundamentally dependent on lipid-lipid and lipid-protein interactions which play a crucial role in regulating cell protein functions and are involved in many diseases when altered. However, the exact properties and the relation among the distinct components that form the biological membrane are not completely understood due the limitations of experimental methods in studying the lipid-protein interactions in living cells. Lately, computer simulations have become a promising tool to understand and clarify experimental results. The goal of our research is to identify specific lipid-protein interactions of biological relevance and work towards large-scale modeling of realistic biological membranes. In this study, we placed ten main classes of eukaryotic membrane proteins with different ratios in a prototypical plasma membrane model, containing various lipid types found in the plasma membranes of eukaryotic cells. The protein-membrane system was prepared using Martini force field. Using molecular dynamic simulations, we intend to investigate how proteins sort lipids, and to identify specific binding partners and lipids of interest for more detailed studies.

EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells.

The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain-binding protein 1-like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1-dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport.

Deficiency of ATP6V1H Causes Bone Loss by Inhibiting Bone Resorption and Bone Formation through the TGF-β1 Pathway.

Vacuolar-type H +-ATPase (V-ATPase) is a highly conserved, ancient enzyme that couples the energy of ATP hydrolysis to proton transport across vesicular and plasma membranes of eukaryotic cells. Previously reported mutations of various V-ATPase subunits are associated with increased bone density. We now show that haploinsufficiency for the H subunit of the V1 domain (ATP6V1H) is associated with osteoporosis in humans and mice. A genome-wide SNP array analysis of 1625 Han Chinese found that 4 of 15 tag SNPs (26.7%) within ATP6V1H were significantly associated with low spine bone mineral density. Atp6v1h+/- knockout mice generated by the CRISPR/Cas9 technique had decreased bone remodeling and a net bone matrix loss. Atp6v1h+/- osteoclasts showed impaired bone formation and increased bone resorption. The increased intracellular pH of Atp6v1h+/- osteoclasts downregulated TGF-β1 activation, thereby reducing induction of osteoblast formation but the bone mineralization was not altered. However, bone formation was reduced more than bone resorption. Our data provide evidence that partial loss of ATP6V1H function results in osteoporosis/osteopenia. We propose that defective osteoclast formation triggers impaired bone formation by altering bone remodeling. In the future, ATP6V1H might, therefore, serve as a target for the therapy of osteoporosis.

Editorial: Membrane domains as new drug targets

Our understanding of the plasma membrane of eukaryotic cells has profoundly changed over the past 40 years (Kusumi et al., 2012). It is now well-understood that at the plasma membrane lipids and proteins are not randomly distributed, but are highly organized in discrete membrane domains with specific compositions such as lipid rafts, tetraspanin-enriched microdomains, caveolae or cortical actin-dependent membrane compartments (Cambi and Lidke, 2012; Kusumi et al., 2012). Membrane domains act as dedicated platforms for a whole range of cellular functions, such as cell proliferation, adhesion, migration, and intracellular trafficking, thus being appealing targets for drug treatment. This possibility has sprouted in numerous research efforts to identify and characterize drugs that selectively target membrane domains. Drugs may block or promote cellular functions by inducing, stabilizing, or disrupting the integrity or structure of these membrane domains.

Microparticle analysis in disorders of hemostasis and thrombosis.

Microparticles (MPs) are submicron vesicles released from the plasma membrane of eukaryotic cells in response to activation or apoptosis. MPs are known to be involved in numerous biologic processes, including inflammation, the immune response, cancer metastasis, and angiogenesis. Their earliest recognized and most widely accepted role, however, is the ability to promote and support the process of blood coagulation. Consequently, there is ongoing interest in studying MPs in disorders of hemostasis and thrombosis. Both phosphatidylserine (PS) exposure and the presence of tissue factor (TF) in the MP membrane may account for their procoagulant properties, and elevated numbers of MPs in plasma have been reported in numerous prothrombotic conditions. To date, however, there are few data on true causality linking MPs to the genesis of thrombosis. A variety of methodologies have been employed to characterize and quantify MPs, although detection is challenging due to their submicron size. Flow cytometry (FCM) remains the most frequently utilized strategy for MP detection; however, it is associated with significant technological limitations. Additionally, preanalytical and analytical variables can influence the detection of MPs by FCM, rendering data interpretation difficult. Lack of methodologic standardization in MP analysis by FCM confounds the issue further, although efforts are currently underway to address this limitation. Moving forward, it will be important to address these technical challenges as a scientific community if we are to better understand the role that MPs play in disorders of hemostasis and thrombosis.

Generation of lymphocytic microparticles and detection of their proapoptotic effect on airway epithelial cells.

Interest in the biological roles of cell membrane-derived vesicles in cell-cell communication has increased in recent years. Microparticles (MPs) are one such type of vesicles, ranging in diameter from 0.1 μm to 1 μm, and typically shed from the plasma membrane of eukaryotic cells undergoing activation or apoptosis. Here we describe the generation of T lymphocyte-derived microparticles (LMPs) from apoptotic CEM T cells stimulated with actinomycin D. LMPs are isolated through a multistep differential centrifugation process and characterized using flow cytometry. This protocol also presents an in situ cell death detection method for demonstrating the proapoptotic effect of LMPs on bronchial epithelial cells derived from mouse primary respiratory bronchial tissue explants. Methods described herein provide a reproducible procedure for isolating abundant quantities of LMPs from apoptotic lymphocytes in vitro. LMPs derived in this manner can be used to evaluate the characteristics of various disease models, and for pharmacology and toxicology testing. Given that the airway epithelium offers a protective physical and functional barrier between the external environment and underlying tissue, use of bronchial tissue explants rather than immortalized epithelial cell lines provides an effective model for investigations requiring airway tract tissue.

Analysis of PI(4,5)P2 Lateral Organization at the Plasma Membrane of Living Cells Through FRET

Phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) is an important component of the inner leaflet of the plasma membrane of eukaryotic cells. Despite the fact that it only comprises approximately 1 mol% of the total membrane phospholipids, this phosphoinosite has been associated with many different cells functions including membrane trafficking, actin cytoskeleton remodeling and cell motility, among others. The physiological functions of this lipid seem to depend on localized concentration fluctuations within the plasma membrane. In fact, the distribution of this lipid in the plasma membrane has been proposed to be heterogeneous, and PI(4,5)P2 clustering is detected on model membranes under specific conditions. Domains highly enriched in PI(4,5)P2 were also reported at the plasma membrane of specific cell types. However, for most cellular models, scarce evidence has been found for PI(4,5)P2 segregation/clustering in the plasma membrane.In this context, our main goal was to study the distribution of PI(4,5)P2 molecules in cells lines where no heterogeneity in PI(4,5)P2 lateral distribution had been previously observed. The distribution of PI(4,5)P2 was assessed from FRET microscopy measurements with pleckstrin homology (PH) domains tagged with different fluorescent proteins. We applied a FRET methodology capable of discriminating between FRET from aggregates/clusters from non-interacting molecules within the plasma membrane of living cells. Our results clearly show distinct PI(4,5)P2 local densities in different cellular models, suggesting different patterns of PI(4,5)P2 lateral distributions within the plasma membrane. In addition, the effect of cholesterol removal on PI(4,5)P2 lateral organization is significantly different in distinct cell lines, suggesting that the role of cholesterol in the formation of PI(4,5)P2 enriched domains varies considerably.This work was supported by FCT - Foundation of Science and Technology (PTDC/QUI-BIQ/119494/2010 and RECI/CTM-POL/0342/2012). M.J.S. and F.F. acknowledge research grants (SFRH/BD/80575/2011 and SFRH/BPD/64320/2009) from FCT.

Control of Membrane Asymmetry by P4-ATPases

The plasma membrane of eukaryotic cells is an asymmetric structure with the cytosolic leaflet highly enriched in phosphatidylserine (PS) and phosphatidylethanolamine (PE), whereas these lipids are rare in the extracellular leaflet. This asymmetric organization is generated by phospholipid flippases in the type IV P-type ATPase (P4-ATPases) family that pump specific phospholipids (e.g. PS and PE) unidirectionally to the cytosolic leaflet. Cells typically express several P4-ATPases that localize to the Golgi, plasma membrane and endosomal system, and differ in substrate specificity. We discovered a crucial role for P4-ATPases in budding protein transport vesicles from the trans-Golgi network (TGN) and early endosomal membranes. Current studies are aimed at understanding how P4-ATPases from Saccharomyces cerevisiae recognize and flip specific phospholipid species to generate membrane asymmetry, and why this plays an important role in budding certain types of transport vesicles. We have succeeded in mapping a number of residues that determine the phospholipid substrate specificity of the P4-ATPases Drs2 and Dnf1 to the first four transmembrane segments. These studies imply that the P4-ATPases use a non-canonical mechanism to recognize and transport substrate relative to closely related Ca++-ATPase. In addition, we can mutationally “tune” the substrate specificity of Drs2 and Dnf1 and use these mutants to probe the cellular requirements for specific P4-ATPase substrates. Neo1 is an essential P4-ATPase in yeast that is closely related to ATP9A and 9B in mammalian cells. Recent studies imply that Neo1 is the primary PE flippase in yeast and a mutation in Drs2 that enhances its recognition of PE can functionally replace Neo1 in vivo.

A Novel Phosphatidylinositol 4,5-Bisphosphate Binding Domain Mediates Plasma Membrane Localization of ExoU and Other Patatin-like Phospholipases

Bacterial toxins require localization to specific intracellular compartments following injection into host cells. In this study, we examined the membrane targeting of a broad family of bacterial proteins, the patatin-like phospholipases. The best characterized member of this family is ExoU, an effector of the Pseudomonas aeruginosa type III secretion system. Upon injection into host cells, ExoU localizes to the plasma membrane, where it uses its phospholipase A2 activity to lyse infected cells. The targeting mechanism of ExoU is poorly characterized, but it was recently found to bind to the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a marker for the plasma membrane of eukaryotic cells. We confirmed that the membrane localization domain (MLD) of ExoU had a direct affinity for PI(4,5)P2, and we determined that this binding was required for ExoU localization. Previously uncharacterized ExoU homologs from Pseudomonas fluorescens and Photorhabdus asymbiotica also localized to the plasma membrane and required PI(4,5)P2 for this localization. A conserved arginine within the MLD was critical for interaction of each protein with PI(4,5)P2 and for localization. Furthermore, we determined the crystal structure of the full-length P. fluorescens ExoU and found that it was similar to that of P. aeruginosa ExoU. Each MLD contains a four-helical bundle, with the conserved arginine exposed at its cap to allow for interaction with the negatively charged PI(4,5)P2. Overall, these findings provide a structural explanation for the targeting of patatin-like phospholipases to the plasma membrane and define the MLD of ExoU as a member of a new class of PI(4,5)P2 binding domains.

Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes.

The virulence of many Gram-positive bacteria depends on cholesterol-dependent cytolysins (CDCs), which form pores in eukaryotic cell plasma membranes. Pyolysin (PLO) from Trueperella pyogenes provided a unique opportunity to explore cellular responses to CDCs because it does not require thiol activation. Sublytic concentrations of PLO stimulated phosphorylation of MAPK ERK and p38 in primary stromal cells, and induced autophagy as determined by protein light-chain 3B cleavage. Although, inhibitors of MAPK or autophagy did not affect PLO-induced cytolysis. However, 10 μM 3-hydroxynaphthalene-2-carboxylic acid-(3,4-dihydroxybenzylidene)-hydrazide (Dynasore), a dynamin guanosine 5′-triphosphatase inhibitor, protected stromal cells against PLO-induced cytolysis as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (85 ± 17% versus 50 ± 9% cell viability), measuring extracellular ATP, and kinetic assays. This was a generalized mechanism because Dynasore also protected HeLa cells against streptolysin O. Furthermore, the effect was reversible, with stromal cell sensitivity to PLO restored within 30 minutes of Dynasore removal. The protective effect of Dynasore was not conferred by dynamin inhibition, induction of ERK phosphorylation, or Dynasore binding to PLO. Rather, Dynasore reduced cellular cholesterol and disrupted plasma membrane lipid rafts, similar to positive control methyl-β-cyclodextrin. Dynasore is a tractable tool to explore the complexity of cholesterol homeostasis in eukaryotic cells and to develop strategies to counter CDCs.—Preta, G., Lotti, V., Cronin, J. G., and Sheldon, I. M. Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes.

Interaction of Clostridium perfringens epsilon-toxin with biological and model membranes: A putative protein receptor in cells

Epsilon-toxin (ETX) is a powerful toxin produced by some strains of Clostridium perfringens (classified as types B and D) that is responsible for enterotoxemia in animals. ETX forms pores through the plasma membrane of eukaryotic cells, consisting of a β-barrel of 14 amphipathic β-strands. ETX shows a high specificity for certain cell lines, of which Madin–Darby canine kidney (MDCK) is the first sensitive cell line identified and the most studied one. The aim of this study was to establish the role of lipids in the toxicity caused by ETX and the correlation of its activity in model and biological membranes. In MDCK cells, using cell counting and confocal microscopy, we have observed that the toxin causes cell death mediated by toxin binding to plasma membrane. Moreover, ETX binds and permeabilizes the membranes of giant plasma membrane vesicles (GPMV). However, little effect is observed on protein-free vesicles. The data suggest the essential role of a protein receptor for the toxin in cell membranes.

Flippases and scramblases in the plasma membrane

Phospholipids in the plasma membrane of eukaryotic cells are asymmetrically distributed between the outer and inner leaflets.1 Phosphatidylcholine and sphingomyelin are located primarily in the outer leaflet, while phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) are restricted to the cytoplasmic side. This asymmetrical phospholipid distribution is disrupted during various biological processes. PtdSer exposed on the outside of activated platelets promotes blood clotting, and on apoptotic cells acts as an “eat me” signal for macrophages. The phospholipid distribution is regulated by lipid transporters in the plasma membrane.1 Scramblases non-specifically and bidirectionally transport phospholipids between the inner and outer leaflets in an ATP-independent manner, while flippases, also known as ATP-dependent aminophospholipid translocases, transport PtdSer and PtdEtn from the extracellular to the cytoplasmic side.1