Plasma Membrane Of Eukaryotic Cells Research Articles

Sorting of GPI-anchored proteins at the trypanosome surface is independent of GPI insertion signals

The segregation of glycosylphosphatidylinositol-anchored proteins (GPI-APs) to distinct domains on the plasma membrane of eukaryotic cells is important for their correct cellular function, but the mechanisms by which GPI-APs are sorted are yet to be fully resolved. An extreme example of this is in African trypanosomes, where the major surface glycoprotein floods the whole cell surface while most GPI-APs are retained in a specialised domain at the base of the flagellum. One possibility is that anchor attachment signals direct differential sorting of proteins. To investigate this, we fused a monomeric reporter to the GPI-anchor insertion signals of trypanosome proteins that are differentially sorted on the plasma membrane. Fusions were correctly anchored by GPI, post-translationally modified, and routed to the plasma membrane, but this delivery was independent of retained signals upstream of the ω site. Instead, ω−minus signal strength appears key to efficacy of GPI addition and to GPI-AP cellular level. Thus, at least in this system, sorting is not encoded at the time of GPI anchor addition or in the insertion sequence retained in processed proteins. We discuss these findings in the context of previously proposed models for sorting mechanisms in trypanosomes.

The multifunction Coxiella effector Vice stimulates macropinocytosis and interferes with the ESCRT machinery

Intracellular bacterial pathogens divert multiple cellular pathways to establish their niche and persist inside their host. Coxiella burnetii, the causative agent of Q fever, secretes bacterial effector proteins via its Type 4 secretion system to generate a Coxiella-containing vacuole (CCV). Manipulation of lipid and protein trafficking by these effectors is essential for bacterial replication and virulence. Here, we have characterized the lipid composition of CCVs and found that the effector Vice interacts with phosphoinositides and membranes enriched in phosphatidylserine and lysobisphosphatidic acid. Remarkably, eukaryotic cells ectopically expressing Vice present compartments that resemble early CCVs in both morphology and composition. We found that the biogenesis of these compartments relies on the double function of Vice. The effector protein initially localizes at the plasma membrane of eukaryotic cells where it triggers the internalization of large vacuoles by macropinocytosis. Then, Vice stabilizes these compartments by perturbing the ESCRT machinery. Collectively, our results reveal that Vice is an essential C. burnetii effector protein capable of hijacking two major cellular pathways to shape the bacterial replicative niche.

Channelrhodopsin-2 Oligomerization in Cell Membrane Revealed by Photo-Activated Localization Microscopy.

Microbial rhodopsins are retinal membrane proteins that found a broad application in optogenetics. The oligomeric state of rhodopsins is important for their functionality and stability. Of particular interest is the oligomeric state in the cellular native membrane environment. Fluorescence microscopy provides powerful tools to determine the oligomeric state of membrane proteins directly in cells. Among these methods is quantitative photoactivated localization microscopy (qPALM) allowing the investigation of molecular organization at the level of single protein clusters. Here, we apply qPALM to investigate the oligomeric state of the first and most used optogenetic tool Channelrhodopsin-2 (ChR2) in the plasma membrane of eukaryotic cells. ChR2 appeared predominantly as a dimer in the cell membrane and did not form higher oligomers. The disulfide bonds between Cys34 and Cys36 of adjacent ChR2 monomers were not required for dimer formation and mutations disrupting these bonds resulted in only partial monomerization of ChR2. The monomeric fraction increased when the total concentration of mutant ChR2 in the membrane was low. The dissociation constant was estimated for this partially monomerized mutant ChR2 as 2.2±0.9 proteins/μm2 . Our findings are important for understanding the mechanistic basis of ChR2 activity as well as for improving existing and developing future optogenetic tools.

Channelrhodopsin‐2 Oligomerization in Cell Membrane Revealed by Photo‐Activated Localization Microscopy

AbstractMicrobial rhodopsins are retinal membrane proteins that found a broad application in optogenetics. The oligomeric state of rhodopsins is important for their functionality and stability. Of particular interest is the oligomeric state in the cellular native membrane environment. Fluorescence microscopy provides powerful tools to determine the oligomeric state of membrane proteins directly in cells. Among these methods is quantitative photoactivated localization microscopy (qPALM) allowing the investigation of molecular organization at the level of single protein clusters. Here, we apply qPALM to investigate the oligomeric state of the first and most used optogenetic tool Channelrhodopsin‐2 (ChR2) in the plasma membrane of eukaryotic cells. ChR2 appeared predominantly as a dimer in the cell membrane and did not form higher oligomers. The disulfide bonds between Cys34 and Cys36 of adjacent ChR2 monomers were not required for dimer formation and mutations disrupting these bonds resulted in only partial monomerization of ChR2. The monomeric fraction increased when the total concentration of mutant ChR2 in the membrane was low. The dissociation constant was estimated for this partially monomerized mutant ChR2 as 2.2±0.9 proteins/μm2. Our findings are important for understanding the mechanistic basis of ChR2 activity as well as for improving existing and developing future optogenetic tools.

Genome-Wide Analysis of the SNARE Family in Cultivated Peanut (Arachis hypogaea L.) Reveals That Some Members Are Involved in Stress Responses.

The superfamily of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediates membrane fusion during vesicular transport between endosomes and the plasma membrane in eukaryotic cells, playing a vital role in plant development and responses to biotic and abiotic stresses. Peanut (Arachis hypogaea L.) is a major oilseed crop worldwide that produces pods below ground, which is rare in flowering plants. To date, however, there has been no systematic study of SNARE family proteins in peanut. In this study, we identified 129 putative SNARE genes from cultivated peanut (A. hypogaea) and 127 from wild peanut (63 from Arachis duranensis, 64 from Arachis ipaensis). We sorted the encoded proteins into five subgroups (Qa-, Qb-, Qc-, Qb+c- and R-SNARE) based on their phylogenetic relationships with Arabidopsis SNAREs. The genes were unevenly distributed on all 20 chromosomes, exhibiting a high rate of homolog retention from their two ancestors. We identified cis-acting elements associated with development, biotic and abiotic stresses in the promoters of peanut SNARE genes. Transcriptomic data showed that expression of SNARE genes is tissue-specific and stress inducible. We hypothesize that AhVTI13b plays an important role in the storage of lipid proteins, while AhSYP122a, AhSNAP33a and AhVAMP721a might play an important role in development and stress responses. Furthermore, we showed that three AhSNARE genes (AhSYP122a, AhSNAP33a and AhVAMP721) enhance cold and NaCl tolerance in yeast (Saccharomyces cerevisiae), especially AhSNAP33a. This systematic study provides valuable information about the functional characteristics of AhSNARE genes in the development and regulation of abiotic stress responses in peanut.

Atp8a1 deletion increases the proliferative activity of hematopoietic stem cells by impairing PTEN function.

The eukaryotic cell plasma membrane contains several asymmetrically distributed phospholipids, which is maintained by the P4-ATPase flippase complex. Herein, we demonstrated the biological effects and mechanisms of asymmetrical loss in hematopoietic stem cells (HSCs). An Atp8a1 knockout mouse model was employed, from which the HSC (long-term HSCs and short-term HSCs) population was analyzed to assess their abundance and function. Additionally, competitive bone marrow transplantation and 5-FU stress assays were performed. RNA sequencing was performed on Hematopoietic Stem and Progenitor Cells, and DNA damage was assayed using immunofluorescence staining and comet electrophoresis. The protein abundance for members of key signaling pathways was confirmed using western blotting. Atp8a1 deletion resulted in slight hyperleukocytosis, associated with the high proliferation of HSCs and BCR/ABL1 transformed leukemia stem cells (LSCs). Atp8a1 deletion increased the repopulation capability of HSCs with a competitive advantage in reconstitution assay. HSCs without Atp8a1 were more sensitive to 5-FU-induced apoptosis. Moreover, Atp8a1 deletion prevented HSC DNA damage and facilitated DNA repair processes. Genes involved in PI3K-AKT-mTORC1, DNA repair, and AP-1 complex signaling were enriched and elevated in HSCs with Atp8a1 deletion. Furthermore, Atp8a1 deletion caused decreased PTEN protein levels, resulting in the activation of PI3K-AKT-mTORC1 signaling, further increasing the activity of JNK/AP-1 signaling and YAP1 phosphorylation. We identified the role of Atp8a1 on hematopoiesis and HSCs. Atp8a1 deletion resulted in the loss of phosphatidylserine asymmetry and intracellular signal transduction chaos.

Structural diversity of photoswitchable sphingolipids for optodynamic control of lipid microdomains

Sphingolipids are a structurally diverse class of lipids predominantly found in the plasma membrane of eukaryotic cells. These lipids can laterally segregate with other rigid lipids and cholesterol into liquid-ordered domains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral organization is of utmost significance. Hence, we made use of the light-induced trans-cis isomerization of azobenzene-modified acyl chains to develop a set of photoswitchable sphingolipids with different headgroups (hydroxyl, galactosyl, phosphocholine) and backbones (sphingosine, phytosphingosine, tetrahydropyran-blocked sphingosine) that are able to shuttle between liquid-ordered and liquid-disordered regions of model membranes upon irradiation with UV-A (λ = 365 nm) and blue (λ = 470 nm) light, respectively. Using combined high-speed atomic force microscopy, fluorescence microscopy, and force spectroscopy, we investigated how these active sphingolipids laterally remodel supported bilayers upon photoisomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that the sphingosine-based (Azo-β-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo-α-Gal-PhCer, Azo-PhCer) photoswitchable lipids promote a reduction in liquid-ordered microdomain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having tetrahydropyran groups that block H-bonding at the sphingosine backbone (lipids named Azo-THP-SM, Azo-THP-Cer) induce an increase in the liquid-ordered domain area when in cis, accompanied by a major rise in height mismatch and line tension. These changes were fully reversible upon blue light-triggered isomerization of the various lipids back to trans, pinpointing the role of interfacial interactions for the formation of stable liquid-ordered domains.

Exploitation of the host exocyst complex by bacterial pathogens.

Intracellular bacterial pathogens remodel the plasma membrane of eukaryotic cells in order to establish infection. A common and well-studied mechanism of plasma membrane remodelling involves bacterial stimulation of polymerization of the host actin cytoskeleton. Here, we discuss recent results showing that several bacterial pathogens also exploit the host vesicular trafficking pathway of 'polarized exocytosis' to expand and reshape specific regions in the plasma membrane during infection. Polarized exocytosis is mediated by an evolutionarily conserved octameric protein complex termed the exocyst. We describe examples in which the bacteria Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and Shigella flexneri co-opt the exocyst to promote internalization into human cells or intercellular spread within host tissues. We also discuss results showing that Legionella pneumophila or S. flexneri manipulate exocyst components to modify membrane vacuoles to favour intracellular replication or motility of bacteria. Finally, we propose potential ways that pathogens manipulate exocyst function, discuss how polarized exocytosis might promote infection and highlight the importance of future studies to determine how actin polymerization and polarized exocytosis are coordinated to achieve optimal bacterial infection.

Collective dynamics in the lipid phases of DMPC/cholesterol bilayers: A neutron spin-echo study.

Cholesterol is essential in cell membranes as it regulates membrane structural and material properties such as phase behavior, elasticity, and viscosity. Cholesterol levels in different organelles vary, from less than 5% in mitochondria to 40% in the plasma membrane of eukaryotic cells. According to an experimental phase diagram of 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC)/cholesterol mixture based on lateral diffusion measurements from early studies, at the above melting temperature, the system undergoes a phase transition from a liquid-disordered (Ld) phase to a liquid-ordered (Lo) phase with a coexisting phase (Ld+Lo) in between upon increasing cholesterol concentration [1]. Our work attempts to understand the collective dynamics of the mixture in each phase and quantify their behavioral trends. We first performed densitometry to determine the mixture's molecular volume and to correct the melting temperature difference between protiated and deuterated DMPC, which was used in neutron experiments. The molecular volume was then implemented in small angle neutron scattering (SANS) experiments and data analysis to extract the area per lipid (AL) and thickness of the lipid mixture in each phase region. Finally, neutron spin echo (NSE) is used to access the dynamic properties by adopting structural information from SANS. The NSE results show the effective bending modulus and membrane viscosity of the Ld phase scale with AL, as observed in our previous study of mixed lipid membranes [2].

Labeling cell surface glycosylphosphatidylinositol-anchored proteins through metabolic engineering using an azide-modified phosphatidylinositol

Glycosylphosphatidylinositol (GPI) anchorage is one of the most common mechanisms to attach proteins to the plasma membrane of eukaryotic cells. GPI-anchored proteins (GPI-APs) play a critical role in many biological processes but are difficult to study. Here, a new method was developed for the effective and selective metabolic engineering and labeling of cell surface GPI-APs with an azide-modified phosphatidylinositol (PI) as the biosynthetic precursor of GPIs. It was demonstrated that this azido-PI derivative was taken up by HeLa cells and incorporated into the biosynthetic pathway of GPIs to present azide-labeled GPI-APs on the live cell surface. The azido group was used as a molecular handle to install other labels through a biocompatible click reaction to enable various biological studies, e.g., fluorescent imaging and protein pull-down, which can help explore the functions of GPI-APs and discover new GPI-APs.

Scratching beyond the surface - minimal actin assemblies as tools to elucidate mechanical reinforcement and shape change.

The interaction between the actin cytoskeleton and the plasma membrane in eukaryotic cells is integral to a large number of functions such as shape change, mechanical reinforcement and contraction. These phenomena are driven by the architectural regulation of a thin actin network, directly beneath the membrane through interactions with a variety of binding proteins, membrane anchoring proteins and molecular motors. An increasingly common approach to understanding the mechanisms that drive these processes is to build model systems from reconstituted lipids, actin filaments and associated actin-binding proteins. Here we review recent progress in this field, with a particular emphasis on how the actin cytoskeleton provides mechanical reinforcement, drives shape change and induces contraction. Finally, we discuss potential future developments in the field, which would allow the extension of these techniques to more complex cellular processes.

Lipid Polarization during Cytokinesis.

The plasma membrane of eukaryotic cells is composed of a large number of lipid species that are laterally segregated into functional domains as well as asymmetrically distributed between the outer and inner leaflets. Additionally, the spatial distribution and organization of these lipids dramatically change in response to various cellular states, such as cell division, differentiation, and apoptosis. Division of one cell into two daughter cells is one of the most fundamental requirements for the sustenance of growth in all living organisms. The successful completion of cytokinesis, the final stage of cell division, is critically dependent on the spatial distribution and organization of specific lipids. In this review, we discuss the properties of various lipid species associated with cytokinesis and the mechanisms involved in their polarization, including forward trafficking, endocytic recycling, local synthesis, and cortical flow models. The differences in lipid species requirements and distribution in mitotic vs. male meiotic cells will be discussed. We will concentrate on sphingolipids and phosphatidylinositols because their transbilayer organization and movement may be linked via the cytoskeleton and thus critically regulate various steps of cytokinesis.

Membrane-mediated dimerization potentiates PIP5K lipid kinase activity.

The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of lipid-modifying enzymes generate the majority of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipids found at the plasma membrane in eukaryotic cells. PI(4,5)P2 lipids serve a critical role in regulating receptor activation, ion channel gating, endocytosis, and actin nucleation. Here, we describe how PIP5K activity is regulated by cooperative binding to PI(4,5)P2 lipids and membrane-mediated dimerization of the kinase domain. In contrast to constitutively dimeric phosphatidylinositol 5-phosphate 4-kinase (PIP4K, type II PIPK), solution PIP5K exists in a weak monomer-dimer equilibrium. PIP5K monomers can associate with PI(4,5)P2-containing membranes and dimerize in a protein density-dependent manner. Although dispensable for cooperative PI(4,5)P2 binding, dimerization enhances the catalytic efficiency of PIP5K through a mechanism consistent with allosteric regulation. Additionally, dimerization amplifies stochastic variation in the kinase reaction velocity and strengthens effects such as the recently described stochastic geometry sensing. Overall, the mechanism of PIP5K membrane binding creates a broad dynamic range of lipid kinase activities that are coupled to the density of PI(4,5)P2 and membrane-bound kinase.

Caveolin-1: A Promising Therapeutic Target for Diverse Diseases.

The plasma membrane of eukaryotic cells contains small flask-shaped invaginations known as caveolae that are involved in the regulation of cellular signaling. Caveolin-1 is a 21-24k- Da protein localized in the caveolar membrane. Caveolin-1 (Cav-1) has been considered as a master regulator among the various signaling molecules. It has been emerging as a chief protein regulating cellular events associated with homeostasis, caveolae formation, and caveolae trafficking. In addition to the physiological role of cav-1, it has a complex role in the progression of various diseases. Caveolin-1 has been identified as a prognosticator in patients with cancer and has a dual role in tumorigenesis. The expression of Cav-1 in hippocampal neurons and synapses is related to neurodegeneration, cognitive decline, and aging. Despite the ubiquitous association of caveolin-1 in various pathological processes, the mechanisms associated with these events are still unclear. Caveolin- 1 has a significant role in various events of the viral cycle, such as viral entry. This review will summarize the role of cav-1 in the development of cancer, neurodegeneration, glaucoma, cardiovascular diseases, and infectious diseases. The therapeutic perspectives involving clinical applications of Caveolin-1 have also been discussed. The understanding of the involvement of caveolin-1 in various diseased states provides insights into how it can be explored as a novel therapeutic target.

Anionic membrane phospholipids: A New Class of Chemokine‐Binding Site Important for both Apoptotic Cell Clearance and Antibiotic Activity by Chemokines

Chemokines are small cytokines that orchestrate immune cell migration by binding to cell surface glycosaminoglycans (GAGs) and activating G protein‐couple receptors (GPCRs) expressed by leukocytes. We have discovered that chemokines selectively bind with high affinity to anionic membrane phospholipids, including phosphatidylserine (PS) and cardiolipin (CL). This adds to GPCRs and GAGs a third class of high affinity membrane binding site for chemokines and expands the scope of chemokine action to new areas of biology dictated by the specific biological niches occupied by their phospholipid binding partners. PS, for example, is usually restricted to the cytosolic face of the phospholipid bilayer of the plasma membrane of healthy eukaryotic cells, but it is flipped to the outer leaflet of the membrane of apoptotic cells and apoptotic extracellular vesicles (ApoEVs), where it acts as an ‘eat‐me’ signal recognized by phagocytes for apoptotic cell clearance. In this regard, we have recently demonstrated that ApoEVs generated in vivo from mouse thymus induce phagocyte migration using endogenous chemokines bound to the vesicle surface via PS. Our results indicate that PS‐bound chemokines on ApoEVs may serve as ‘find‐me’ signals that recruit phagocytes toward apoptotic cells for the removal of dying cells. This is a new PS‐dependent mechanism for chemokine delivery by extracellular vesicles. On the other hand, CL is absent in the plasma membrane of eukaryotic cells but it is a major and naturally exposed anionic phospholipid in the bacterial plasma membrane. As such, we have found that chemokines act as potent direct bacteria‐killing agents by binding to bacterial CL. This finding identifies the molecular mechanism for antimicrobial action by chemokines and launch new avenues of research in antimicrobial therapies and bacteria‐host interactions. In short, we have identified chemokines as the first example of a family of soluble cytokines whose functions are regulated by direct interaction with membrane phospholipids.

Asymmetric phase diagram of a binary lipid mixture

The plasma membranes of eukaryotic cells are asymmetric with respect to their lipid composition. Model membrane studies consistently find that simplified outer leaflet mixtures tend to separate into coexisting liquid phases in symmetric bilayers, while inner leaflet mixtures do not. Among the major unanswered questions is how the phase behavior of the two leaflets is coupled in an asymmetric bilayer. Mean field theory has been used to calculate asymmetric phase diagrams, where phase boundaries and tieline orientations depend on the relative strength of in-plane and out-of-plane lipid interaction energies.

Characteristic lengths of transmembrane peptides controlling their tilt and lateral distribution between membrane domains.

Lipids and proteins of plasma membranes of eukaryotic cells are supposed to form protein-lipid domains, characterized by a different molecular order, bilayer thickness, and elastic parameters. Several mechanisms of preferable distribution of transmembrane proteins to the ordered or disordered membrane domains have been revealed. The mismatch between the length of the protein transmembrane domain and hydrophobic thickness of the lipid bilayer is considered to be an important driving force of protein lateral sorting. Utilizing the continuum theory of elasticity, we analyzed optimal configurations and preferable membrane domains for single-pass transmembrane peptides of various hydrophobic lengths and effective molecular shapes. We obtained that short transmembrane peptides stand perpendicularly to the membrane plane. The exceedance of a certain characteristic length leads to the tilt of the peptide. This length depends on the bilayer thickness. Thus, in the membrane with coexisting ordered (thicker) and disordered (thinner) phases tilting of the peptide in each phase is governed by its individual characteristic length. The lateral distribution of the peptides between ordered and disordered membrane domains is shown to be described by two additional characteristic lengths. The exceedance of the smaller one drives the peptide towards a more ordered and thicker membrane, while the exceedance of the larger characteristic length switches the preferable membrane domain from ordered and thicker to disordered and thinner. Thus, membrane proteins with long enough transmembrane domains are predicted to accumulate in the thinner disordered membrane as compared to the thicker ordered bilayer. For hourglass-like and barrel-like shaped transmembrane peptides the specific regime of sorting was obtained: the peptides distributed almost equally between the phases in a wide range of peptide lengths. This finding allowed explaining the experimental data on lateral distribution of transmembrane peptide tLAT.

NEW GENES AND DISEASES: EP.301 The alpha2-subunit of the AP2 clathrin adaptor as the causal gene in an atypical myopathy with granulofilamentous inclusions

Clathrin-mediated endocytosis is the best characterized process for the entry of proteins and lipids at the plasma membrane of eukaryotic cells. We have previously shown that at costameres, muscle adhesion and force transmission sites, clathrin recruited by the AP2 adaptor forms flat plaques required for actin and desmin intermediate filament organization. Here, we describe a patient presenting a progressive muscular atrophy with loss of ambulation, complete tetraplegia, ophthalmoplegia, severe scoliosis and lower limb contractures and no cardiac involvement. Electron microscopy of muscle biopsy revealed inclusions composed of filamentous material, vacuoles, honeycomb structures and thin granular material surcharge. This case was classified as a myofibrillar myopathy with granulofilamentous inclusion bodies despite no mutation in DES, FHL1, CRYAB and other usual suspect genes. Whole exome sequencing identified two compound heterozygote single-nucleotide variants in the AP2A2 gene encoding the AP2 a2-subunit. Immunofluorescent labeling on muscle sections displayed intracellular aggregates positive for AP2, clathrin and desmin. Depletion of total AP2 in mouse muscle induced a dystrophic phenotype with severe force loss, subsarcolemmal disorganization, vacuoles and neuromuscular junction defects while specifically depleting AP2 a2-subunit induced small subsarcolemmal disorganization and light neuromuscular junction defects. Lastly, transferrin uptake assays of endocytosis function in the patient fibroblasts showed a decrease in endocytosis rates compared to fibroblasts from his healthy parents. Altogether, our results suggest that AP2A2 could be the causal gene in this family and AP2A2 mutations leading to a severe but atypical myopathy. Mutations in AP2A2 could represent the first case in a wider family of atypical myopathies with desmin inclusions caused by mutations in genes encoding proteins of the endocytosis machinery.

Phenothiazines alter plasma membrane properties and sensitize cancer cells to injury by inhibiting annexin-mediated repair

Repair of damaged plasma membrane in eukaryotic cells is largely dependent on the binding of annexin repair proteins to phospholipids. Changing the biophysical properties of the plasma membrane may provide means to compromise annexin-mediated repair and sensitize cells to injury. Since, cancer cells experience heightened membrane stress and are more dependent on efficient plasma membrane repair, inhibiting repair may provide approaches to sensitize cancer cells to plasma membrane damage and cell death. Here, we show that derivatives of phenothiazines, which have widespread use in the fields of psychiatry and allergy treatment, strongly sensitize cancer cells to mechanical-, chemical-, and heat-induced injury by inhibiting annexin-mediated plasma membrane repair. Using a combination of cell biology, biophysics, and computer simulations, we show that trifluoperazine acts by thinning the membrane bilayer, making it more fragile and prone to ruptures. Secondly, it decreases annexin binding by compromising the lateral diffusion of phosphatidylserine, inhibiting the ability of annexins to curve and shape membranes, which is essential for their function in plasma membrane repair. Our results reveal a novel avenue to target cancer cells by compromising plasma membrane repair in combination with noninvasive approaches that induce membrane injuries.

Ceramide structure dictates glycosphingolipid nanodomain assembly and function

Gangliosides in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. How gangliosides are dynamically organized and how they respond to ligand binding is poorly understood. Using fluorescence anisotropy imaging of synthetic, fluorescently labeled GM1 gangliosides incorporated into the plasma membrane of living cells, we found that GM1 with a fully saturated C16:0 acyl chain, but not with unsaturated C16:1 acyl chain, is actively clustered into nanodomains, which depends on membrane cholesterol, phosphatidylserine and actin. The binding of cholera toxin B-subunit (CTxB) leads to enlarged membrane domains for both C16:0 and C16:1, owing to binding of multiple GM1 under a toxin, and clustering of CTxB. The structure of the ceramide acyl chain still affects these domains, as co-clustering with the glycosylphosphatidylinositol (GPI)-anchored protein CD59 occurs only when GM1 contains the fully saturated C16:0 acyl chain, and not C16:1. Thus, different ceramide species of GM1 gangliosides dictate their assembly into nanodomains and affect nanodomain structure and function, which likely underlies many endogenous cellular processes.