Mammalian Cell Membranes Research Articles

Asymmetric distribution of aminophospholipids stiffens fluid membranes.

Cellular envelopes contain a large number of lipid species that distribute asymmetrically between the two leaflets of the bilayer. For example, mammalian plasma membranes are composed of an outer leaflet enriched in cholinephospholipids, while the majority of the aminophospholipids are confined to the inner leaflet. Membrane asymmetry can theoretically lead to distinct effects regarding its elastic behavior, either due to lipid specific properties (e.g. size, shape), or simply lipid over/under-crowding of a given leaflet. Corresponding experimental data are scarce. This prompted us to determine the structure and bending rigidity of minimal mammalian plasma membrane mimics composed of milk sphingomyelin (MSM), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), and palmitoyl oleoyl phosphatidylserine (POPS) using small-angle neutron/X-ray scattering and neutron spin echo spectroscopy. Most strikingly, we observed an anomalous stiffening of freely-floating fluid vesicles (size ∼ 100 nm) in case of inner leaflets composed of POPE/POPS mixtures and outer leaflets enriched in POPC. That is, the bending rigidities of POPCout/(POPE/POPS)in vesicles did not only exceed those of their scrambled (symmetric) analogs, but also those of symmetric vesicles mimicking either their inner and outer leaflets. Addition MSM in the outer leaflet reduced but not abolished this effect, possibly due to hydrocarbon chain interdigitation. To reconcile our findings, we speculate that lipid shape and charge asymmetry may increase the weight of short-wavelength undulatory modes in membranes by inducing a coupling at distances smaller than the membrane thickness.

A united-atom representation for sphingolipids in the CHARMM force field.

With the development of the CHARMM force field in the 1970s and 1980s came a software package that was able to model the complexity of macromolecules and biomolecular systems. The CHARMM family has since expanded and improved to include such intricacies as cellular membranes. Sphingolipids were originally found in brain extracts and neurons in the late 1800s and have since been found to be common in mammalian plasma membranes. In recent years, these lipids have been included into the CHARMM36; however, there is still more to these lipids that have yet to be implemented in CHARMM. One such inclusion currently being implemented by the authors of this poster is that of a united-atom representation of these sphingolipids. Currently, lipids in CHARMM36 can be modeled using an ‘all-atom’ representation in which all atoms of the lipids are modeled as interaction sites or a ‘united-atom’ representation in which the hydrogens on the lipid tails are combined onto their connected carbons for computational ease. With the recent implementation of a united-atom representation for phospholipids in CHARMM (C36UAr), the authors are working on extending this for sphingolipids. Although an all-atom force field is useful in many cases, a united-atom force field can also be beneficial, such as in modeling large membrane systems where the atomic details of the lipids can be slightly relaxed for computational ease. Thus, the authors’ goal is to create and implement a C36UAr for sphingolipids. The authors hope that this representation will be used by future researchers to study the functions, properties, and applications of cell membranes.

Number asymmetry: The hidden dimension of mammalian plasma membranes.

The transbilayer distribution of lipids in mammalian plasma membranes (PMs) is functionally important and incompletely understood. It has been generally assumed that the two PM leaflets contain similar numbers of phospholipids (PLs) due to the constraint that their areas must be evenly matched in a bilayer. Contrary to this widely held assumption, our recent detailed lipidomics analysis of human erythrocytes reveals a large phospholipid imbalance between PM leaflets, with the cytoplasmic leaflet possessing almost 2-fold more PLs than the exoplasmic one. This surprising finding challenges our understanding of living membrane organization and structure. Extensive atomistic simulations of a PM model guided by the lipidomics data reveal that a large PL imbalance between the leaflets can be sustained via highly asymmetric distribution of cholesterol. We also demonstrate that cholesterol increases membrane tolerance for PL imbalances in model membranes and cells. Driven by preferential interactions with saturated lipids and its tendency to ‘fill gaps’, we show that cholesterol is poised for enrichment in the exoplasmic leaflet of the plasma membrane and confirm this prediction in live red blood cells using a novel FRET-based assay. The resulting PL and cholesterol number asymmetries in the PM give rise to unique biophysical properties including differential permeability of the two bilayer leaflets and a substantial differential stress. Being under tension, the cytoplasmic leaflet has deep-reaching hydrophobic defects and imaging of living mammalian cells confirms their role in recruiting lipidated proteins to the plasma membrane. This number asymmetry of major membrane constituents thus presents a new and largely unexplored dimension of membrane organization.

Molecular dynamics simulations of Hepatitis C viroporin and lipid membranes.

Mammalian cell membranes contain thousands of different lipid species with important roles in cellular processes that involve interactions with proteins. Disruption of lipid metabolism is deleterious to the cell and a hallmark of viral infections. One factor that causes this is the interactions of uniquely structured viral proteins with membrane lipids during virus morphogenesis. These interactions drive distinct protein and lipid interactions that aid virus propagation and modify the local membrane environment. Hepatitis C virus (HCV) is notorious for causing fat mismanagement in the liver, that can often develop into non-alcoholic fatty liver diseases (NAFLDs). These can progress to more severe conditions such as cirrhosis and hepatic carcinoma. p7 is a non-structural HCV protein critical for virus assembly in the endoplasmic reticulum, close to sites of lipid droplet formation. We hypothesize that p7 induces local changes in membrane composition that may lead to lipid droplets with different characteristics in lipid composition and structural properties, which contribute to fat mismanagement in the long run. As a first step, all-atom molecular dynamics were run to examine interactions between a p7 monomer and model lipid bilayers. This study aims to determine differences in protein-lipid interactions in membrane models with different lipid composition, and to quantify membrane response to protein binding. Specifically, we characterize protein binding time and conformation, frequency of contact between specific amino acids and lipid species, and changes to the local membrane environment from a total of over 7us of simulation trajectories. Protein residues in the N-terminus consistently come in contact with the bilayer first, and a more charged membrane surface results in a flatter protein bound conformation that allows for partial protein insertion.

Membrane order in pollen tubes-Methodology and unexpected findings.

The plasma membrane of mammalian cells is made up of two co-existing liquid phases, liquid ordered (Lo) phase and liquid disordered (Ld) phase. In pollen tubes that grow 3-20 μm/min upon germination, little is known about the membrane order and the existence of ordered membrane domains. Accordingly, we stained pollen tubes with the solvatochromic probe di-4-ANEPPDHQ, which undergoes a red emission shift in Lo-phase compared to Ld-phase membranes, because the latter are more hydrated. Growing pollen tubes were imaged live using widefield fluorescence microscopy with the emission split into two channels recorded simultaneously. Z-series were captured, deconvolved and the relative abundance of Lo- versus Ld-phase was quantified by generalised polarisation (GP). The plasma membrane was first outlined by marking sequential points, which were used as a basis for finding the most intense pixels performing searches along short line segments perpendicular to the local orientation of the plasma membrane. Additional points were then interpolated and similarly optimized. An area outside the pollen tube was automatically defined and used for background subtraction. The mapped plasma membrane was used to automatically identify the tip of the pollen tube and three distance maps were generated (i) from the tip along the plasma membrane, (ii) from tip inside the pollen tube and (iii) inwards from the plasma membrane. These maps were combined to delineate specific regions of interest. The membrane order of apical vesicles (AV) was compared to that of three biologically relevant zones along the plasma membrane. Interestingly, the highest membrane order was observed in the AV and the tip zone of the plasma membrane, whereas the order of the plasma membrane along the tube was lower. Pollen tubes thus differ from other eukaryotic cells, whose plasma membranes consistently has the highest order.

A conformational switch in clathrin light chain regulates lattice structure and endocytosis at the plasma membrane of mammalian cells.

The nanoscale localizations, interactions, and conformations of endocytic proteins are key regulators of clathrin-mediated endocytosis. Although super-resolution microscopy has revealed the detailed organization of endocytic proteins, the resolution of super-resolution imaging cannot assess molecular interaction and conformational changes. Fluorescence resonance energy transfer (FRET) occurs when dyes are separated by less than 10 nm for most fluorescent proteins pairs. Thus, it can be used to map close-range molecular complexes and dynamics. Yet, to understand how endocytosis works, measurements from FRET must be correlated to the distinct stages of endocytosis. To accomplish this, we developed a new correlative lifetime-based FRET (FLIM-FRET) and platinum replica transmission electron microscopy (PREM) method, named FRET-CLEM. Here, FRET-based atomic distances can be mapped directly to individual cellular structures visualized in EM at the plasma membrane. We used this method to measure the conformational changes in clathrin light chain (CLC), a component of the clathrin triskelion and assembled clathrin lattice. CLC conformational changes have been proposed to regulate the assembly of clathrin in solution. However, CLC conformational changes and their effects on clathrin lattice growth, curvature, and endocytosis at the membrane of living cells are unknown. Using FRET-CLEM, we discovered that CLC undergoes a conformational switch as clathrin lattices curve. Preventing this conformational switch with acute chemical tools increased clathrin lattice sizes and inhibited endocytosis. Therefore, a specific conformational switch in CLC regulates lattice curvature and endocytosis in mammalian cells. These new correlative light and EM data will help develop a complete mechanistic model of endocytosis. More generally, FRET-CLEM can map molecular interactions and conformational changes at targeted membrane-associated proteins at identified cellular compartments including exocytic sites and neuronal synapses.

How ostreolysin A detects SM/cholesterol in membranes.

Some pore-forming toxins target cells by binding specific lipids on the plasma membrane. Structural investigation of these protein-lipid interactions can provide insight into how membrane lipids are presented to proteins on the extracellular surface of cells. We use toxin proteins that recognize distinct regulatory pools of cholesterol in mammalian plasma membranes to better understand cholesterols membrane organization. The fungal toxin Ostreolysin A (OlyA) specifically binds sphingomyelin (SM) only when SM is complexed with cholesterol in the SM-sequestered pool. We’ve rigorously tested the lipid specificity of OlyA in reconstituted and cellular systems and discovered a single mutation in OlyA that abolishes SM/cholesterol specificity. This mutant, OlyA (E69A), binds SM containing membranes regardless of cholesterols presence. High-resolution X-ray structures of WT and mutant OlyA have led to a working model proposing these proteins recognize different membrane conformations of SM. Unfortunately, our solution structures lack direct information about sphingomyelins conformation in a membrane environment. Using single-particle Cryo-EM, we’ve now solved structures of OlyA bound to SM containing membranes with and without cholesterol. These structures provide the first direct insights into how WT and mutant OlyA recognize SM in these two forms. They also provide high-resolution information about the protein-protein interactions that drive pore assembly and shed light on how protein-lipid interactions initiate this process.

Untangling the rivalry between cholesterol and antimicrobial peptides.

Antimicrobial peptides (AMPs) are promising candidates for next generation antimicrobial therapeutics. In mammalian cell membranes cholesterol plays a key regulatory function in antibiotic drug resistance and the immune response. However, the mechanism of action and the selectivity of AMPs towards bacterial membranes are not yet well understood. We hypothesized that differences in biophysical properties of mammalian versus bacterial membranes give rise to differences in peptide-membrane interactions which underlie the AMP mechanism and selectivity. Solid-state 2H NMR spectroscopy was employed to study di-monounsaturated phosphatidylcholine and phosphatidylethanolaminelipids (DOPC versus DOPE) bilayers in the presence of the antimicrobial peptide LL37 (2−4 mol%) and cholesterol (20−30% mol) as model systems mimicking mammalian and bacterial membranes. The lipid systems were probed with POPC-d31 (10 mol%) where the residual quadrupolar couplings yielded segmental order parameters (SCD) for the individual acyl segments. The LL37-peptide decreased the quadrupolar splittings indicating it increased the area per lipid, while cholesterol showed the opposite. The mean-torque model was used to calculate the key bilayer properties of area per lipid and bilayer thickness [2]. The model-free functional dependence of spin-lattice relaxation rates on SCD (square-law plots) indicated greater bending rigidity with cholesterol for both DOPC and DOPE membranes. By contrast LL37 decreased the bending rigidity of the membranes. Additionally all-atom molecular dynamics simulations for bilayers comprising POPE/DMPG/cardiolipin (90:5:5) and DMPC/cholesterol (90:10) were conducted with LL37 peptides on the bilayer surface to capture the molecular-level interactions. We observed partial insertion of the LL37-peptides in the POPE system, while the peptides were repelled from the membrane in the DMPC/cholesterol system. These observations reveal the surprising rivalry between cholesterol and AMPs which underlies AMP mechanism and selectivity.

Lipid packing is disrupted in copolymeric nanodiscs compared with intact membranes

Discoidal lipid-protein nanoparticles known as nanodiscs are widely used tools in structural and membrane biology. Amphipathic, synthetic copolymers have recently become an attractive alternative to membrane scaffold proteins for the formation of nanodiscs. Such copolymers can directly intercalate into, and form nanodiscs from, intact membranes without detergents. Although these copolymer nanodiscs can extract native membrane lipids, it remains unclear whether native membrane properties are also retained. To determine the extent to which bilayer lipid packing is retained in nanodiscs, we measured the behavior of packing-sensitive fluorescent dyes in various nanodisc preparations compared with intact lipid bilayers. We analyzed styrene-maleic acid (SMA), diisobutylene-maleic acid (DIBMA), and polymethacrylate (PMA) as nanodisc scaffolds at various copolymer-to-lipid ratios and temperatures. Measurements of Laurdan spectral shifts revealed that dimyristoyl-phosphatidylcholine (DMPC) nanodiscs had increased lipid headgroup packing compared with large unilamellar vesicles (LUVs) above the lipid melting temperature for all three copolymers. Similar effects were observed for DMPC nanodiscs stabilized by membrane scaffolding protein MSP1E1. Increased lipid headgroup packing was also observed when comparing nanodiscs with intact membranes composed of binary mixtures of 1-palmitoyl-2-oleoyl-phosphocholine (POPC) and di-palmitoyl-phosphocholine (DPPC), which show fluid-gel-phase coexistence. Similarly, Laurdan reported increased headgroup packing in nanodiscs for biomimetic mixtures containing cholesterol, most notable for relatively disordered membranes. The magnitudes of these ordering effects were not identical for the various copolymers, with SMA being the most and DIBMA being the least perturbing. Finally, nanodiscs derived from mammalian cell membranes showed similarly increased lipid headgroup packing. We conclude that nanodiscs generally do not completely retain the physical properties of intact membranes.

Inactivation of potassium channels by ceramide in rat pancreatic β-cells

Lipid regulation of ion channels is a fundamental mechanism in physiological processes as of neurotransmitter release and hormone secretion. Ceramide is a bioactive lipid proposed as a regulator of several voltage-gated ion channels including potassium channels (Kv). It is generated either de novo or by sphingomyelin (SM) hydrolysis in membranes of mammalian cells. In pancreatic β-cells, ceramide is the main sphingolipid associated with lipotoxicity and likely involved in cell dysfunction. Despite of the wealth of information regarding regulation of potassium channels by ceramides, the regulation of Kv channels by accumulated ceramide in native pancreatic β-cells has not been investigated. To do so, we used either the C2-ceramide, a cell-permeable short-chain analogue, or a sphingomyelinase (SMase C), a hydrolase causing ceramide to elevate from an endogenous production, in pancreatic β-cells of rat. C2-ceramide markedly accelerates steady-state current inactivation according to kinetic changes in the channel machinery. Interestingly, only C2-ceramide accelerates current inactivation while SMase C decreases both, peak-current and step-current amplitude supporting differential effects of ceramide derivatives. A specific inhibitor of the Kv2.1 channel (GxTX-1E), readily inhibits a fraction of the Kv channel current while no further inhibition by C2-ceramide superfusion can be observed supporting Kv2.1 channel involvement in the ceramide inhibition. Thus, intramembrane ceramide accumulation, as a lipidic metabolite released under cell-stress conditions, may alter pancreatic β-cell repolarization and secretion. These results may provide a new insight regarding lipid-protein regulation and advance our understanding in ceramide actions on Kv channels in pancreatic β-cells.

Designer co-beta-peptide copolymer selectively targets resistant and biofilm Gram-negative bacteria

New antimicrobials are urgently needed to combat Gram-negative bacteria, particularly multi-drug resistant (MDR) and phenotypically resistant biofilm species. At present, only sequence-defined alpha-peptides (e.g. polymyxin B) can selectively target Gram-negative bacterial lipopolysaccharides. We show that a copolymer, without a defined sequence, shows good potency against MDR Gram-negative bacteria including its biofilm form. The tapered blocky co-beta-peptide with controlled N-terminal hydrophobicity (#4) has strong interaction with the Gram-negative bacterial lipopolysaccharides via its backbone through electrostatic and hydrogen bonding interactions but not the Gram-positive bacterial and mammalian cell membranes so that this copolymer is non-toxic to these two latter cell types. The new #4 co-beta-peptide selectively kills Gram-negative bacteria with low cytotoxicity both in vitro and in a mouse biofilm wound infection model. This strategy provides a new concept for the design of Gram-negative selective antimicrobial peptidomimetics against MDR and biofilm species.

A mini review on the role of phosphatidylcholine metabolism in obesity

Phosphatidylcholine (PC) is one of the major building blocks of biological membranes in mammalian cells. In PC, two fatty acyl chains are linked to the sn-1 and sn-2 positions of the glycerol backbone, and a head group containing a phosphate and choline is linked to the sn-3 position. The length and saturation of the fatty acyl chains of PC vary significantly and determine the biophysical and chemical properties of PC. In mammalian cells, active metabolism of PC takes place primarily through de novo synthesis, the remodelling cycle of deacylation and reacylation, and further modification of lysophosphatidylcholine. PC metabolism undergoes remarkable changes in obesity. In this mini review, we discuss PC metabolism and summarize recent advances about the important involvement of PC metabolism in obesity and obesity-related diseases.

Development of a novel fluorescence assay for studying lipid bilayer perturbation induced by amyloidogenic peptides using cell plasma membrane vesicles

Numerous pathophysiological conditions are associated with the misfolding and aggregation of proteins into insoluble amyloid fibrils. The mechanisms by which this process leads to cellular dysfunction remain elusive, though several hypotheses point toward the perturbation of the cell plasma membrane by pre-fibrillar intermediates and/or amyloid growth. However, current models to study membrane perturbations are largely limited to synthetic lipid vesicles and most of experimental approaches cannot be transposed to complex cell-derived plasma membrane systems. Herein, vesicles originating from the plasma membrane of erythrocytes and β-pancreatic cells were used to study the perturbations induced by an amyloidogenic peptide, the islet amyloid polypeptide (IAPP). These biologically relevant lipid vesicles displayed a characteristic clustering in the presence of the amyloidogenic peptide, which was able to rupture membranes. By exploiting Förster resonance energy transfer (FRET), a rapid, simple, and potentially high-throughput assay to detect membrane perturbations of intact mammalian cell plasma membrane vesicles was implemented. The FRET kinetics of membrane perturbations closely correlated with the kinetics of thioflavin-T fluorescence associated with amyloid formation. This novel kinetics assay expands the toolbox available to study amyloid-associated membrane damage, bridging the gap between synthetic lipid vesicles and living cells.

Influence of the Nature of the Lipid Building Blocks on the Second-Order Nonlinear Optical Responses of an Embedded Di-8-ANEPPS Probe.

The extensive collection of lipids found in cell membranes is justified by the fact that each lipid contributes to their overall structure, dynamics, and properties and so to the biological processes taking place within them. It also showcases that, in order to deepen our understanding of membranes, we need to have a tool to differentiate lipid bilayers of varying composition. In this work, we investigate a suite of single-component saturated glycerophospholipids varying only in their headgroup structure by analyzing the second harmonic generation (SHG) nonlinear optical (NLO) response of a probe, di-8-ANEPPS, embedded into the membranes. The seven hydrophilic heads chosen (phosphatidylcholine (PC), phosphatidylethanolamine (PE), diaglycerol (GL), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidyc acid (PA)) represent all the major headgroups that are part of mammalian plasma membranes and provide an assortment of neutral, zwiterrionic, and charged species. First, molecular dynamics simulations revealed that the lipidic arrangement is strongly sensitive to the nature of the hydrophilic head and less to the variety in the hydrophobic region. Membranes exhibiting drastically opposite structural properties can be pointed out: 1,2-dihexadecanoyl-rac-glycerol (DPGL) is the thickest and most ordered and aligned system, whereas 1,2-diacyl-sn-glycero-3-phospho-(1'-sn-glycerol) (DPPG) is thinnest and least ordered and aligned system. The structural analyses are then confronted with the molecular NLO responses, β, computed at the time-dependent density functional theory (TDDFT) level. As the orientation of the chromophore is impacted by the various degrees of order within the lipid bilayers, the diagonal component of the β tensor parallel to the bilayer normal, βZZZ, is as well. In the end, this computational approach provides insights into the link between lipid building blocks and the NLO responses of the embedded dye.

Assessing the role of membrane lipids in the action of ruthenium(III) anticancer compounds.

This work addresses the possible role of the cell membrane in the molecular mechanism of action of two salan-type ruthenium complexes that were previously shown to be active against human tumor cells, namely [Ru(III)(L1)(PPh3)Cl] and [Ru(III)(L2)(PPh3)Cl] (where L1 is 6,6'-(1R,2R)-cyclohexane-1,2-diylbis(azanediyl)bis(methylene)bis(3-methoxyphenol); and L2 is 2,2'-(1R,2R)-cyclohexane-1,2-diylbis(azanediyl)bis(methylene)bis(4-methoxyphenol)). One-component membrane models were first used, a disordered fluid bilayer of dioleoylphosphatodylcholine (DOPC), and an ordered rigid gel bilayer of dipalmitoylphosphatidylcholine. In addition, two quaternary mixtures of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol were used to mimic the lipid composition either of mammalian plasma membrane (1:1:1:1mol ratio) or of a cancer cell line membrane (36.2:23.6:6.8:33.4mol ratio). The results show that both salan ligands L1 and L2 bind relatively strongly to DOPC bilayers, but without significantly affecting their structure. The ruthenium complexes have moderate affinity for DOPC. However, their impact on the membranes was notable, leading to a significant increase in the permeability of the lipid vesicles. None of the compounds compromised liposome integrity, as revealed by dynamic light scattering. Fluorescence spectroscopy studies revealed changes in the biophysical properties of all membrane models analyzed in the presence of the two complexes, which promoted an increased fluidity and water penetration into the lipid bilayer in the one-component systems. In the quaternary mixtures, one of the complexes had an analogous effect (increasing water penetration), whereas the other complex reorganized the liquid ordered and liquid disordered domains. Thus, small structural differences in the metal ligands may lead to different outcomes. To better understand the effect of these complexes in cancer cells, the membrane dipole potential was also measured. For both Ru complexes, an increase in the dipole potential was observed for the cancer cell membrane model, while no alteration was detected on the non-cancer plasma membrane model. Our results show that the action of the Ru(III) complexes tested involves changes in the biophysical properties of the plasma membrane, and that it also depends on membrane lipid composition, which is frequently altered in cancer cells when compared to their normal counterparts.

Molecular Dynamics of Glycolipids in Liposomes.

Glycosphingolipids (GSLs) in the mammalian plasma membrane are essential for various biological events as they form glycolipid-rich membrane domains, such as lipid rafts. GSLs consist of a certain oligosaccharide head group and a ceramide tail with various lengths of acyl chains. The structure of the head group as well as the carbon number and degree of the unsaturation of the acyl chain are known to regulate the membrane distributions and interleaflet couplings of GSLs by altering physicochemical properties, such as dynamics, interactions, and cluster sizes. This chapter provides the detailed use of time-resolved fluorescence measurement for investigating the membrane properties of lactosylceramide (LacCer)-enriched domains in bilayer membranes. LacCer belongs to the neutral GSLs and is believedin forming a highly ordered phase in model membranes and biological membranes, while the details of the domain remain unclear. Here, we suggest using trans-parinaric acid (tPA) and tPA-LacCer fluorescent probes to reveal the dynamics and size of the GSL domains since they prefer to bedistributed in the GSL-rich ordered phase. The fluorescence lifetime in the nanosecond timescale reveals the difference in the surrounding membrane environments, which relates to hydrocarbon chain ordering, membrane hydration, and submicrometer domain size. The fluorescence lifetime of these probes can thus provide important information on submicron- to nano-scale small GSL domains not only in model membranes but also in biological membranes.

A Near InfraRed Emissive Chemosensor for Zn2+ and Phosphate Derivatives Based on a Di-(2-picolyl)amine-styrylflavylium Push-Pull Fluorophore.

A new Near InfraRed (NIR) fluorescent chemosensor for metal ions and anions is herein presented. The fluorophore is based on a styrylflavylium dye, a synthetic analogue of the natural anthocyanin family, with a di-(2-picolyl)amine (DPA) moiety as the metal chelating unit. The substitution pattern of the styrylflavylium core (with tertiary amines on positions 7 and 4') shifts the optical properties of the dye towards the NIR region of the electronic spectra, due to a strong push-pull character over the π-conjugated system. The NIR chemosensor is highly sensitive to the presence of Zn2+, which induces a strong CHelation Enhanced Fluorescence (CHEF) effect upon binding to the DPA unit (2.7 fold increase). The strongest competing ion is Cu2+, with a complete fluorescence quenching, while other metals induce lower responses on the optical properties of the chemosensor. Subsequent anion screening of the Zn2+-chemosensor coordination compound has demonstrated a distinct selectivity towards adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphate (ADP), with high association constants (K ~ 106 M-1) and a strong CHEF effect (2.4 and 2.9 fold fluorescence increase for ATP and ADP, respectively). Intracellular studies with the Zn2+-complexed sensor showed strong luminescence in the cellular membrane of Gram- bacteria (E. coli) and mitochondrial membrane of mammalian cells (A659), which highlights its possible application for intracellular labelling.

Visualizing NBD-lipid Uptake in Mammalian Cells by Confocal Microscopy.

Eukaryotic cells use a series of membrane transporters to control the movement of lipids across their plasma membrane. Several tools and techniques have been developed to analyze the activity of these transporters in the plasma membrane of mammalian cells. Among them, assays based on fluorescence microscopy in combination with fluorescent lipid probes are particularly suitable, allowing visualization of lipid internalization in living cells. Here, we provide a step-by-step protocol for mammalian cell culture, lipid probe preparation, cell labeling, and confocal imaging to monitor lipid internalization by lipid flippases at the plasma membrane based on lipid probes carrying a fluorophore at a short-chain fatty acid. The protocol allows studying a wide range of mammalian cell lines, to test the impact of gene knockouts on lipid internalization at the plasma membrane and changes in lipid uptake during cell differentiation. Key features Visualization and quantification of lipid internalization by lipid flippases at the plasma membrane based on confocal microscopy. Assay is performed on living adherent mammalian cells in culture. The protocol can be easily modified to a wide variety of mammalian cell lines.

GAPR-1 Interferes with Condensate Formation of Beclin 1 in Saccharomyces cerevisiae

Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1) acts as a negative regulator of autophagy by interacting with Beclin 1 at Golgi membranes in mammalian cells. The molecular mechanism of this interaction is largely unknown. We recently showed that human GAPR-1 (hGAPR-1) has amyloidogenic properties resulting in the formation of protein condensates upon overexpression in Saccharomyces cerevisiae. Here we show that human Beclin 1 (hBeclin 1) has several predicted amyloidogenic regions and that overexpression of hBeclin 1-mCherry in yeast also results in the formation of fluorescent protein condensates. Surprisingly, co-expression of hGAPR-1-GFP and hBeclin 1-mCherry results in a strong reduction of hBeclin 1 condensates. Mutations of the known interaction site on the hGAPR-1 and hBeclin 1 surface abolished the effect on condensate formation during co-expression without affecting the condensate formation properties of the individual proteins. Similarly, a hBeclin 1-derived B18 peptide that is known to bind hGAPR-1 and to interfere with the interaction between hGAPR-1 and hBeclin 1, abolished the reduction of hBeclin 1 condensates by co-expression of hGAPR-1. These results indicate that the same type of protein–protein interactions interfere with condensate formation during co-expression of hGAPR-1 and hBeclin 1 as previously described for their interaction at Golgi membranes. The amyloidogenic properties of the B18 peptide were, however, important for the interaction with hGAPR-1, as mutant peptides with reduced amyloidogenic properties also showed reduced interaction with hGAPR-1 and reduced interference with hGAPR-1/hBeclin 1 condensate formation. We propose that amyloidogenic interactions take place between hGAPR-1 and hBeclin 1 prior to condensate formation.

Conformational Flexibility Is a Key Determinant for the Lytic Activity of the Pore-Forming Protein, Cytolysin A.

Several bacterial infections are mediated by pore-forming toxins (PFTs), a subclass of proteins that oligomerize on mammalian cell membranes forming lytic nanopores. Cytolysin A (ClyA), an α-PFT, undergoes a dramatic conformational change restructuring its two membrane-binding motifs (the β-tongue and the N-terminus helix), during pore formation. A complete molecular picture for this key transition and the driving force behind the secondary structure change upon membrane binding remain elusive. Using all-atom molecular dynamics (MD) simulations of the ClyA monomer and string method based free energy computations with path collective variables, we illustrate that an unfolded β-tongue motif is an on-pathway intermediate during the transition to the helix-turn-helix motif of the protomer. An aggregate of 28 μs of all-atom thermal unfolding MD simulations of wild-type ClyA and its single point mutants reveal that the membrane-binding motifs of the ClyA protein display high structural flexibility in water. However, point mutations in these motifs lead to a distinct reduction in the flexibility, especially in the β-tongue, thereby stabilizing the pretransition secondary structure. Resistance to unfolding was further corroborated by MD simulations of the β-tongue mutant motif in the membrane. Combined with the thermal unfolding simulations, we posit that the β-tongue as well as N-terminal mutants that lower the tendency to unfold and disorder the β-tongue are detrimental to pore formation by ClyA and its lytic activity. Erythrocyte turbidity and vesicle leakage assays indeed reveal a loss of activity for the β-tongue mutant, and delayed kinetics for the N-terminus mutants. On the other hand, a point mutation in the extracellular domain that did not abrogate lytic activity displayed similar unfolding characteristics as the wild type. Thus, attenuation of conformational flexibility in membrane-binding motifs correlates with reduced lytic and leakage activity. Combined with secondary structure changes observed in the membrane bound states, our study shows that the tendency to unfold in the β-tongue region is a critical step in the conformational transition and bistability of the ClyA protein and mutants that disrupt this tendency reduced pore formation. Overall, our finding suggests that inherent flexibility in the protein could play a wider and hitherto unrecognized role in membrane-mediated conformational transitions of PFTs and other membrane protein transformations.