Curved Membrane Surfaces Research Articles

Detection of Highly Curved Membrane Surfaces Using a Cyclic Peptide Derived from Synaptotagmin-I

The generation of highly curved membranes is essential to cell growth, division, and movement. Recent research in the field is focused to answer questions related to the consequences of changes in the topology of the membrane once it is created, broadly termed as membrane curvature sensing. Most probes that are used to study curvature sensing are intact membrane active proteins such as DP1/Yop1p, ArfGAP1, BAR domains, and Synaptotagmin-I (Syt1). Taking a cue from nature, we created the cyclic peptide C2BL3C based on the membrane penetration C2B loop 3 of Syt1 via "Click" chemistry. Using a combination of spectroscopic techniques, we investigated the peptide-lipid interactions of this peptide with synthetic phospholipid vesicles and exosomes from rat blood plasma. We found that the macrocycle peptide probe was selective for lipid vesicles with highly curved surfaces (d < 100 nm). These results suggested that C2BL3C functions as a selective detector of highly curved phospholipid bilayers.

Lipid bilayer condition abnormalities following Macrovipera lebetina obtusa snake envenomation

Viper bites is an endemic public health problem in Armenia, even in the cities. Human envenomation is often characterized by clotting disorders, hypofibrinogenemia, and local tissue necrosis. In this original study, we assess some changes of cell membranes plastic properties (namely, its microviscosity, thickness, permeability) in a rat envenomation model using the biophysical approaches. We describe the interaction of Macrovipera lebetina obtusa (MLO) venom with giant unilamellar vesicles (GUVs) composed of the native phospholipid mixtures visualized through fluorescent microscopy. GUVs with a mean diameter of 30 μm have a minimum curvature and mimic cell membranes in this respect.The membrane fluorescence probe, ANS and pyrene, were used to assess the state of membrane and specifically mark the phospholipid domains. Independent of their lipid composition, GUVs were enlarged in size as venom-dependent lipid hydrolysis proceeded. Except of the visible morphological changes, ANS and pyrene also allows us to quantify the fluidity changes in the membrane by measuring of the fluorescence intensity. The presence of viper venom in GUVs media reveals a noticeable decreasing of membrane fluidity compare the control, while the binding of fluorophores with GUVs modified by venom lead to appearance of channel activity. These studies also emphasize the importance of a membrane surface curvature for its interaction with enzymatic components of venom.

Molecular events associated with Macrovipera lebetina obtusa and Montivipera raddei venom intoxication and condition of biomembranes

Studies on the interaction of snake venom and organized lipid interfaces have been conducted using a variety of systems, including BLMs, SUVs and GUVs. The present study was undertaken to elucidate how the plastic properties (namely, its microviscosity, thickness, permeability) of model membranes from native lipids of different tissues of rats change in the course of Macrovipera lebetina obtusa (MLO), Montivipera raddei (MR) and Naja kaouthia (NK) venoms processing. The presence of viper venom in organism leads to increasing of the electrical resistance of BLMs from liver and muscle lipids approximately on a sequence, while the BLMs from brain lipids have not shown noticeable differences of plastic properties compared to the control. Giant unilamellar vesicles (GUVs) with a mean diameter of 30μm have a minimum curvature and mimic cell membranes in this respect. Snake venom was added to the sample chamber before the vesicles were formed. The membrane fluorescence probes, ANS and pyrene, were used to assess the state of the membrane and specifically mark the phospholipid domains. Fluorescent spectra were acquired on a Varian fluorometer instrument. ANS and pyrene allow us to quantify the fluidity changes in the membrane by measuring of the fluorescence intensity. The presence of viper venom in GUVs media reveals a noticeable decreasing of membrane fluidity compared to the control, while the binding of fluorophores with GUVs modified by venom leads to the appearance of channel activity. These studies also emphasize the importance of a membrane surface curvature for its interaction with enzymatic components of venom.

Association of the Endosomal Sorting Complex ESCRT-II with the Vps20 Subunit of ESCRT-III Generates a Curvature-sensitive Complex Capable of Nucleating ESCRT-III Filaments

The scission of membranes necessary for vesicle biogenesis and cytokinesis is mediated by cytoplasmic proteins, which include members of the ESCRT (endosomal sorting complex required for transport) machinery. During the formation of intralumenal vesicles that bud into multivesicular endosomes, the ESCRT-II complex initiates polymerization of ESCRT-III subunits essential for membrane fission. However, mechanisms underlying the spatial and temporal regulation of this process remain unclear. Here, we show that purified ESCRT-II binds to the ESCRT-III subunit Vps20 on chemically defined membranes in a curvature-dependent manner. Using a combination of liposome co-flotation assays, fluorescence-based liposome interaction studies, and high-resolution atomic force microscopy, we found that the interaction between ESCRT-II and Vps20 decreases the affinity of ESCRT-II for flat lipid bilayers. We additionally demonstrate that ESCRT-II and Vps20 nucleate flexible filaments of Vps32 that polymerize specifically along highly curved membranes as a single string of monomers. Strikingly, Vps32 filaments are shown to modulate membrane dynamics in vitro, a prerequisite for membrane scission events in cells. We propose that a curvature-dependent assembly pathway provides the spatial regulation of ESCRT-III to fuse juxtaposed bilayers of elevated curvature.

Going on a (Membrane) Bender to the F-BAR

This study is a personal favorite because it simplified the field and proposed an elegant model of how actin and membranes work together in endocytic trafficking. A group of proteins containing FCH domains (Fes/CIP4 homology) were discovered to have extended homology with the banana-shaped alpha helical BAR domain and renamed F-BAR proteins. F-BAR and BAR proteins bind to and bend curved membrane surfaces and participate in budding and tubulation events. Dynamin, a large GTPase that forms helical cages around tubulated membranes, is the main partner of BAR and F-BAR proteins. The actin cytoskeleton opposes scission of the tubulated membranes pending arrival of an appropriate signal. The idea that dynamin is the most important partner of F-BARs and that the trio of actin, F-BAR, and dynamin sculpt and cut membrane tubules in this elegant way remains important for future research and puts into context the barrage of sometimes disparate studies on these proteins. This PaperPick refers to and the Actin Cytoskeleton Cooperatively Regulate Plasma Membrane Invagination by BAR and F-BAR Proteins by T. Itoh, K.S. Erdmann, A. Roux, B. Habermann, H. Werner, and P. De Camilli, published in December, 2005. F-BAR Proteins: Coordinating the Effects of Actin and Dynamin on Endocytosis Pietro De Camilli discusses how his group came to define the F-BAR domain, its role in endocytosis, and where subsequent work has taken the field.

Characterization of biofouling on hollow fiber membranes using confocal laser scanning microcscopy and image analysis

The composition and structure of membrane biofouling in water treatment can be studied using specific stains (fluorochromes) and confocal laser scanning microscopy (CLSM). An image analysis program (CMem) has been developed to quantify parameters describing the biofilm structure on curved membrane surfaces, such as hollow fiber membranes. Two CLSM images stacks were acquired: one of the stained biofilm and one of the membrane underneath it. The curved reference surface in a three-dimensional stack of CLSM images was located, based upon detection of membrane autofluorescence/reflection signals. The stained components identified in the biofilm stack were then related to the membrane surface. Structural parameters such as thickness, volume of biomass, porosity and roughness of biofilms on curved membrane surfaces were computed.

Curvature Dynamics of α-Synuclein Familial Parkinson's Disease Mutants: Molecular Dynamics Simulations of the Micelle- and Membrane-Bound Forms

α-synuclein remains a protein of interest due to its propensity to form fibrillar aggregates in neurodegenerative disease and its putative function in synaptic vesicle regulation. We have performed a series of all-atom molecular dynamics simulations of wild-type α-synuclein and the three Parkinson's disease familial mutants, A30P, A53T, and E46K, bound to an SDS detergent micelle. Our analysis explains how α-synuclein adapts to, and alters, highly curved membrane surfaces through helical bending. We find that α-synuclein binding induces significant deformation in the micelle, flattening the structure and decreasing its surface area. Similar effects on biological membranes would relieve curvature stress, ameliorating the propensity to fuse, and would perhaps explain α-synuclein's role in stabilizing synaptic vesicles. Consistent with the experimentally determined behavior of A30P, the proline dramatically destabilizes the secondary structure, inducing reversible unfolding up- and down-stream of the substitution. The E46K mutation provides an additional electrostatic interaction between the protein and micelle, offering an explanation for the mutants increased affinity. In the case of the A53T mutant, recent NMR data suggested that an enthalpic interaction might be responsible for a slight rigidification of the helical structure. We show that the equilibrium structure of A53T contains a very tight hydrogen-bond between the threonine's hydroxyl and the backbone carbonyl of Val49. We speculate as to the potential effects of these dynamic and structural changes, on α-synuclein's role in neural signaling. In order to assess the applicability of these results to biological membranes, we have performed simulations of the wild-type and mutants on a DOPS bilayer. These simulations reproduce the major conclusions of the micelle-bound simulations, suggesting that the detergent micelle is an adequate model system.

Mechanism of ATP-binding Cassette Transporter A1-mediated Cellular Lipid Efflux to Apolipoprotein A-I and Formation of High Density Lipoprotein Particles

The ATP-binding cassette transporter A1 (ABCA1) plays a critical role in the biogenesis of high density lipoprotein (HDL) particles and in mediating cellular cholesterol efflux. The mechanism by which ABCA1 achieves these effects is not established, despite extensive investigation. Here, we present a model that explains the essential features, especially the effects of ABCA1 activity in inducing apolipoprotein (apo) A-I binding to cells and the compositions of the discoidal HDL particles that are produced. The apo A-I/ABCA1 reaction scheme involves three steps. First, there is binding of a small regulatory pool of apo A-I to ABCA1, thereby enhancing net phospholipid translocation to the plasma membrane exofacial leaflet; this leads to unequal lateral packing densities in the two leaflets of the phospholipid bilayer. Second, the resultant membrane strain is relieved by bending and by creation of exovesiculated lipid domains. The formation of highly curved membrane surface promotes high affinity binding of apo A-I to these domains. Third, this pool of bound apo A-I spontaneously solubilizes the exovesiculated domain to create discoidal nascent HDL particles. These particles contain two, three, or four molecules of apo A-I and a complement of membrane phospholipid classes together with some cholesterol. A key feature of this mechanism is that membrane bending induced by ABCA1 lipid translocase activity creates the conditions required for nascent HDL assembly by apo A-I. Overall, this mechanism is consistent with the known properties of ABCA1 and apo A-I and reconciles many of the apparently discrepant findings in the literature.

COMPUTER SIMULATIONS OF MEMBRANE-LYTIC PEPTIDES: PERSPECTIVES IN DRUG DESIGN

Structure activity relationships were investigated for membrane-lytic peptides (MLP) Ltc1 and Ltc2a from the latarcin family. The peptides were studied via long-term molecular dynamics (MD) simulations in different membrane environments (detergent micelles, mixed lipid bilayers mimiking eukaryotic and bacterial membranes). The calculated structure of Ltc2a in sodium dodecyl sulfate micelle agrees well with the data obtained by (1)H-NMR spectroscopy. This validates the applied modeling approach. The binding mode of MLPs is governed by several factors: (i) the membrane surface curvature; (ii) the conformational plasticity and hydrophobic organization of the peptide, which depend on the arrangement of charged, non-polar and helix-breaking residues in the amino acid sequence. In contrast to Ltc1, insertion of Ltc2a into model membranes induces significant changes in dynamic behavior of lipids in the contact region. Such a prominent membrane destabilization correlates with high membrane-lytic activity of Ltc2a. In all cases the "membrane response" has a local character and is caused by formation of specific peptide-lipid contacts. Results of MD simulations of Ltc2a in model membranes were used to develop a number of its analogs with predefined activity.

Diacylglycerols as activators of protein kinase C (Review)

Diacylglycerols are generated in the membrane as the result of extracellular signals and are able to stimulate the activity of protein kinase C, acting as membrane second messengers. Diacylglycerols are recognized by protein kinases C through the C1 domain and established models propose that they will stabilize the translocation of the protein to the membrane. However, diacylglycerols also act by modulating the physical properties of the membrane, thus favouring the translocation of the enzyme. This is done through alteration of the membrane surface curvature, dehydration of the surface and the separation of phospholipid surface groups. Good correlations have been observed between the physical state of the membrane and protein kinase C activity.

Cationic triple-chain amphiphiles facilitate vesicle fusion compared to double-chain or single-chain analogues

Cationic, triple-chain amphiphiles promote vesicle fusion more than structurally related double-chain or single-chain analogues. Two types of vesicle fusion experiments were conducted, mixing of oppositely charged vesicles and acid-triggered self-fusion of vesicles composed of cationic amphiphile and anionic cholesteryl hemisuccinate (CHEMS). Vesicle fusion was monitored by standard fluorescence assays for intermembrane lipid mixing, aqueous contents mixing and leakage. Differential scanning calorimetry was used to show that triple-chain amphiphiles lower the lamellar–inverse hexagonal (L α–H II) phase transition temperature for dipalmitoleoylphosphatidylethanolamine. The triple-chain amphiphiles may enhance vesicle fusion because they can stabilize the inversely curved membrane surfaces of the fusion intermediates, however, other factors such as extended conformation, packing defects, chain motion, or surface dehydration may also contribute. From the perspective of drug delivery, the results suggest that vesicles containing cationic, triple-chain amphiphiles (and cationic, cone-shaped amphiphiles in general) may be effective as fusogenic delivery capsules.

The Mechanism of Oxidation-Induced Low-Density Lipoprotein Aggregation: An Analogy to Colloidal Aggregation and Beyond?

Atherosclerosis is a disease initiated by lipoprotein aggregation and deposition in artery walls. In this study, the de novo low-density lipoprotein aggregation process was examined. Nine major intermediates were identified in two stages of the aggregation process. In the aggregation stage, low-density lipoprotein molecules aggregate and form nucleation units. The nucleation units chain together and form linear aggregates. The linear aggregates branch and interact with one another, forming fractals. In the fusion stage, spatially adjacent nucleation units in the fractal fuse into curved membrane surfaces, which, in turn, fuse into multilamellar or unilamellar vesicles. Alternatively, some adjacent nucleation units in the fractals assemble in a straight line and form rods. Subsequently, the rods flatten out into rough and then into smooth ribbons. Occasionally, tubular membrane vesicles are formed from the fractals. The aggregation stage seems to be analogous to colloidal aggregation and amyloid fiber formation. The fusion stage seems to be characteristic of the lipid-rich lipoproteins and is beyond colloidal aggregation and amyloid fiber formation.

Cholesterol Organization in Membranes at Low Concentrations: Effects of Curvature Stress and Membrane Thickness

Cholesterol is often found distributed nonrandomly in domains in biological and model membranes and has been reported to be distributed heterogeneously among various intracellular membranes. Although a large body of literature exists on the organization of cholesterol in plasma membranes or membranes with high cholesterol content, very little is known about organization of cholesterol in membranes containing low amounts of cholesterol. Using a fluorescent cholesterol analog (25-[ N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol, or NBD-cholesterol), we have previously shown that cholesterol may exhibit local organization even at very low concentrations in membranes, which could possibly be attributable to transbilayer tail-to-tail dimers. This is supported by similar observations reported by other groups using cholesterol or dehydroergosterol, a naturally occurring fluorescent cholesterol analog which closely mimics cholesterol. In this paper, we have tested the basic features of cholesterol organization in membranes at low concentrations using spectral features of dehydroergosterol. More importantly, we have investigated the role of membrane surface curvature and thickness on transbilayer dimer arrangement of cholesterol using NBD-cholesterol. We find that dimerization is not favored in membranes with high curvature. However, cholesterol dimers are observed again if the curvature stress is relieved. Further, we have monitored the effect of membrane thickness on the dimerization process. Our results show that the dimerization process is stringently controlled by a narrow window of membrane thickness. Interestingly, this type of local organization of NBD-cholesterol at low concentrations is also observed in sphingomyelin-containing membranes. These results could be significant in membranes that have very low cholesterol content, such as the endoplasmic reticulum and the inner mitochondrial membrane, and in trafficking and sorting of cellular cholesterol.

Membrane Interactions of a Novel Viral Enterotoxin: Rotavirus Nonstructural Glycoprotein NSP4

The rotavirus enterotoxin, NSP4, is a novel secretory agonist that also plays a role in the unique rotavirus morphogenesis that involves a transient budding of newly made immature viral particles into the endoplasmic reticulum. NSP4 and an active peptide corresponding to NSP4 residues 114 to 135 (NSP4(114-135)) mobilize intracellular calcium and induce secretory chloride currents when added exogenously to intestinal cells or mucosa. Membrane-NSP4 interactions may contribute to these alterations; however, details of a lipid-binding domain are unresolved. Therefore, circular dichroism was used to determine (i) the interaction(s) of NSP4 and NSP4(114-135) with model membranes, (ii) the conformational changes elicited in NSP4 upon interacting with membranes, (iii) if NSP4(114-135) is a membrane interacting domain, and (iv) the molar dissociation constant (K(d)) of NSP4(114-135) with defined lipid vesicles. Circular dichroism revealed for the first time that NSP4 and NSP4(114-135) undergo secondary structural changes upon interaction with membrane vesicles. This interaction was highly dependent on both the membrane surface curvature and the lipid composition. NSP4 and NSP4(114-135) preferentially interacted with highly curved, small unilamellar vesicle membranes (SUV), but significantly less with low-curvature, large unilamellar vesicle membranes (LUV). Binding to SUV, but not LUV, was greatly enhanced by negatively charged phospholipids. Increasing the SUV cholesterol content, concomitant with the presence of negatively charged phospholipids, further potentiated the interaction of NSP4(114-135) with the SUV membrane. The K(d) of NSP4(114-135) was determined as well as partitioning of NSP4(114-135) with SUVs in a filtration-binding assay. These data confirmed NSP4 and its active peptide interact with model membranes that mimic caveolae.

Interaction of the N-terminus of sterol carrier protein 2 with membranes: role of membrane curvature

Although neither the physiological function nor the mechanism of action of sterol carrier protein 2 (SCP(2)) is yet completely clear, it is thought that SCP(2) interacts with membranes to elicit its biological effects. The results presented here show that the SCP(2) N-terminus, composed of two amphipathic alpha-helices, interacted preferentially with highly curved but not lower-curvature membranes containing anionic phospholipid. CD spectra of SCP(2) showed up to 1. 2-fold increased alpha-helical content, on the interaction of SCP(2) with small unilamellar vesicles (SUV) (median radius 10-14 nm) but less with large unilamellar vesicles (LUV) (median radius 52-60 nm). Although enhanced interaction with the SUV membranes was due in part to the radius of curvature and to the greater exposure of acidic phospholipid in the outer leaflet of the bilayer, simply increasing the molar percentage of acidic phospholipid in the LUV membranes had much less effect on SCP(2) binding. A similar preferential interaction was observed with highly curved SUV as opposed to LUV for the SCP(2) N-terminal peptide (1-32)SCP(2) as well as structurally modified peptides in the order (1-32)SCP(2)=(10-32)SCP(2)>(1-24)SCP(2)>>(1-E20-32)SCP(2). The CD results were confirmed with an independent filtration binding assay, which showed that SCP(2) bound 5-fold more to SUV than LUV, whereas its N-terminal peptides bound up to 4-fold better in the order (1-32)SCP(2)=(10-32)SCP(2)>(1-24)SCP(2)>(1-E20-32)SCP(2). Finally, cholesterol potentiated the binding of SCP(2) and N-terminal peptides to anionic-phospholipid-containing SUV but not LUV. These findings were consistent with the SCP(2) N-terminus being a membrane-binding domain that was highly dependent on membrane surface curvature as well as on lipid composition.

Simulation of dynamics in NMR spectra of rotating solids

A general formalism, suitable for the simulation of dynamically modulated nuclear magnetic resonance lineshapes, is presented. The relaxation function containing all the observable components of transverse magnetization is generated by successive multiplication by a complex propagator matrix. Its elements are determined by the spin Hamiltonians at each site visited by the spins, and by the conditional probability function which describes the motion of the spins between these sites. Chemical exchange, diffusion and sample rotation are accounted for by employing appropriate discretization schemes in each case. Frequency spectra are calculated by Fourier transformation of the relaxation function, evaluated at intervals of Δ, where Δ is equal to the inverse sweep width. The method is entirely general but is particularly efficient in the slow motional regime. It is applied to evaluate 2H magic angle spinning (MAS) NMR lineshapes for deuterons undergoing 180° flip motion and 31P MAS lineshapes for phospholipids undergoing lateral diffusion across curved membrane surfaces.

Polyunsaturation in cell membranes and lipid bilayers and its effects on membrane proteins.

The effect of variation of the degree of cis-unsaturation on cell membrane protein functioning was investigated using a model lipid bilayer system and protein kinase C (PKC). This protein is a key element of signal transduction. Furthermore it is representative of a class of extrinsic membrane proteins that show lipid dependent interactions with cell membranes. To test for dependence of activity on the phospholipid unsaturation, experiments were devised using a vesicle assay system consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) in which the unsaturation was systematically varied. Highly purified PKC alpha and epsilon were obtained using the baculovirus-insect cell expression system. It was shown that increased PC unsaturation elevated the activity of PKC alpha. By contrast, increasing the unsaturation of PS decreased the activity of PKC alpha, and to a lesser extent PKC epsilon. This result immediately rules out any single lipid bilayer physical parameter, such as lipid order, underlying the effect. It is proposed that while PC unsaturation effects are explainable on the basis of a contribution to membrane surface curvature stress, the effects of PS unsaturation may be due to specific protein-lipid interactions. Overall, the results indicate that altered phospholipid unsaturation in cell membranes that occurs in certain disease states such as chronic alcoholism, or by dietary manipulations, are likely to have profound effects on signal transduction pathways involving PKC and similar proteins.

Modulation of membrane surface curvature by peptide-lipid interactions.

Recent reports on the interaction of cardiotoxin and melittin with phospholipid model membranes are reviewed and analyzed. These types of peptide toxins are able to modulate lipid surface curvature and polymorphism in a highly lipid-specific way. It is demonstrated that the remarkable variety of effects of melittin on the organization of different membrane phospholipids can be understood in a relatively simple model, based on the shape-structure concept of lipid polymorphism and taking into account the position of the peptide molecule with respect to the lipids. Based on the strong preference of the peptides for negatively charged lipids and the structural consequences thereof, and on preliminary studies of signal peptide-lipid interaction, a role of inverted or concave lipid structures in the process of protein translocation across membranes is suggested.

Transverse nuclear spin relaxation in phospholipid bilayer membranes

Experimental proof is presented that some of the motions responsible for transverse relation (T/sub 2/) in deuterium magnetic resonance (/sup 2/H NMR) experiments on acyl chains of a model membrane in the liquid-crystalline phase are extremely slow on the /sup 2/H NMR time scale being characterized by a correlation time tau/sub 2/ >> 10/sup -5/ s. The experiments used to investigate these slow motions involve a form of the Carr-Purcell-Meiboom-Gill pulse sequence modified so as to be suitable for /sup 2/H NMR. The most plausible mechanism responsible for T/sub 2/ relaxation is the gradual change in the average molecular orientation due to lateral diffusion of the phospholipid molecules along curved membrane surfaces. A procedure for separating contributions to T/sub 2/ relaxation due to slow and fast motions is described.

A simple model for calcium induced exocytosis

We have developed a simple model showing how the presence or absence of Ca 2+ can determine whether an uncurved or curved membrane surface is favored energetically. The model shows why fusion of vesicles with the presynaptic membrane is favored in the presence of calcium and why the budding off of vesicles is favored in the absence of calcium inside of the presynaptic membrane. The model accurately predicts the radius of a synaptic vesicle using known properties of lipids and suggests consequences of temperature change, varied stimulation rate and addition of calcium by artificial means on rates of transmitter release.