The influence of curvature on membrane domains

An interdependence between local curvature and domain formation has been observed in both cell and model membranes. An implication of this observation is that domain formation in model membranes may be modulated by membrane curvature. In this paper, small-angle neutron scattering (SANS) is used to examine the influence of membrane curvature (i.e., vesicle size) on the formation of membrane domains. It is found that, although vesicle size and polydispersity are not significantly altered by the formation of membrane domains, the area fraction occupied by domains depends on the overall vesicle size. In particular, increasing membrane curvature (i.e., decreasing vesicle size) results in increased area fractions of membrane domains.     

The fusion peptide of Semliki Forest virus associates with sterol-rich membrane domains

Semliki Forest virus (SFV) is an enveloped alphavirus whose membrane fusion is triggered by low pH and promoted by cholesterol and sphingolipid in the target membrane. Fusion is mediated by E1, a viral membrane protein containing the putative fusion peptide. Virus mutant studies indicate that SFV's cholesterol dependence is controlled by regions of E1 outside of the fusion peptide. Both E1 and E1*, a soluble ectodomain form of E1, interact with membranes in a reaction dependent on low pH, cholesterol, and sphingolipid and form highly stable homotrimers. Here we have used detergent extraction and gradient floatation experiments to demonstrate that E1* associated selectively with detergent-resistant membrane domains (DRMs or rafts). In contrast, reconstituted full-length E1 protein or influenza virus fusion peptide was not associated with DRMs. Methyl beta-cyclodextrin quantitatively extracted both cholesterol and E1* from membranes in the absence of detergent, suggesting a strong association of E1* with sterol. Monoclonal antibody studies demonstrated that raft association was mediated by the proposed E1 fusion peptide. Thus, although other regions of E1 are implicated in the control of virus cholesterol dependence, once the SFV fusion peptide inserts in the target membrane it has a high affinity for membrane domains enriched in cholesterol and sphingolipid.     

Amphipathic motifs in BAR domains are essential for membrane curvature sensing

BAR (Bin/Amphiphysin/Rvs) domains and amphipathic α‐helices (AHs) are believed to be sensors of membrane curvature thus facilitating the assembly of protein complexes on curved membranes. Here, we used quantitative fluorescence microscopy to compare the binding of both motifs on single nanosized liposomes of different diameters and therefore membrane curvature. Characterization of members of the three BAR domain families showed surprisingly that the crescent‐shaped BAR dimer with its positively charged concave face is not able to sense membrane curvature. Mutagenesis on BAR domains showed that membrane curvature sensing critically depends on the N‐terminal AH and furthermore that BAR domains sense membrane curvature through hydrophobic insertion in lipid packing defects and not through electrostatics. Consequently, amphipathic motifs, such as AHs, that are often associated with BAR domains emerge as an important means for a protein to sense membrane curvature. Measurements on single liposomes allowed us to document heterogeneous binding behaviour within the ensemble and quantify the influence of liposome polydispersity on bulk membrane curvature sensing experiments. The latter results suggest that bulk liposome‐binding experiments should be interpreted with great caution.     

Coassembly of flotillins induces formation of membrane microdomains, membrane curvature, and vesicle budding

Endocytosis has a crucial role in many cellular processes. The best-characterized mechanism for endocytosis involves clathrin-coated pits [1], but evidence has accumulated for additional endocytic pathways in mammalian cells [2]. One such pathway involves caveolae, plasma-membrane invaginations defined by caveolin proteins. Plasma-membrane microdomains referred to as lipid rafts have also been associated with clathrin-independent endocytosis by biochemical and pharmacological criteria [3]. The mechanisms, however, of nonclathrin, noncaveolin endocytosis are not clear [4, 5]. Here we show that coassembly of two similar membrane proteins, flotillin1 and flotillin2 [6-8], is sufficient to generate de novo membrane microdomains with some of the predicted properties of lipid rafts [9]. These microdomains are distinct from caveolin1-positive caveolae, are dynamic, and bud into the cell. Coassembly of flotillin1 and flotillin2 into microdomains induces membrane curvature, the formation of plasma-membrane invaginations morphologically similar to caveolae, and the accumulation of intracellular vesicles. We propose that flotillin proteins are defining structural components of the machinery that mediates a clathrin-independent endocytic pathway. Key attributes of this machinery are the dependence on coassembly of both flotillins and the inference that flotillin microdomains can exist in either flat or invaginated states.     

BAR domains and membrane curvature: bringing your curves to the BAR

BAR (bin, amphiphysin and Rvs161/167) domains are a unique class of dimerization domains, whose dimerization interface is edged by a membrane-binding surface. In its dimeric form, the membrane-binding interface is concave, and this gives the ability to bind better to curved membranes, i.e. to sense membrane curvature. When present at higher concentrations, the domain can stabilize membrane curvature, generating lipid tubules. This domain is found in many contexts in a wide variety of proteins, where the dimerization and membrane-binding function of this domain is likely to have a profound effect on protein activity. If these proteins function as predicted, then there will be membrane subdomains based on curvature, and thus there is an additional layer of compartmentalization on membranes. These and other possible functions of the BAR domain are discussed.     

The sticholysin family of pore-forming toxins induces the mixing of lipids in membrane domains

Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.     

C2 domain membrane penetration by group IVA cytosolic phospholipase A2 induces membrane curvature changes

Group IVA cytosolic phospholipase A₂ (cPLA₂α) is an 85 kDa enzyme that regulates the release of arachidonic acid (AA) from the sn-2 position of membrane phospholipids. It is well established that cPLA₂α binds zwitterionic lipids such as phosphatidylcholine in a Ca²⁺-dependent manner through its N-terminal C2 domain, which regulates its translocation to cellular membranes. In addition to its role in AA synthesis, it has been shown that cPLA₂α promotes tubulation and vesiculation of the Golgi and regulates trafficking of endosomes. Additionally, the isolated C2 domain of cPLA₂α is able to reconstitute Fc receptor-mediated phagocytosis, suggesting that C2 domain membrane binding is sufficient for phagosome formation. These reported activities of cPLA₂α and its C2 domain require changes in membrane structure, but the ability of the C2 domain to promote changes in membrane shape has not been reported. Here we demonstrate that the C2 domain of cPLA₂α is able to induce membrane curvature changes to lipid vesicles, giant unilamellar vesicles, and membrane sheets. Biophysical assays combined with mutagenesis of C2 domain residues involved in membrane penetration demonstrate that membrane insertion by the C2 domain is required for membrane deformation, suggesting that C2 domain-induced membrane structural changes may be an important step in signaling pathways mediated by cPLA₂α.     

Clustering induces redistribution of syndecan-4 core protein into raft membrane domains

Syndecan-4 is a heparan sulfate-carrying core protein that has been directly implicated in fibroblast growth factor 2 (FGF2) signaling. Recent studies have suggested that many signaling proteins localize to the raft compartment of the plasma cell membrane. To establish whether syndecan-4 is present in the raft compartment, we have studied the distribution of the core protein and an Fc receptor (FcR)-syndecan-4 chimera prior to and following clustering with FGF2 or antibodies. Whereas unclustered syndecan-4 was present predominantly in the non-raft membrane compartment, clustering induced extensive syndecan-4 redistribution to the rafts as demonstrated by the sucrose gradient centrifugation and life confocal microscopy. Although syndecan-4 and caveolin-1 moved in tandem, syndecan-4 was not present in caveolae, a major subset of raft compartments. We conclude that syndecan-4 clustering induces its redistribution to the non-caveolae raft compartment. This process may play an important role in syndecan-4-mediation of FGF2 signaling.     

The cyclic antimicrobial peptide RTD-1 induces stabilized lipid-peptide domains more efficiently than its open-chain analogue

The effects of a mammalian cyclic antimicrobial peptide, rhesus theta defensin 1 (RTD-1) and its open chain analogue (oRTD-1), on the phase behaviour and structure of model membrane systems (dipalmitoyl phosphatidylcholine, DPPC and dipalmitoyl phosphatidylglycerol, DPPG) were studied. The increased selectivity of RTD-1 for anionic DPPG over zwitterionic DPPC was shown by differential scanning calorimetry. RTD-1, at a molar peptide-lipid ratio of 1:100, induced considerable changes in the phase behaviour of DPPG, but not of DPPC. The main transition temperature, Tm, was unchanged, but additional phase transitions appeared above Tm. oRTD-1 induced similar effects. However, the effects were not observable below a peptide:lipid molar ratio of 1:50, which correlates with the weaker biological activity of oRTD-1. Small- and wide-angle X-ray scattering revealed for DPPG the appearance of additional structural features induced by RTD-1 above Tm, which were interpreted as correlated lamellar structures, with increased order of the fatty acyl side chains of the lipid. It is proposed that after initial electrostatic interaction of the cationic rim of the peptide with the anionic DPPG headgroups, leading to stabilized lipid-peptide clusters, the hydrophobic face of the peptide assists in its interaction with the fatty acyl side chains eventually leading to membrane disruption.     

Factors influencing local membrane curvature induction by N-BAR domains as revealed by molecular dynamics simulations

N-BAR domains are protein modules that bind to and induce curvature in membranes via a charged concave surface and N-terminal amphipathic helices. Recently, molecular dynamics simulations have demonstrated that the N-BAR domain can induce a strong local curvature that matches the curvature of the BAR domain surface facing the bilayer. Here we present further molecular dynamics simulations that examine in greater detail the roles of the concave surface and amphipathic helices in driving local membrane curvature. We find that the strong curvature induction observed in our previous simulations requires the stable presentation of the charged concave surface to the membrane and is not driven by the membrane-embedded amphipathic helices. Nevertheless, without these amphipathic helices embedded in the membrane, the N-BAR domain does not maintain a close association with the bilayer, and fails to drive membrane curvature. Increasing the membrane negative charge through the addition of PIP₂ facilitates closer association with the membrane in the absence of embedded helices. At sufficiently high concentrations, amphipathic helices embedded in the membrane drive membrane curvature independently of the BAR domain.     

穿膜肽的结构特点和穿膜机制

穿膜肽（penetratin）是果蝇的触角足同源异形域的DNA结合结构域第三个片段的商品名,由16个氨基酸残基组成,它可以介导多种疏水大分子进入活体细胞质内而不破坏细胞膜的完整性;其最大的特点是可以介导多种大分子进入细胞内,并且无需外源能量,分子作为整体插入细胞内,穿膜过程不需解折叠。该发现开辟了药物进入细胞的新的介导途径。该文介绍穿膜肽的结构特点、穿膜机制、应用及局限性。     

A peptide derived from tenascin-C induces beta1 integrin activation through syndecan-4

Tenascin-C (TN-C) is unique for its cell adhesion modulatory function. We have shown that TNIIIA2, a synthetic 22-mer peptide derived from TN-C, stimulated beta1 integrin-mediated cell adhesion of nonadherent and adherent cell types, by inducing activation of beta1 integrin. The active site of TNIIIA2 appeared cryptic in the TN-C molecule but was exposed by MMP-2 processing of TN-C. The following results suggest that cell surface heparan sulfate (HS) proteoglycan (HSPG), including syndecan-4, participated in TNIIIA2-induced beta1 integrin activation: 1) TNIIIA2 bound to cell surface HSPG via its HS chains, as examined by photoaffinity labeling; 2) heparitinase I treatment of cells abrogated beta1 integrin activation induced by TNIIIA2; 3) syndecan-4 was isolated by affinity chromatography using TNIIIA2-immobilized beads; 4) small interfering RNA-based down-regulation of syndecan-4 expression reduced TNIIIA2-induced beta1 integrin activation, and consequent cell adhesion to fibronectin; 5) overexpression of syndecan-4 core protein enhanced TNIIIA2-induced activation of beta1 integrin. However, treatments that targeted the cytoplasmic region of syndecan-4, including ectopic expression of its mutant truncated with the cytoplasmic domains and treatment with protein kinase Calpha inhibitor G?6976, did not influence the TNIIIA2 activity. These results suggest that a TNIIIA2-related matricryptic site of the TN-C molecule, exposed by MMP-2 processing, may have bound to syndecan-4 via its HS chains and then induced conformational change in beta1 integrin necessary for its functional activation. A lateral interaction of beta1 integrin with the extracellular region of the syndecan-4 molecule may be involved in this conformation change.     

BAR domains, amphipathic helices and membrane-anchored proteins use the same mechanism to sense membrane curvature

The internal membranes of eukaryotic cells are all twists and bends characterized by high curvature. During recent years it has become clear that specific proteins sustain these curvatures while others simply recognize membrane shape and use it as “molecular information” to organize cellular processes in space and time. Here we discuss this new important recognition process termed membrane curvature sensing (MCS). First, we review a new fluorescence-based experimental method that allows characterization of MCS using measurements on single vesicles and compare it to sensing assays that use bulk/ensemble liposome samples of different mean diameter. Next, we describe two different MCS protein motifs (amphipathic helices and BAR domains) and suggest that in both cases curvature sensitive membrane binding results from asymmetric insertion of hydrophobic amino acids in the lipid membrane. This mechanism can be extended to include the insertion of alkyl chain in the lipid membrane and consequently palmitoylated and myristoylated proteins are predicted to display similar curvature sensitive binding. Surprisingly, in all the aforementioned cases, MCS is predominantly mediated by a higher density of binding sites on curved membranes instead of higher affinity as assumed so far. Finally, we integrate these new insights into the debate about which motifs are involved in sensing versus induction of membrane curvature and what role MCS proteins may play in biology.     

Influence of N-acylation of a peptide derived from human lactoferricin on membrane selectivity

Increasing numbers of bacterial strains being resistant to conventional antibiotics emphasize the urgent need for new antimicrobial agents. One strategy is based on host defence peptides that can be found in every organism including humans. We have studied the antimicrobial peptide LF11, derived from the pepsin cleavage product of human lactoferrin, known for its antimicrobial and lipid A-binding activity, and peptide C12LF11, the N-lauryl-derivative of LF11, which has owing to the attached hydrocarbon chain an additional hydrophobic segment. The influence of this hydrocarbon chain on membrane selectivity was studied using model membranes composed of dipalmitoylphosphatidylglycerol (DPPG), mimicking bacterial plasma membranes, and of dipalmitoylphosphatidylcholine (DPPC), a model system for mammalian membranes. A variety of biophysical techniques was applied. Thereby, we found that LF11 did not affect DPPC bilayers and showed only moderate effects on DPPG membranes in accordance with its non-hemolytic and weak antimicrobial activity. In contrast, the introduction of the N-lauryl group caused significant changes in the phase behaviour and lipid chain packing in both model membrane systems. These findings correlate with the in vitro tests on methicillin resistant S. aureus, E. coli, P. aeruginosa and human red blood cells, showing increased biological activity of C12LF11 towards these test organisms. This provides evidence that both electrostatic and hydrophobic interactions are crucial for biological activity of antimicrobial peptides, whereas a certain balance between the two components has to be kept, in order not to loose the specificity for bacterial membranes.     

A 3D analysis of yeast ER structure reveals how ER domains are organized by membrane curvature

We analyzed the structure of yeast endoplasmic reticulum (ER) during six sequential stages of budding by electron tomography to reveal a three-dimensional portrait of ER organization during inheritance at a nanometer resolution. We have determined the distribution, dimensions, and ribosome densities of structurally distinct but continuous ER domains during multiple stages of budding with and without the tubule-shaping proteins, reticulons (Rtns) and Yop1. In wild-type cells, the peripheral ER contains cytoplasmic cisternae, many tubules, and a large plasma membrane (PM)-associated ER domain that consists of both tubules and fenestrated cisternae. In the absence of Rtn/Yop1, all three domains lose membrane curvature, ER ribosome density changes, and the amount of PM-associated ER increases dramatically. Deletion of Rtns/Yop1 does not, however, prevent bloated ER tubules from being pulled from the mother cisterna into the bud and strongly suggests that Rtns/Yop1 stabilize/maintain rather than generate membrane curvature at all peripheral ER domains in yeast.     

Influence of N-acylation of a peptide derived from human lactoferricin on membrane selectivity

Increasing numbers of bacterial strains being resistant to conventional antibiotics emphasize the urgent need for new antimicrobial agents. One strategy is based on host defence peptides that can be found in every organism including humans. We have studied the antimicrobial peptide LF11, derived from the pepsin cleavage product of human lactoferrin, known for its antimicrobial and lipid A-binding activity, and peptide C12LF11, the N-lauryl-derivative of LF11, which has owing to the attached hydrocarbon chain an additional hydrophobic segment. The influence of this hydrocarbon chain on membrane selectivity was studied using model membranes composed of dipalmitoylphosphatidylglycerol (DPPG), mimicking bacterial plasma membranes, and of dipalmitoylphosphatidylcholine (DPPC), a model system for mammalian membranes. A variety of biophysical techniques was applied. Thereby, we found that LF11 did not affect DPPC bilayers and showed only moderate effects on DPPG membranes in accordance with its non-hemolytic and weak antimicrobial activity. In contrast, the introduction of the N-lauryl group caused significant changes in the phase behaviour and lipid chain packing in both model membrane systems. These findings correlate with the in vitro tests on methicillin resistant S. aureus, E. coli, P. aeruginosa and human red blood cells, showing increased biological activity of C12LF11 towards these test organisms. This provides evidence that both electrostatic and hydrophobic interactions are crucial for biological activity of antimicrobial peptides, whereas a certain balance between the two components has to be kept, in order not to loose the specificity for bacterial membranes.     

BH3 death domain peptide induces cell type-selective mitochondrial outer membrane permeability

The BH3 domain is essential for the release of cytochrome c from mitochondria by pro-apoptotic Bcl-2 family proteins during apoptosis. This study tested the hypothesis that a Bax peptide that includes the BH3 domain can permeabilize the mitochondrial outer membrane and release cytochrome c in the absence of a permeability transition at the mitochondrial inner membrane. BH3 peptide (0.1-60 microm) released cytochrome c from mitochondria in the presence of physiological concentrations of ions in a cell type-selective manner, whereas a BH3 peptide with a single amino acid substitution was ineffective. The release of cytochrome c by BH3 peptide correlated with the presence of endogenous Bax at the mitochondria and its integral membrane insertion. Cytochrome c release was accompanied by adenylate kinase release, was not associated with mitochondrial swelling or substantial loss of electrical potential across the inner membrane, and was unaffected by inhibitors of the permeability transition pore. Cytochrome c release was, however, inhibited by Bcl-2. Although energy-coupled respiration was inhibited after the release of cytochrome c, mitochondria maintained membrane potential in the presence of ATP due to the reversal of the ATP synthase. Overall, results support the hypothesis that BH3 peptide releases cytochrome c by a Bax-dependent process that is independent of the mitochondrial permeability transition pore but regulated by Bcl-2.     

Thermodynamics of membrane domains

The concept of lipid rafts and the intense work toward their characterization in biological membranes has spurred a renewed interest in the understanding of domain formation, particularly in the case of cholesterol-containing membranes. The thermodynamic principles underlying formation of domains, rafts, or cholesterol/phospholipid complexes are reviewed here, along with recent work in model and biological membranes. A major motivation for this review was to present those concepts in a way appropriate for the broad readership that has been drawn to the field. Evidence from a number of different techniques points to the conclusion that lipid-lipid interactions are generally weak; therefore, in most cases, massive phase separations are not to be expected in membranes. On the contrary, small, dynamic lipid domains, possibly stabilized by proteins are the most likely outcome. The results on mixed lipid bilayers are used to discuss recent experiments in biological membranes. The clear indication is that proteins partition preferentially into fluid, disordered lipid domains, which is contrary to their localization in ordered, cholesterol/sphingomyelin rafts inferred from detergent extraction experiments on cell membranes. Globally, the evidence appears most consistent with a membrane model in which the majority of the lipid is in a liquid-ordered phase, with dispersed, small, liquid-disordered domains, where most proteins reside. Co-clustering of proteins and their concentration in some membrane areas may occur because of similar preferences for a particular domain but also because of simultaneous exclusion from other lipid phases. Specialized structures, such as caveolae, which contain high concentrations of cholesterol and caveolin are not necessarily similar to bulk liquid-ordered phase.