Membrane events involved in myoblast fusion

Myoblast fusion has been studied in cultures of chick embryonic muscle utilizing ultrastructural techniques. The multinucleated muscle cells (myotubes) are generated by the fusion of two plasma membranes from adjacent cells, apparently by forming a single bilayer that is particle-free in freeze-fracture replicas. This single bilayer subsequently collapses, and cytoplasmic continuity is established between the cells. The fusion between the two plasma membranes appears to take place primarily within particle-free domains (probably phospholipid enriched), and cytoplasmic unilamellar, particle-free vesicles are occasionally associated with these regions. These vesicles structurally resemble phospholipid vesicles (liposomes). They are present in normal myoblasts, but they are absent in certain fusion-arrested myoblast popluations, such as those treated with either 5-bromo-deoxyuridine (BUdR), cycloheximide (CHX), or pospholipase C (PLC). The unilamellar, particle-free vesicles are present in close proximity to the plasma membranes, and physical contact is observed frequently between the vesicle membrane and the plasma membrane. The regions of vesicle membrane-plasma membrane interaction are characteristically free of intramembrane particles. A model for myoblast fusion is presented that is based onan interpretation of these observations. This model suggests that the cytoplasmic vesicles initiate the generation of particle-depleted membrane domains, both being essential components in the fusion process.     

Diffusion as a probe of peptide-induced membrane domain formation

Recently, we have shown that association with an antimicrobial peptide (AMP) can drastically alter the diffusion behavior of the constituent lipids in model membranes (Biochemistry 49, 4672-4678). In particular, we found that the diffusion time of a tracer fluorescent lipid through a confocal volume measured via fluorescence correlation spectroscopy (FCS) is distributed over a wide range of time scales, indicating the formation of stable and/or transient membrane species that have different mobilities. A simple estimate, however, suggested that the slow diffusing species are too large to be attributed to AMP oligomers or pores that are tightly bound to a small number of lipids. Thus, we tentatively ascribed them to membrane domains and/or clusters that possess distinctively different diffusion properties. In order to further substantiate our previous conjecture, herein we study the diffusion behavior of the membrane-bound peptide molecules using the same AMPs and model membranes. Our results show, in contrast to our previous findings, that the diffusion times of the membrane-bound peptides exhibit a much narrower distribution that is more similar to that of the lipids in peptide-free membranes. Thus, taken together, these results indicate that while AMP molecules prompt domain formation in membranes, they are not tightly associated with the lipid domains thus formed. Instead, they are likely located at the boundary regions separating various domains and acting as mobile fences.     

Lipid domains in bacterial membranes

The recent development of specific probes for lipid molecules has led to the discovery of lipid domains in bacterial membranes, that is, of membrane areas differing in lipid composition. A view of the membrane as a patchwork is replacing the assumption of lipid homogeneity inherent in the fluid mosaic model of Singer and Nicolson (Science 1972, 175: 720–731). If thus membranes have complex lipid structure, questions arise about how it is generated and maintained, and what its function might be. How do lipid domains relate to the functionally distinct regions in bacterial cells as they are identified by protein localization techniques? This review assesses the current knowledge on the existence of cardiolipin (CL) and phosphatidylethanolamine (PE) domains in bacterial cell membranes and on the specific cellular localization of certain membrane proteins, which include phospholipid synthases, and discusses possible mechanisms, both chemical and physiological, for the formation of the lipid domains. We propose that bacterial membranes contain a mosaic of microdomains of CL and PE, which are to a significant extent self‐assembled according to their respective intrinsic chemical characteristics. We extend the discussion to the possible relevance of the domains to specific cellular processes, including cell division and sporulation.     

Detecting the presence of membrane domains using DSC

Both biological and model liposomal membranes have unequal distribution of molecular components in the plane of the membrane. There is increasing interest to determine the composition and properties of membrane domains enriched with specific molecular components. Several methodologies have been applied to study this. Each has its own advantages and provides a particular kind of information. In the present article, we will focus on the application of differential scanning calorimetry to the determination of the distribution of molecules into membrane domains with particular emphasis on protein and peptide-induced domain formation.     

A glycosynapse in myelin?

Myelin, the multilayered membrane which surrounds nerve axons, is the only example of a membranous structure where contact between extracellular surfaces of membrane from the same cell occurs. The two major glycosphingolipids (GSLs) of myelin, galactosylceramide (GalC) and its sulfated form, galactosylceramide I(3)-sulfate (SGC), can interact with each other by trans carbohydrate-carbohydrate interactions across apposed membranes. They occur in detergent-insoluble lipid rafts containing kinases and thus may be located in membrane signaling domains. These signaling domains may contact each other across apposed extracellular membranes, thus forming glycosynapses in myelin. Multivalent forms of these carbohydrates, GalC/SGC-containing liposomes, or galactose conjugated to albumin, have been added to cultured oligodendrocytes (OLs) to mimic interactions which might occur between these signaling domains when OL membranes or the extracellular surfaces of myelin come into contact. These interactions between multivalent carbohydrate and the OL membrane cause co-clustering or redistribution of myelin GSLs, GPI-linked proteins, several transmembrane proteins, and signaling proteins to the same membrane domains. They also cause depolymerization of the cytoskeleton, indicating that they cause transmission of a signal across the membrane. Their effects have similarities to those of anti-GSL antibodies on OLs, shown by others, suggesting that the multivalent carbohydrate interacts with GalC/SGC in the OL membrane. Communication between the myelin sheath and the axon regulates both axonal and myelin function and is necessary to prevent neurodegeneration. Participation of transient GalC and SGC interactions in glycosynapses between the apposed extracellular surfaces of mature compact internodal myelin might allow transmission of signals throughout the myelin sheath and thus facilitate myelin-axonal communication.     

Hydrophobic Compounds Reshape Membrane Domains

Cell membranes have a complex lateral organization featuring domains with distinct composition, also known as rafts, which play an essential role in cellular processes such as signal transduction and protein trafficking. In vivo, perturbations of membrane domains (e.g., by drugs or lipophilic compounds) have major effects on the activity of raft-associated proteins and on signaling pathways, but they are difficult to characterize because of the small size of the domains, typically below optical resolution. Model membranes, instead, can show macroscopic phase separation between liquid-ordered and liquid-disordered domains, and they are often used to investigate the driving forces of membrane lateral organization. Studies in model membranes have shown that some lipophilic compounds perturb membrane domains, but it is not clear which chemical and physical properties determine domain perturbation. The mechanisms of domain stabilization and destabilization are also unknown. Here we describe the effect of six simple hydrophobic compounds on the lateral organization of phase-separated model membranes consisting of saturated and unsaturated phospholipids and cholesterol. Using molecular simulations, we identify two groups of molecules with distinct behavior: aliphatic compounds promote lipid mixing by distributing at the interface between liquid-ordered and liquid-disordered domains; aromatic compounds, instead, stabilize phase separation by partitioning into liquid-disordered domains and excluding cholesterol from the disordered domains. We predict that relatively small concentrations of hydrophobic species can have a broad impact on domain stability in model systems, which suggests possible mechanisms of action for hydrophobic compounds in vivo.     

How the molecular features of glycosphingolipids affect domain formation in fluid membranes

Glycosphingolipids, sphingomyelin and cholesterol are often all found in the detergent resistant fraction of biological membranes and are therefore recognized as raft components, but they do not necessarily co-localize in the same lateral domains. From cell biological studies it is evident that different sphingolipid species can be found in different lateral regions within the same cellular membrane. Biophysical studies have shown that their tendency to co-localize with each other and with other membrane components is largely governed by structural features of all lipids present. Glycosphingolipids form gel-phase like domains in fluid lipid bilayers. Sphingomyelin readily associates with cholesterol, forming liquid-ordered phase domains, but glycosphingolipids do not readily form cholesterol-enriched domains by themselves. However, mixed sphingomyelin- and glycosphingolipid-rich domains appear to incorporate cholesterol. Recent studies indicate that the ceramide backbone structure as well as the number of sugar units and presence of charge in the glycosphingolipid head group will influence the partitioning of these lipids between lateral membrane domains. The properties of the domains will be largely influenced by the presence of glycosphingolipids, which have very high melting temperatures. The lateral partitioning of glycosphingolipid molecular species has only recently been studied more intensively, and a lot remains to be done in this field of research.     

Molecular mechanisms of steric pressure generation and membrane remodeling by disordered proteins

Cellular membranes, which are densely crowded by proteins, take on an elaborate array of highly curved shapes. Steric pressure generated by protein crowding plays a significant role in shaping membrane surfaces. It is increasingly clear that many proteins involved in membrane remodeling contain substantial regions of intrinsic disorder. These domains have large hydrodynamic radii, suggesting that they may contribute significantly to steric congestion on membrane surfaces. However, it has been unclear to what extent they are capable of generating steric pressure, owing to their conformational flexibility. To address this gap, we use a recently developed sensor based on Förster resonance energy transfer to measure steric pressure generated at membrane surfaces by the intrinsically disordered domain of the endocytic protein, AP180. We find that disordered domains generate substantial steric pressure that arises from both entropic and electrostatic components. Interestingly, this steric pressure is largely invariant with the molecular weight of the disordered domain, provided that coverage of the membrane surface is held constant. Moreover, equivalent levels of steric pressure result in equivalent degrees of membrane remodeling, regardless of protein molecular weight. This result, which is consistent with classical polymer scaling relationships for semi-dilute solutions, helps to explain the molecular and physical origins of steric pressure generation by intrinsically disordered domains. From a physiological perspective, these findings suggest that a broad range of membrane-associated disordered domains are likely to play a significant and previously unknown role in controlling membrane shape.     

Protein-promoted membrane domains

The current notion of biological membranes encompasses a very complex structure, made of dynamically changing compartments or domains where different membrane components partition. These domains have been related to important cellular functions such as membrane sorting, signal transduction, membrane fusion, neuronal maturation, and protein activation. Many reviews have dealt with membrane domains where lipid-lipid interactions direct their formation, especially in the case of raft domains, so in this review we considered domains induced by integral membrane proteins. The nature of the interactions involved and the different mechanisms through which membrane proteins segregate lipid domains are presented, in particular with regard to those induced by the nAChR. It may be concluded that coupling of favourable lipid-lipid and lipid-protein interactions is a general condition for this phenomenon to occur.     

Membrane recognition by phospholipid-binding domains

Many different globular domains bind to the surfaces of cellular membranes, or to specific phospholipid components in these membranes, and this binding is often tightly regulated. Examples include pleckstrin homology and C2 domains, which are among the largest domain families in the human proteome. Crystal structures, binding studies and analyses of subcellular localization have provided much insight into how members of this diverse group of domains bind to membranes, what features they recognize and how binding is controlled. A full appreciation of these processes is crucial for understanding how protein localization and membrane topography and trafficking are regulated in cells.     

Role of ceramide in membrane protein organization investigated by combined AFM and FCS

Ceramide-induced alterations in the lateral organization of membrane proteins can be involved in several biological contexts, ranging from apoptosis to viral infections. In order to investigate such alterations in a simple model, we used a combined approach of atomic force microscopy, scanning fluorescence correlation spectroscopy and confocal fluorescence imaging to study the partitioning of different membrane components in sphingomyelin/dioleoyl-phosphatidylcholine/cholesterol/ceramide supported bilayers. Such model membranes exhibit coexistence of liquid-disordered, liquid-ordered (raft-like) and ceramide-rich lipid phases. Our results show that components with poor affinity toward the liquid-ordered phase, such as several fluorescent lipid analogues or the synaptic protein Synaptobrevin 2, are excluded from ceramide-rich domains. Conversely, we show for the first time that the raft-associated protein placental alkaline phosphatase (GPI-PLAP) and the ganglioside GM1 are enriched in such domains, while exhibiting a strong decrease in lateral diffusion. Analogue modulation of the local concentration and dynamics of membrane proteins/receptors by ceramide can be of crucial importance for the biological functions of cell membranes.     

Role of ceramide in membrane protein organization investigated by combined AFM and FCS

Ceramide-induced alterations in the lateral organization of membrane proteins can be involved in several biological contexts, ranging from apoptosis to viral infections. In order to investigate such alterations in a simple model, we used a combined approach of atomic force microscopy, scanning fluorescence correlation spectroscopy and confocal fluorescence imaging to study the partitioning of different membrane components in sphingomyelin/dioleoyl-phosphatidylcholine/cholesterol/ceramide supported bilayers. Such model membranes exhibit coexistence of liquid-disordered, liquid-ordered (raft-like) and ceramide-rich lipid phases. Our results show that components with poor affinity toward the liquid-ordered phase, such as several fluorescent lipid analogues or the synaptic protein Synaptobrevin 2, are excluded from ceramide-rich domains. Conversely, we show for the first time that the raft-associated protein placental alkaline phosphatase (GPI-PLAP) and the ganglioside G_M1 are enriched in such domains, while exhibiting a strong decrease in lateral diffusion. Analogue modulation of the local concentration and dynamics of membrane proteins/receptors by ceramide can be of crucial importance for the biological functions of cell membranes.     

The molecular basis of differential subcellular localization of C2 domains of protein kinase C-alpha and group IVa cytosolic phospholipase A2

The C2 domain is a Ca(2+)-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. C2 domains are unique among membrane targeting domains in that they show a wide range of lipid selectivity for the major components of cell membranes, including phosphatidylserine and phosphatidylcholine. To understand how C2 domains show diverse lipid selectivity and how this functional diversity affects their subcellular targeting behaviors, we measured the binding of the C2 domains of group IVa cytosolic phospholipase A(2) (cPLA(2)) and protein kinase C-alpha (PKC-alpha) to vesicles that model cell membranes they are targeted to, and we monitored their subcellular targeting in living cells. The surface plasmon resonance analysis indicates that the PKC-alpha C2 domain strongly prefers the cytoplasmic plasma membrane mimic to the nuclear membrane mimic due to high phosphatidylserine content in the former and that Asn(189) plays a key role in this specificity. In contrast, the cPLA(2) C2 domain has specificity for the nuclear membrane mimic over the cytoplasmic plasma membrane mimic due to high phosphatidylcholine content in the former and aromatic and hydrophobic residues in the calcium binding loops of the cPLA(2) C2 domain are important for its lipid specificity. The subcellular localization of enhanced green fluorescent protein-tagged C2 domains and mutants transfected into HEK293 cells showed that the subcellular localization of the C2 domains is consistent with their lipid specificity and could be tailored by altering their in vitro lipid specificity. The relative cell membrane translocation rate of selected C2 domains was also consistent with their relative affinity for model membranes. Together, these results suggest that biophysical principles that govern the in vitro membrane binding of C2 domains can account for most of their subcellular targeting properties.     

Role of lipid rafts in agrin-elicited acetylcholine receptor clustering

Emerging concepts of membrane organization point to the compartmentalization of the plasma membrane into distinct lipid microdomains. This lateral segregation within cellular membranes is based on cholesterol-sphingolipid-enriched microdomains or lipid rafts which can move laterally and assemble into large-scale domains to create plasma membrane specialized cellular structures at specific cell locations. Such domains are likely involved in the genesis of the postsynaptic specialization at the neuromuscular junction, which requires the accumulation of acetylcholine receptors (AChRs), through activation of the muscle specific kinase MuSK by the neurotropic factor agrin and the reorganization of the actin cytoskeleton. We used C2C12 myotubes as a model system to investigate whether agrin-elicited AChR clustering correlated with lipid rafts. In a previous study, using two-photon Laurdan confocal imaging, we showed that agrin-induced AChR clusters corresponded to condensed membrane domains: the biophysical hallmark of lipid rafts [F. Stetzkowski-Marden, K. Gaus, M. Recouvreur, A. Cartaud, J. Cartaud, Agrin elicits membrane condensation at sites of acetylcholine receptor clusters in C2C12 myotubes, J. Lipid Res. 47 (2006) 2121-2133]. We further demonstrated that formation and stability of AChR clusters depend on cholesterol. We also reported that three different extraction procedures (Triton X-100, pH 11 or isotonic Ca++, Mg++ buffer) generated detergent resistant membranes (DRMs) with similar cholesterol/GM1 ganglioside content, which are enriched in several signalling postsynaptic components, notably AChR, the agrin receptor MuSK, rapsyn and syntrophin. Upon agrin engagement, actin and actin-nucleation factors such as Arp2/3 and N-WASP were transiently recovered within raft fractions suggesting that the activation by agrin can trigger actin polymerization. Taken together, the present data suggest that AChR clustering at the neuromuscular junction relies upon a mechanism of raft coalescence driven by agrin-elicited actin polymerization.     

Domains in biological membranes

Our current view on biological membranes has gradually evolved from the influential fluid mosaic model of the early 1970s to a distinctively more complex picture. Biological membranes are now assumed to encompass multiple membrane domains and a plethora of protein-lipid and protein-protein interactions that compartmentalize and temporarily order what has originally been envisioned to be mostly random. In this minireview, we will first highlight some structural principles that govern membrane domain formation and permit a classification of membrane domains. We will then focus on the still controversial issue of lipid-based membrane domains, or lipid rafts, and discuss recent advances in detecting these enigmatic structures in living cells. Finally we will evaluate biochemical approaches to characterize lipid rafts and discuss their contribution to the emerging topic of lipid raft diversity     

Dynamics and size of cross-linking-induced lipid nanodomains in model membranes

Changes of membrane organization upon cross-linking of its components trigger cell signaling response to various exogenous factors. Cross-linking of raft gangliosides GM1 with cholera toxin (CTxB) was shown to cause microscopic phase separation in model membranes, and the CTxB-GM1 complexes forming a minimal lipid raft unit are the subject of ongoing cell membrane research. Yet, those subdiffraction sized rafts have never been described in terms of size and dynamics. By means of two-color z-scan fluorescence correlation spectroscopy, we show that the nanosized domains are formed in model membranes at lower sphingomyelin (Sph) content than needed for the large-scale phase separation and that the CTxB-GM1 complexes are confined in the domains poorly stabilized with Sph. Förster resonance energy transfer together with Monte Carlo modeling of the donor decay response reveal the domain radius of ～8 nm, which increases at higher Sph content. We observed two types of domains behaving differently, which suggests a dual role of the cross-linker: first, local transient condensation of the GM1 molecules compensating for a lack of Sph and second, coalescence of existing nanodomains ending in large-scale phase separation.     

The Fluid–Mosaic model of cell membranes: A brief introduction,historical features,some general principles,and its adaptation to current information

The Fluid–Mosaic Membrane (FMM) model was originally proposed as a general, nanometer-scale representation of cell membranes (Singer and Nicolson, 1972). The FMM model was based on some general principles, such as thermodynamic considerations, intercalation of globular proteins into a lipid bilayer, independent protein and lipid dynamics, cooperativity and other characteristics. Other models had trimolecular structures or membrane globular lipoprotein units. These latter models were flawed, because they did not allow autonomous lipids, membrane domains or discrete lateral dynamics. The FMM model was also consistent with membrane asymmetry, cis- and trans-membrane linkages and associations of membrane components into multi-molecular complexes and domains. It has remained useful for explaining the basic organizational principles and properties of various biological membranes. New information has been added, such as membrane-associated cytoskeletal assemblies, extracellular matrix interactions, transmembrane controls, specialized lipid-protein domains that differ in compositions, rotational and lateral mobilities, lifetimes, functions, and other characteristics. The presence of dense, structured membrane domains has reduced significantly the extent of fluid-lipid membrane areas, and the FMM model is now considered to be more mosaic and dense than the original proposal.     

Targeting membrane proteins to liquid-ordered phases: molecular self-organization explored by fluorescence correlation spectroscopy

The complex and dynamic architecture of biological membranes comprises of various heterogeneities, some of which may include lipid-based and/or protein-based microdomains called "rafts". Due to interactions among membrane components, several types of domains can form with different characteristics and mechanisms of formation. Model membranes, such as giant unilamellar vesicles (GUVs), provide a key system to study lipid-lipid and lipid-protein interactions, which are potentially relevant to raft formation, by (single-molecule) optical microscopy. Here, we review studies of combined confocal imaging and fluorescence correlation spectroscopy (FCS) on lipid dynamics and organization in domains assembled in GUVs, prepared from various lipid mixtures, which are relevant to the problem of raft formation. Finally, we summarize the results on lipid-protein interactions, which govern the targeting of several putative raft- and non-raft-associated membrane proteins to domain-exhibiting GUVs.     

Lateral redistribution of transmembrane proteins and liquid-ordered domains in lipid membranes with inhomogeneous curvature

A number of processes in living cells are accompanied by significant changes of the geometric curvature of lipid membranes. In turn, heterogeneity of the lateral curvature can lead to spatial redistribution of membrane components, most important of which are transmembrane proteins and liquid-ordered lipid-protein domains. These components have a so-called hydrophobic mismatch: the length of the transmembrane domain of the protein, or the thickness of the bilayer of the domain differ from the thickness of the surrounding membrane. In this work we consider redistribution of membrane components with hydrophobic mismatch in membranes with non-uniform geometric curvature. Dependence of the components’ energy on the curvature is calculated in terms of theory of elasticity of liquid crystals adapted to lipid membranes. According to the calculations, transmembrane proteins prefer regions of the membrane with zero curvature. Liquid-ordered domains having a size of a few nm distribute mainly into regions of the membrane with small negative curvature appearing in the cell plasma membrane in the process of endocytosis. The distribution of domains of a large radius is determined by a decrease of their perimeter upon bending; these domains distribute into membrane regions with relatively large curvature.     

Tilted peptides: a structural motif involved in protein membrane insertion?

Tilted peptides are short hydrophobic protein fragments characterized by an asymmetric distribution of their hydrophobic residues when helical. They are able to interact with a hydrophobic/hydrophilic interface (such as a lipid membrane) and to destabilize the organized system into which they insert. They were detected in viral fusion proteins and in proteins involved in different biological processes involving membrane insertion or translocation of the protein in which they are found. In this paper, we have analysed different protein domains related to membrane insertion with regard to their tilted properties. They are the N-terminal signal peptide of the filamentous haemagglutinin (FHA), a Bordetella pertussis protein secreted in high amount and the hydrophobic domain from proteins forming pores (i.e. ColIa, Bax and Bcl-2). From the predictions and the experimental approaches, we suggest that tilted peptides found in those proteins could have a more general role in the mechanism of insertion/translocation of proteins into/across membranes. For the signal sequences, they could help the protein machinery involved in protein secretion to be more active. In the case of toroidal pore formation, they could disturb the lipids, facilitating the insertion of the other more hydrophilic helices.