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1.
We review structure and dynamic measurements of biomembranes by atomic force microscopy (AFM). We focus mainly on studies involving supported lipid bilayers (SLBs), particularly formation by vesicle rupture on flat and corrugated surfaces, nucleation and growth of domains in phase-separated systems, anesthetic-lipid interactions, and protein/peptide interactions in multicomponent systems. We show that carefully designed experiments along with real-time AFM imaging with superior lateral and z resolution (0.1 nm) have revealed quantitative details of the mechanisms and factors controlling vesicle rupture, domain shape and size, phase transformations, and some model biological interactions. The AFM tip can also be used as a mechanical transducer and incorporated in electrochemical measurements of membrane components; therefore, we touch on these important applications in both model and cell membranes. 相似文献
2.
Atomic Force Microscopy (AFM) has become an invaluable tool for studying the micro- and nanoworlds. As a stand-alone, high-resolution imaging technique and force transducer, it defies most other surface instrumentation in ease of use, sensitivity and versatility. The main strength of AFM relies on the possibility to operate in an aqueous environment on a wide variety of biological samples, from single molecules – DNA or proteins – to macromolecular assemblies like biological membranes. Understanding the effect of mechanical stress on membranes is of primary importance in biophysics, since cells are known to perform their function under a complex combination of forces. In the later years, AFM-based Force-Spectroscopy (AFM-FS) has provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Lipid membranes are electrostatically charged entities that physiologically coexist with electrolyte solutions. Thus, specific interactions with ions are a matter of considerable interest. The distribution of ions in the solution and their interaction with the membranes are factors that substantially modify the structure and dynamics of the cell membranes. Furthermore, signaling processes are modified by the membrane capability of retaining ions. Supported Lipid Bilayers (SLBs) are a versatile tool to investigate phospholipid membranes mimicking biological surfaces. In the present contribution, we review selected experiments on the mechanical stability of SLBs as models of lipid membranes by means of AFM-FS, with special focus on the effect of cations and ionic strength in the overall nanomechanical stability. 相似文献
3.
Romina F. Vázquez Erasmo Ovalle-García Armando Antillón Iván Ortega-Blake Laura S. Bakás Carlos Muñoz-Garay Sabina M. Maté 《生物化学与生物物理学报:生物膜》2021,1863(1):183467
Sphingolipids-enriched rafts domains are proposed to occur in plasma membranes and to mediate important cellular functions. Notwithstanding, the asymmetric transbilayer distribution of phospholipids that exists in the membrane confers the two leaflets different potentials to form lateral domains as next to no sphingolipids are present in the inner leaflet. How the physical properties of one leaflet can influence the properties of the other and its importance on signal transduction across the membrane are questions still unresolved. In this work, we combined AFM imaging and Force spectroscopy measurements to assess domain formation and to study the nanomechanical properties of asymmetric supported lipid bilayers (SLBs) mimicking membrane rafts. Asymmetric SLBs were formed by incorporating N-palmitoyl-sphingomyelin (16:0SM) into the outer leaflet of preformed 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Cholesterol SLBs through methyl-β-cyclodextrin–mediated lipid exchange. Lipid domains were detected after incorporation of 16:0SM though their phase state varied from gel to liquid ordered (Lo) phase if the procedure was performed at 24 or 37 °C, respectively. When comparing symmetric and asymmetric Lo domains, differences in size and morphology were observed, with asymmetric domains being smaller and more interconnected. Both types of Lo domains showed similar mechanical stability in terms of rupture forces and Young's moduli. Notably, force curves in asymmetric domains presented two rupture events that could be attributed to the sequential rupture of a liquid disordered (Ld) and a Lo phase. Interleaflet coupling in asymmetric Lo domains could also be inferred from those measurements. The experimental approach outlined here would significantly enhance the applicability of membrane models. 相似文献
4.
Wide-angle X-ray diffraction studies have indicated that rough and smooth microsomal membranes from bean cotyledons acquire increasing proportions of gel phase lipid at physiological temperature as the tissue senesces. In addition, for both types of membrane the lipid phase transition temperature, defined as the highest temperature at which gel phase lipid can be detected, progressively rises with advancing senescence. Liposomes prepared from total lipid extracts of the membranes show a similar increase in transition temperature with age, indicating that separation of the polar lipids into distinct gel and liquid-crystalline domains is not attributable to peculiar protein-lipid interactions. Liposomes prepared from purified phospholipid fractions of the membranes show little change in transition temperature with age, indicating that the altered phase properties of the lipid do not reflect an increase in fatty acid saturation. However, the formation of gel phase lipid that occurs naturally during senescence can be stimulated by preparing liposomes from a mixture of the phospholipid fraction from young membrane and the neutral lipid fraction from old membrane. By adding the separated components of the neutral lipid fraction to purified phospholipid it was found that sterol esters and several unidentified lipids are able to raise the transition temperature of the polar lipids. Sterols have no effect on the phospholipid transition temperature. The data have been interpreted as indicating that several neutral lipids, which presumably increase in abundance with advancing senescence, induce a lateral phase separation of the polar lipids resulting in distinct gel and liquid-crystalline domains of lipid in the senescent membranes. 相似文献
5.
Goksu EI Vanegas JM Blanchette CD Lin WC Longo ML 《Biochimica et biophysica acta》2009,1788(1):254-266
We review structure and dynamic measurements of biomembranes by atomic force microscopy (AFM). We focus mainly on studies involving supported lipid bilayers (SLBs), particularly formation by vesicle rupture on flat and corrugated surfaces, nucleation and growth of domains in phase-separated systems, anesthetic-lipid interactions, and protein/peptide interactions in multicomponent systems. We show that carefully designed experiments along with real-time AFM imaging with superior lateral and z resolution (0.1 nm) have revealed quantitative details of the mechanisms and factors controlling vesicle rupture, domain shape and size, phase transformations, and some model biological interactions. The AFM tip can also be used as a mechanical transducer and incorporated in electrochemical measurements of membrane components; therefore, we touch on these important applications in both model and cell membranes. 相似文献
6.
Nathan J. Wittenberg Timothy W. Johnson Luke R. Jordan Xiaohua Xu Arthur E. Warrington Moses Rodriguez Sang-Hyun Oh 《Journal of visualized experiments : JoVE》2014,(87)
Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified. 相似文献
7.
The organization of the plasma membrane is regulated by the dynamic equilibrium between the liquid ordered (Lo) and liquid disordered (Ld) phases. The abundance of the Lo phase is assumed to be a consequence of the interaction between cholesterol and the other lipids, which are otherwise in either the Ld or gel (So) phase. The characteristic lipid packing in these phases results in significant differences in their respective lateral dynamics. In this study, imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) is applied to monitor the diffusion within supported lipid bilayers (SLBs) as functions of temperature and composition. We show that the temperature dependence of membrane lateral diffusion, which is parameterized by the Arrhenius activation energy (EArr), can resolve the sub-resolution phase behavior of lipid mixtures. The FCS diffusion law, a novel membrane heterogeneity ruler implemented in ITIR-FCS, is applied to show that the domains in the So–Ld phase are static and large while they are small and dynamic in the Lo–Ld phase. Diffusion measurements and the subsequent FCS diffusion law analyses at different temperatures show that the modulation in membrane dynamics at high temperature (313 K) is a cumulative effect of domain melting and rigidity relaxation. Finally, we extend these studies to the plasma membranes of commonly used neuroblastoma, HeLa and fibroblast cells. The temperature dependence of membrane dynamics for neuroblastoma cells is significantly different from that of HeLa or fibroblast cells as the different cell types exhibit a high level of compositional heterogeneity. 相似文献
8.
The current view of the biological membrane is that in which lipids and proteins mutually interact to accomplish membrane functions. The lateral heterogeneity of the lipid bilayer can induce partitioning of membrane-associated proteins, favoring protein-protein interaction and influence signaling and trafficking. The Atomic Force Microscope allows to study the localization of membrane-associated proteins with respect to the lipid organization at the single molecule level and without the need for fluorescence staining. These features make AFM a technique of choice to study lipid/protein interactions in model systems or native membranes. Here we will review the technical aspects inherent to and the main results obtained by AFM in the study of protein partitioning in lipid domains concentrating in particular on GPI-anchored proteins, lipidated proteins, and transmembrane proteins. Whenever possible, we will also discuss the functional consequences of what has been imaged by Atomic Force Microscopy. 相似文献
9.
《生物化学与生物物理学报:生物膜》2023,1865(1):184070
The fusion between two lipid membranes is a ubiquitous mechanism in cell traffic and pathogens invasion. Yet it is not well understood how two distinct bilayers overcome the energy barriers towards fusion and reorganize themselves to form a unique continuous bilayer. The magnitudes and numbers of these energy barriers are themselves an open question. To tackle these issues, we developed a new tool that allows to control the forces applied between two supported lipid bilayers (SLBs) deposited on superparamagnetic beads. By applying a magnetic field, the beads self-organize along field lines in chains of beads and compress the two membranes on the contact zone. Using the diffusion of fluorescently labelled lipids from one bilayer to the other allows us to identify fusion of the bilayers in contact. We applied increasing forces on SLBs and increased the occurrence of fusion. This experimental system allows the simultaneous study of tens of facing bilayers in a single experiment and mitigates the stochasticity of the fusion process. It is thus a powerful tool to test the various parameters involved in the membrane fusion process. 相似文献
10.
《生物化学与生物物理学报:生物膜》2019,1861(9):1523-1532
The biological membrane surrounding milk fat globules (MFGM) exhibits lateral phase separation of lipids, interpreted as gel or liquid-ordered phase sphingomyelin-rich (milk SM) domains dispersed in a fluid continuous lipid phase. The objective of this study was to investigate whether changes in the phase state of milk SM-rich domains induced by temperature (T < Tm or T > Tm) or cholesterol affected the Young modulus of the lipid membrane. Supported lipid bilayers composed of MFGM polar lipids, milk SM or milk SM/cholesterol (50:50 mol) were investigated at 20 °C and 50 °C using atomic force microscopy (AFM) and force spectroscopy. At 20 °C, gel-phase SM-rich domains and the surrounding fluid phase of the MFGM polar lipids exhibited Young modulus values of 10–20 MPa and 4–6 MPa, respectively. Upon heating at 50 °C, the milk SM-rich domains in MFGM bilayers as well as pure milk SM bilayers melted, leading to the formation of a homogeneous membrane with similar Young modulus values to that of a fluid phase (0–5 MPa). Upon addition of cholesterol to the milk SM to reach 50:50 mol%, membranes in the liquid-ordered phase exhibited Young modulus values of a few MPa, at either 20 or 50 °C. This indicated that the presence of cholesterol fluidized milk SM membranes and that the Young modulus was weakly affected by the temperature. These results open perspectives for the development of milk polar lipid based vesicles with modulated mechanical properties. 相似文献
11.
An experimental study of phosphocholine membranes made from one lipid, from mixtures of DPPC and DLPC, and also from lipids and small amounts of alamethicin is presented. We used atomic force microscopy to investigate the spatial organization and structure of lipid domains and also of the defects induced by the peptide. Alamethicin was found to alter the state of lipids in the gel state in a way that domains of fluid lipids are formed close to the defects. Differential calorimetry revealed phase characteristics of the lipid mixtures and the effect of small amounts of alamethicin on the phase behavior. It was also shown that the sound velocity profiles of the membranes suspensions can be well calculated from the heat capacity traces of the samples. This result confirms the correlation between the mechanical properties and the specific heat of membrane systems. 相似文献
12.
Most phospholipids constituting biological membranes are chiral molecules with a hydrophilic head group and hydrophobic alkyl chains, rendering biphasic property characteristic of membrane lipids. Some lipids assemble into small domains via chirality-dependent homophilic and heterophilic interactions, the latter of which sometimes include cholesterol to form lipid rafts and other microdomains. On the other hand, lipid mediators and hormones derived from chiral lipids are recognized by specific membrane or nuclear receptors to induce downstream signaling. It is crucial to clarify the physicochemical properties of the lipid self-assembly for the study of the functions and behavior of biological membranes, which often become elusive due to effects of membrane proteins and other biological events. Three major lipids with different skeletal structures were discussed: sphingolipids including ceramides, phosphoglycerolipids, and cholesterol. The physicochemical properties of membranes and physiological functions of lipid enantiomers and diastereomers were described in comparison to natural lipids. When each enantiomer formed a self-assembly or interacted with achiral lipids, both lipid enantiomers exhibited identical membrane physicochemical properties, while when the enantiomer interacted with chiral lipids or with the opposite enantiomer, mixed membranes exhibited different properties. For example, racemic membranes comprising native sphingomyelin and its antipode exhibited phase segregation due to their strong homophilic interactions. Therefore, lipid enantiomers and diastereomers can be good probes to investigate stereospecific lipid-lipid and lipid-protein interactions occurring in biological membranes. 相似文献
13.
Effective antimicrobial peptides (AMPs) distinguish between the host and microbial cells, show selective antimicrobial activity and exhibit a fast killing mechanism. Although understanding the structure-function characteristics of AMPs is important, the impact of the peptides on the architecture of membranes with different lipid compositions is also critical in understanding the molecular mechanism and specificity of membrane destabilisation. In this study, the destabilisation of supported lipid bilayers (SLBs) by the AMP aurein 1.2 was quantitatively analysed by dual polarisation interferometry. The lipid bilayers were formed on a planar silicon oxynitride chip, and composed of mixed synthetic lipids, or Escherichiacoli lipid extract. The molecular events leading sequentially from peptide adsorption to membrane lysis were examined in real time by changes in bilayer birefringence (lipid molecular ordering) as a function of membrane-bound peptide mass. Aurein 1.2 bound weakly without any change in membrane ordering at low peptide concentration (5 μM), indicating a surface-associated state without significant perturbation in membrane structure. At 10 μM peptide, marked reversible changes in molecular ordering were observed for all membranes except DMPE/DMPG. However, at 20 μM aurein 1.2, removal of lipid molecules, as determined by mass loss with a concomitant decrease in birefringence during the association phase, was observed for DMPC and DMPC/DMPG SLBs, which indicates membrane lysis by aurein. The membrane destabilisation induced by aurein 1.2 showed cooperativity at a particular peptide/lipid ratio with a critical mass/molecular ordering value. Furthermore, the extent of membrane lysis for DMPC/DMPG was nearly double that for DMPC. However, no lysis was observed for DMPC/DMPG/cholesterol, DMPE/DMPG and E. coli SLBs. The extent of birefringence changes with peptide mass suggested that aurein 1.2 binds to the membrane without inserting through the bilayer and membrane lysis occurs through detergent-like micellisation above a critical P/L ratio. Real-time quantitative analysis of the structural properties of membrane organisation has allowed the membrane destabilisation process to be resolved into multiple steps and provides comprehensive information to determine the molecular mechanism of aurein 1.2 action. 相似文献
14.
《Molecular membrane biology》2013,30(5):144-154
AbstractConstituting functional interactions between proteins and lipid membranes is one of the essential features of cellular membranes. The major challenge of quantitatively studying these interactions in living cells is the multitude of involved components that are difficult, if not impossible, to simultaneously control. Therefore, there is great need for simplified but still sufficiently detailed model systems to investigate the key constituents of biological processes. To specifically focus on interactions between membrane proteins and lipids, several membrane models have been introduced which recapitulate to varying degrees the complexity and physicochemical nature of biological membranes. Here, we summarize the presently most widely used minimal model membrane systems, namely Supported Lipid Bilayers (SLBs), Giant Unilamellar Vesicles (GUVs) and Giant Plasma Membrane Vesicles (GPMVs) and their applications for protein-membrane interactions. 相似文献
15.
There has been increasing interest in recent years in describing the lateral organization of membranes and the formation of membrane domains. Much of the focus in this area has been on the formation of cholesterol-rich domains in mammalian membranes. However, it is likely that there are domains in all biological membranes. One of the challenges has been to define the chemical composition, lifetime and size of these domains. There is evidence that bacteria have domains that are enriched in cardiolipin. In addition, the formation of lipid domains can be induced in bacteria by clustering negatively charged lipids with polycationic substances. Many antimicrobial compounds have multiple positive charges. Such polycationic compounds can sequester anionic lipids to induce lipid phase separation. The molecular interactions among lipids and their lateral packing density will be different in a domain from its environment. This will lead to phase boundary defects that will lower the permeability barrier between the cell and its surroundings. The formation of these clusters of anionic lipids may also alter the stability or composition of existing membrane domains that may affect bacterial function. Interestingly many antimicrobial agents are polycationic and therefore likely have some effect in promoting lipid phase segregation between anionic and zwitterionic lipids. However, this mechanism is expected to be most important for substances with sequential positive charges contained within a flexible molecule that can adapt to the arrangement of charged groups on the surface of the bacterial cell. When this mechanism is dominant it can allow the prediction of the bacterial species that will be most affected by the agent as a consequence of the nature of the lipid composition of the bacterial membrane. 相似文献
16.
There has been increasing interest in recent years in describing the lateral organization of membranes and the formation of membrane domains. Much of the focus in this area has been on the formation of cholesterol-rich domains in mammalian membranes. However, it is likely that there are domains in all biological membranes. One of the challenges has been to define the chemical composition, lifetime and size of these domains. There is evidence that bacteria have domains that are enriched in cardiolipin. In addition, the formation of lipid domains can be induced in bacteria by clustering negatively charged lipids with polycationic substances. Many antimicrobial compounds have multiple positive charges. Such polycationic compounds can sequester anionic lipids to induce lipid phase separation. The molecular interactions among lipids and their lateral packing density will be different in a domain from its environment. This will lead to phase boundary defects that will lower the permeability barrier between the cell and its surroundings. The formation of these clusters of anionic lipids may also alter the stability or composition of existing membrane domains that may affect bacterial function. Interestingly many antimicrobial agents are polycationic and therefore likely have some effect in promoting lipid phase segregation between anionic and zwitterionic lipids. However, this mechanism is expected to be most important for substances with sequential positive charges contained within a flexible molecule that can adapt to the arrangement of charged groups on the surface of the bacterial cell. When this mechanism is dominant it can allow the prediction of the bacterial species that will be most affected by the agent as a consequence of the nature of the lipid composition of the bacterial membrane. 相似文献
17.
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. 相似文献
18.
Biological membranes define not only the cell boundaries but any compartment within the cell. To some extent, the functionality of membranes is related to the elastic properties of the lipid bilayer and the mechanical and hydrophobic matching with functional membrane proteins. Supported lipid bilayers (SLBs) are valid biomimetic systems for the study of membrane biophysical properties. Here, we acquired high-resolution topographic and quantitative mechanics data of phase-separated SLBs using a recent atomic force microscopy (AFM) imaging mode based on force measurements. This technique allows us to quantitatively map at high resolution the mechanical differences of lipid phases at different loading forces. We have applied this approach to evaluate the contribution of the underlying hard support in the determination of the elastic properties of SLBs and to determine the adequate indentation range for obtaining reliable elastic moduli values. At ~200 pN, elastic forces dominated the force-indentation response and the sample deformation was <20% of the bilayer thickness, at which the contribution of the support was found to be negligible. The obtained Young's modulus (E) of 19.3 MPa and 28.1 MPa allowed us to estimate the area stretch modulus (k(A)) as 106 pN/nm and 199 pN/nm and the bending stiffness (k(c)) as 18 k(B)T and 57 k(B)T for the liquid and gel phases, respectively. 相似文献
19.
Line-active molecules (“linactants”) that bind to the boundary interface between different fluid lipid domains in membranes have a strong potential as regulators of the lateral heterogeneity that is important for many biological processes. Here, we use molecular dynamics simulations in combination with a coarse-grain model that retains near-atomic resolution to identify lipid species that can act as linactants in a model membrane that is segregated into two lipid domains of different fluidity. Our simulations predict that certain hybrid saturated/unsaturated chain lipids can bind to the interface and lower the line tension, whereas cone-shaped lysolipids have a less pronounced effect. 相似文献
20.
By means of scanning force and fluorescence microscopy of artificial membranes immobilized on mica surfaces, the lateral organization of the annexin A2/S100A10 heterotetramer (annexin A2t) and its influence on the lateral organization of the lipids within the membrane have been elucidated. Planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) were prepared on atomically flat mica surfaces by the spreading of unilamellar vesicles. Fluorescence images of fluorescently labeled annexin A2t and scanning force microscopy images of nonlabeled protein bound to POPC/POPS bilayers show the formation of micrometer-sized lateral protein domains in the presence of 1 mM CaCl2. By means of scanning force microscopy, not only protein domains became discernible but also small membrane domains, which were attributed to POPS-enriched areas. A depletion of these POPS domains was observed in the vicinity of annexin A2t protein domains. These results indicate that annexin A2t is a peripheral membrane-binding complex capable of inducing lipid segregation. 相似文献