18 research outputs found
Structure and Nanomechanics of Model Membranes by Atomic Force Microscopy and Spectroscopy: Insights into the Role of Cholesterol and Sphingolipids
Biological membranes mediate several biological processes that are directly associated with their physical properties but sometimes difficult to evaluate. Supported lipid bilayers (SLBs) are model systems widely used to characterize the structure of biological membranes. Cholesterol (Chol) plays an essential role in the modulation of membrane physical properties. It directly influences the order and mechanical stability of the lipid bilayers, and it is known to laterally segregate in rafts in the outer leaflet of the membrane together with sphingolipids (SLs). Atomic force microscope (AFM) is a powerful tool as it is capable to sense and apply forces with high accuracy, with distance and force resolution at the nanoscale, and in a controlled environment. AFM-based force spectroscopy (AFM-FS) has become a crucial technique to study the nanomechanical stability of SLBs by controlling the liquid media and the temperature variations. In this contribution, we review recent AFM and AFM-FS studies on the effect of Chol on the morphology and mechanical properties of model SLBs, including complex bilayers containing SLs. We also introduce a promising combination of AFM and X-ray (XR) techniques that allows for in situ characterization of dynamic processes, providing structural, morphological, and nanomechanical information
Quantifying the tuneable interactions between colloid supported lipid bilayers
Colloid supported lipid bilayers (CSLBs) are formed via the rupture and fusion of lipid vesicles to coat spherical colloidal particles. CSLBs are an emerging vector for the controlled self-assembly of colloids due to the ability to include additives into the bilayer, which influence the (a)specific interactions between particles. To evaluate the specificity of CSLB assembly, first a fundamental study on the tunability of the colloidal interaction and resulting colloidal stability of CSLBs without specific interactions is reported here. It was found that both fluid and gel CSLBs showed significant clustering and attraction, while the addition of steric stabilizers induced a profound increase in stability. The interactions were rendered attractive again by the introduction of depletion forces via the addition of free non-adsorbing polymers. The compositions of fluid and gel CSLBs with 5% membrane stabiliser were concluded to be optimal for further studies where both colloidal stability, and contrasting membrane fluidity are required. These experimental findings were confirmed semi-quantitatively by predictions using numerical self-consistent mean-field theory lattice computations
Quantifying the tuneable interactions between colloid supported lipid bilayers
Colloid supported lipid bilayers (CSLBs) are formed via the rupture and fusion of lipid vesicles to coat spherical colloidal particles. CSLBs are an emerging vector for the controlled self-assembly of colloids due to the ability to include additives into the bilayer, which influence the (a)specific interactions between particles. To evaluate the specificity of CSLB assembly, first a fundamental study on the tunability of the colloidal interaction and resulting colloidal stability of CSLBs without specific interactions is reported here. It was found that both fluid and gel CSLBs showed significant clustering and attraction, while the addition of steric stabilizers induced a profound increase in stability. The interactions were rendered attractive again by the introduction of depletion forces via the addition of free non-adsorbing polymers. The compositions of fluid and gel CSLBs with 5% membrane stabiliser were concluded to be optimal for further studies where both colloidal stability, and contrasting membrane fluidity are required. These experimental findings were confirmed semi-quantitatively by predictions using numerical self-consistent mean-field theory lattice computations
Structural organization of model membranes: a complementary approach combining atomic force microscopy and X-ray techniques
[eng] Biological membranes (BMs) are self-sealing boundaries, which confine the permeability barriers of cells and organelles and provide the means to compartmentalize functions. Apart from being crucial for the cell structure, they provide a support matrix for all the proteins inserted in the cell, acting as channels to exchange mass, energy and information with the environment. BMs mediate several biological functions, such as trafficking, cell division, endocytosis and exocytosis, demanding strong conformational changes of the lipid membrane like fusion, fission or tubes growth. These mechanical requirements are only possible due to the organization of the chemical composition of the lipids into the membrane of each organelle, which is directly linked to the organelle function. Thanks to the dynamic behavior of the membrane, lateral and transverse forces within the membrane are significant and change rapidly as the membrane is bent or stretched, and as new constituents are added, removed or chemically modified. Differences in structure between the two leaflets and between different areas of the bilayer can be associate to membrane deformation to alter the activities of membrane binding proteins. It is then the correlation between the composition and the packing of the lipids what essentially governs the membrane physicochemical and mechanical properties.
Considering the complex chemical diversity of BMs, model bilayers systems are frequently used to study membrane properties and biological processes. Because of the micro and nanoscale range of domains in BMs, and the consequent need of local techniques to explore BMs at the nanometric level, supported bilayer systems are very manageable platforms, since they retain two-dimensional order and lateral mobility and offer excellent environments for the insertion of membrane proteins. In particular, supported lipid bilayers (SLBs) facilitate the use of surface analytical techniques, being ideal models to study the lipid lateral interactions, the growth of lipid domains, as well as interactions between the lipid membrane and proteins, peptides and drugs, cell signaling, etc.
Several reports demonstrate the wide variety of useful techniques to study supported and non-supported lipid membranes. Thanks to the possibility of working under controlled environment and with distance and force resolution at the nanoscale, atomic force microscopy (AFM) is nowadays a well-established technique for both imaging the morphology and probing the local physical and mechanical properties of SLBs by means of force spectroscopy. However, the resolution given by AFM might be inferior to the one achievable with X-ray (XR) and neutron techniques. In particular, XR techniques such as XR reflectivity (XRR) and grazing incidence XR diffraction (GIXD) are powerful tools to characterize surfaces below the nanoscale, providing structural information in the reciprocal space through the interaction between XR and the sample electronic structure. Still, since these techniques do not involve any mechanical interaction with the specimen, mechanical properties cannot be evaluated with XR.
The general objective of this thesis is to investigate the physicochemical and structural properties of model lipid membranes combining atomic force microscopy (AFM) and spectroscopy (AFM-FS) and X-Ray techniques. The AFM provides the morphological and mechanical information of the SLBs, whereas the XR gives more understandings on the electronic structure of the bilayers. We also propose advanced methodologies based on AFM and XR as well as the coupling of both techniques for local in situ experiments. These technical progresses allow us to study not only the diversity on the chemical composition of the bilayers, but also the effect of small molecules or peptides to the membrane physical and structural properties. In addition, by means of AFM and AFM-FS we also characterize vesicular systems that are not composed by phospholipid molecules, which have a technological application: to act as nanocarriers for drug delivery.[cat] Les membranes biològiques (BMs) són fronteres autosegellants, que limiten les barreres permeables de les cèl·lules i els orgànuls i proporcionen els mitjans necessaris per compartir funcions. A part de ser crucials per l’estructura cel·lular, proporcionen una matriu de suport per a totes les proteïnes que es troben inserides a la cèl·lula, actuant com canals per l’intercanvi de massa, energia i informació amb l’exterior. Les BMs intervenen en moltes funcions biològiques, com el tràfic, la divisió cel·lular, l’endocitosi i l’exocitosi, que exigeixen canvis conformacionals durs en la membrana lipídica com la fusió, la fissió o el creixement de tubs. La correlació entre la composició i l’empaquetament dels lípid regeix les propietats fisicoquímiques de la membrana i la seva estructura mecànica
Considerant la complexa diversitat química de les BMs, sistemes de membranes model són utilitzats sovint per estudiar propietats de membrana. Degut a la heterogeneïtat de les BMs i la conseqüent necessitat de tècniques locals per a explorar BMs a escala nanomètrica, sistemes de bicapes suportades, com les bicapes de lípids suportades (SLBs), s’han proposat com models, ja que conserven l’ordre bidimensional i la mobilitat lateral, oferint ambients excel·lents per a la inserció de proteïnes de membrana.
Diversos informes demostren la gran varietat de tècniques útils per estudiar membranes lipídiques suportades i sense suport. Gràcies a la possibilitat de treballar sota un ambient controlat i amb una resolució nanomètrica en distància i força, la microscòpia de forces atòmiques (AFM) és, avui en dia, una tècnica ben establerta tant per a obtenir una imatge de la morfologia com per mesurar les propietats locals físiques i mecàniques de les SLBs mitjançant modes d’espectroscòpia de forces (AFM-FS). De totes formes, la resolució que s’obté amb l’AFM és inferior a la que es pot obtenir amb tècniques de raigs X (XR) i neutrons.
L’objectiu general d’aquesta tesis és investigar les propietats fisicoquímiques i estructurals de membranes lipídiques model combinant tècniques d’AFM, d’AFM-FS i de XR. També proposem metodologies avançades basades en AFM-FS i XR, així com l’acoblament de les dues tècniques per dur a terme experiments locals in situ. A més, amb AFM i AFM-FS també hem caracteritzat sistemes vesiculars que no contenen fosfolípids, els quals tenen una aplicació tecnològica: actuar com a nanotransportadors per al lliurament de fàrmacs
Impact of Galactosylceramide on the nanomechanical properties lipid bilayer models: AFM-force spectroscopy study
Galactosylceramides (GalCer) are glycosphingolipids bound to a monosaccharide group, responsible for inducing extensive hydrogen bonds that yield their alignment and accumulation in the outer leaflet of the biological membrane together with cholesterol (Chol) in rafts. In this work, the influence of GalCer on the nanomechanical properties of supported lipid bilayer (SLB) based on DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DLPC (1,2-didodecanoyl-sn-glycero-3-phosphocoline) as model systems was assesed. Phosphatidylcholine (PC):GalCer SLBs were characterized by means of differential scanning calorimetry (DSC) and atomic force microscopy (AFM), in both imaging and force spectroscopy (AFM-FS) modes. Comparing both PC systems, we determined that the behaviour of SLBs mixtures is governed by the PC phase-like state at the working temperature. While a phase segregated system is observed for DLPC:GalCer SLBs, GalCer is found to be dissolved in the DPPC SLBs for GalCer contents up to 20 mol %. In both systems, the incorporation of GalCer intensifies the nanomechanical properties of SLBs. Interestingly, segregated domains of exceptionally high mechanical stability are formed in DLPC:GalCer SLBs. Finally, the role of 20 mol % Chol in GalCer organization and function in the membranes was assessed. Both PC model systems displayed phase segregation and remarkable nanomechanical stability when GalCer and Chol coexist in the SLBs
Impact of Galactosylceramide on the nanomechanical properties lipid bilayer models: AFM-force spectroscopy study
Galactosylceramides (GalCer) are glycosphingolipids bound to a monosaccharide group, responsible for inducing extensive hydrogen bonds that yield their alignment and accumulation in the outer leaflet of the biological membrane together with cholesterol (Chol) in rafts. In this work, the influence of GalCer on the nanomechanical properties of supported lipid bilayer (SLB) based on DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DLPC (1,2-didodecanoyl-sn-glycero-3-phosphocoline) as model systems was assesed. Phosphatidylcholine (PC):GalCer SLBs were characterized by means of differential scanning calorimetry (DSC) and atomic force microscopy (AFM), in both imaging and force spectroscopy (AFM-FS) modes. Comparing both PC systems, we determined that the behaviour of SLBs mixtures is governed by the PC phase-like state at the working temperature. While a phase segregated system is observed for DLPC:GalCer SLBs, GalCer is found to be dissolved in the DPPC SLBs for GalCer contents up to 20 mol %. In both systems, the incorporation of GalCer intensifies the nanomechanical properties of SLBs. Interestingly, segregated domains of exceptionally high mechanical stability are formed in DLPC:GalCer SLBs. Finally, the role of 20 mol % Chol in GalCer organization and function in the membranes was assessed. Both PC model systems displayed phase segregation and remarkable nanomechanical stability when GalCer and Chol coexist in the SLBs
Impact of Galactosylceramide on the nanomechanical properties lipid bilayer models: AFM-force spectroscopy study
Galactosylceramides (GalCer) are glycosphingolipids bound to a monosaccharide group, responsible for inducing extensive hydrogen bonds that yield their alignment and accumulation in the outer leaflet of the biological membrane together with cholesterol (Chol) in rafts. In this work, the influence of GalCer on the nanomechanical properties of supported lipid bilayer (SLB) based on DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DLPC (1,2-didodecanoyl-sn-glycero-3-phosphocoline) as model systems was assesed. Phosphatidylcholine (PC):GalCer SLBs were characterized by means of differential scanning calorimetry (DSC) and atomic force microscopy (AFM), in both imaging and force spectroscopy (AFM-FS) modes. Comparing both PC systems, we determined that the behaviour of SLBs mixtures is governed by the PC phase-like state at the working temperature. While a phase segregated system is observed for DLPC:GalCer SLBs, GalCer is found to be dissolved in the DPPC SLBs for GalCer contents up to 20 mol %. In both systems, the incorporation of GalCer intensifies the nanomechanical properties of SLBs. Interestingly, segregated domains of exceptionally high mechanical stability are formed in DLPC:GalCer SLBs. Finally, the role of 20 mol % Chol in GalCer organization and function in the membranes was assessed. Both PC model systems displayed phase segregation and remarkable nanomechanical stability when GalCer and Chol coexist in the SLBs
Structure and Nanomechanics of Model Membranes by Atomic Force Microscopy and Spectroscopy: Insights into the Role of Cholesterol and Sphingolipids
Biological membranes mediate several biological processes that are directly associated with their physical properties but sometimes difficult to evaluate. Supported lipid bilayers (SLBs) are model systems widely used to characterize the structure of biological membranes. Cholesterol (Chol) plays an essential role in the modulation of membrane physical properties. It directly influences the order and mechanical stability of the lipid bilayers, and it is known to laterally segregate in rafts in the outer leaflet of the membrane together with sphingolipids (SLs). Atomic force microscope (AFM) is a powerful tool as it is capable to sense and apply forces with high accuracy, with distance and force resolution at the nanoscale, and in a controlled environment. AFM-based force spectroscopy (AFM-FS) has become a crucial technique to study the nanomechanical stability of SLBs by controlling the liquid media and the temperature variations. In this contribution, we review recent AFM and AFM-FS studies on the effect of Chol on the morphology and mechanical properties of model SLBs, including complex bilayers containing SLs. We also introduce a promising combination of AFM and X-ray (XR) techniques that allows for in situ characterization of dynamic processes, providing structural, morphological, and nanomechanical information
Structure and Nanomechanics of Model Membranes by Atomic Force Microscopy and Spectroscopy: Insights into the Role of Cholesterol and Sphingolipids
Biological membranes mediate several biological processes that are directly associated with their physical properties but sometimes difficult to evaluate. Supported lipid bilayers (SLBs) are model systems widely used to characterize the structure of biological membranes. Cholesterol (Chol) plays an essential role in the modulation of membrane physical properties. It directly influences the order and mechanical stability of the lipid bilayers, and it is known to laterally segregate in rafts in the outer leaflet of the membrane together with sphingolipids (SLs). Atomic force microscope (AFM) is a powerful tool as it is capable to sense and apply forces with high accuracy, with distance and force resolution at the nanoscale, and in a controlled environment. AFM-based force spectroscopy (AFM-FS) has become a crucial technique to study the nanomechanical stability of SLBs by controlling the liquid media and the temperature variations. In this contribution, we review recent AFM and AFM-FS studies on the effect of Chol on the morphology and mechanical properties of model SLBs, including complex bilayers containing SLs. We also introduce a promising combination of AFM and X-ray (XR) techniques that allows for in situ characterization of dynamic processes, providing structural, morphological, and nanomechanical information
Bioinspired Scaffolding by Supramolecular Amines Allows the Formation of One- and Two-Dimensional Silica Superstructures
Silica materials attract an increasing amount of interest in (fundamental) research, and find applications in, for example, sensing, catalysis, and drug delivery. As the properties of these (nano)materials not only depend on their chemistry but also their size, shape, and surface area, the controllable synthesis of silica is essential for tailoring the materials to specific applications. Advantageously, bioinspired routes for silica production are environmentally friendly and straightforward since the formation process is spontaneous and proceeds under mild conditions. These strategies mostly employ amine-bearing phosphorylated (bio)polymers. In this work, we expand this principle to supramolecular polymers based on the water-soluble cationic cyanine dye Pinacyanol acetate. Upon assembly in water, these dye molecules form large, polyaminated, supramolecular fibers. The surfaces of these fibers can be used as a scaffold for the condensation of silicic acid. Control over the ionic strength, dye concentration, and silicic acid saturation yielded silica fibers with a diameter of 25 nm and a single, 4 nm pore. Unexpectedly, other unusual superstructures, namely, nummulites and spherulites, are also observed depending on the ionic strength and dye concentration. Transmission and scanning electron microscopy (TEM and SEM) showed that these superstructures are formed by aligned silica fibers. Close examination of the dye scaffold prior silicification using small-angle X-ray scattering (SAXS), and UV/Vis spectroscopy revealed minor influence of the ionic strength and dye concentration on the morphology of the supramolecular scaffold. Total internal reflection fluorescence (TIRF) during silicification unraveled that if the reaction is kept under static conditions, only silica fibers are obtained. Experiments performed on the dye scaffold and silica superstructures evidenced that the marked structural diversity originates from the arrangement of silica/dye fibers. Under these mild conditions, external force fields can profoundly influence the morphology of the produced silica