by Keyword: bilayer

Woythe L, Porciani D, Harzing T, van Veen S, Burke DH, Albertazzi L, (2023). Valency and affinity control of aptamer-conjugated nanoparticles for selective cancer cell targeting Journal Of Controlled Release 355, 228-237

Nanoparticles (NPs) are commonly functionalized using targeting ligands to drive their selective uptake in cells of interest. Typical target cell types are cancer cells, which often overexpress distinct surface receptors that can be exploited for NP therapeutics. However, these targeted receptors are also moderately expressed in healthy cells, leading to unwanted off-tumor toxicities. Multivalent interactions between NP ligands and cell receptors have been investigated to increase the targeting selectivity towards cancer cells due to their non-linear response to receptor density. However, to exploit the multivalent effect, multiple variables have to be considered such as NP valency, ligand affinity, and cell receptor density. Here, we synthesize a panel of aptamer-functionalized silica-supported lipid bilayers (SSLB) to study the effect of valency, aptamer affinity, and epidermal growth factor receptor (EGFR) density on targeting specificity and selectivity. We show that there is an evident interplay among those parameters that can be tuned to increase SSLB selectivity towards high-density EGFR cells and reduce accumulation at non-tumor tissues. Specifically, the combination of high-affinity aptamers and low valency SSLBs leads to increased high-EGFR cell selectivity. These insights provide a better understanding of the multivalent interactions of NPs with cells and bring the nanomedicine field a step closer to the rational design of cancer nanotherapeutics.Copyright © 2023. Published by Elsevier B.V.

JTD Keywords: aptamer avidity and affinity, delivery, microscopy, multivalency, multivalent, nanoparticle targeting, silica -supported lipid bilayers, Aptamer avidity and affinity, Multivalency, Nanoparticle targeting, Silica-supported lipid bilayers, Supported lipid-bilayers, Tumor targeting

Avalos-Padilla Y, Georgiev VN, Ewins E, Robinson T, Orozco E, Lipowsky R, Dimova R, (2023). Stepwise remodeling and subcompartment formation in individual vesicles by three ESCRT-III proteins Iscience 26, 105765

The endosomal sorting complex required for transport (ESCRT) is a multi-protein machinery involved in several membrane remodeling processes. Different approaches have been used to resolve how ESCRT proteins scission membranes. However, the underlying mechanisms generating membrane deformations are still a matter of debate. Here, giant unilamellar vesicles, microfluidic technology, and micropipette aspiration are combined to continuously follow the ESCRT-III-mediated membrane remodeling on the single-vesicle level for the first time. With this approach, we identify different mechanisms by which a minimal set of three ESCRT-III proteins from Entamoeba histolytica reshape the membrane. These proteins modulate the membrane stiffness and spontaneous curvature to regulate bud size and generate intraluminal vesicles even in the absence of ATP. We demonstrate that the bud stability depends on the protein concentration and membrane tension. The approaches introduced here should open the road to diverse applications in synthetic biology for establishing artificial cells with several membrane compartments.© 2022 The Author(s).

JTD Keywords: bilayer, curvature, diffusion-coefficients, identification, membrane-scission, phase-diagram, reveals, sorting complex, structural basis, Biophysics, Biotechnology, Cell biology, Giant vesicles, Membranes

Joseph A, Wagner AM, Garay-Sarmiento M, Aleksanyan M, Haraszti T, Söder D, Georgiev VN, Dimova R, Percec V, Rodriguez-Emmenegger C, (2022). Zwitterionic Dendrimersomes: A Closer Xenobiotic Mimic of Cell Membranes Advanced Materials 34, 2206288

Building functional mimics of cell membranes is an important task toward the development of synthetic cells. So far, lipid and amphiphilic block copolymers are the most widely used amphiphiles with the bilayers by the former lacking stability while membranes by the latter are typically characterized by very slow dynamics. Herein, we introduce a new type of Janus dendrimer containing a zwitterionic phosphocholine hydrophilic headgroup (JDPC ) and a 3,5-substituted dihydrobenzoate-based hydrophobic dendron. JDPC self-assembles in water into zwitterionic dendrimersomes (z-DSs) that faithfully recapitulate the cell membrane in thickness, flexibility, and fluidity, while being resilient to harsh conditions and displaying faster pore closing dynamics in the event of membrane rupture. This enables the fabrication of hybrid DSs with components of natural membranes, including pore-forming peptides, structure-directing lipids, and glycans to create raft-like domains or onion vesicles. Moreover, z-DSs can be used to create active synthetic cells with life-like features that mimic vesicle fusion and motility as well as environmental sensing. Despite their fully synthetic nature, z-DSs are minimal cell mimics that can integrate and interact with living matter with the programmability to imitate life-like features and beyond. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

JTD Keywords: biological-membranes, bottom-up synthetic biology, chain, hybrid vesicles, hydroethidine, organization, polymersome, proteins, stability, synthetic cells, thickness, vesicle fusion, vesicle motility, vesicles, zwitterionic dendrimersomes, Biosensor, Biosensors, Bottom-up synthetic biology, Hybrid vesicles, Lipid-bilayers, Synthetic cells, Vesicle fusion, Vesicle motility, Zwitterionic dendrimersomes

Bar L, Perissinotto F, Redondo-Morata L, Giannotti MI, Goole J, Losada-Pérez P, (2022). Interactions of hydrophilic quantum dots with defect-free and defect containing supported lipid membranes Colloids And Surfaces B-Biointerfaces 210, 112239

Quantum dots (QDs) are semiconductor nanoparticles with unique optical and electronic properties, whose interest as potential nano-theranostic platforms for imaging and sensing is increasing. The design and use of QDs requires the understanding of cell-nanoparticle interactions at a microscopic and nanoscale level. Model systems such as supported lipid bilayers (SLBs) are useful, less complex platforms mimicking physico-chemical properties of cell membranes. In this work, we investigated the effect of topographical homogeneity of SLBs bearing different surface charge in the adsorption of hydrophilic QDs. Using quartz-crystal microbalance, a label-free surface sensitive technique, we show significant differences in the interactions of QDs onto homogeneous and inhomogeneous SLBs formed following different strategies. Within short time scales, QDs adsorb onto topographically homogeneous, defect-free SLBs is driven by electrostatic interactions, leading to no layer disruption. After prolonged QD exposure, the nanomechanical stability of the SLB decreases suggesting nanoparticle insertion. In the case of inhomogeneous, defect containing layers, QDs target preferentially membrane defects, driven by a subtle interplay of electrostatic and entropic effects, inducing local vesicle rupture and QD insertion at membrane edges. © 2021

JTD Keywords: adsorption, atomic force microscopy, bilayer formation, gold nanoparticles, hydrophilic quantum dots, lipid membrane defects, model, nanomechanics, quartz crystal microbalance with dissipation, size, supported lipid bilayers, surfaces, Atomic force microscopy, Atomic-force-microscopy, Cytology, Defect-free, Electronic properties, Electrostatics, Hydrophilic quantum dot, Hydrophilic quantum dots, Hydrophilicity, Hydrophilics, Lipid bilayers, Lipid membrane defect, Lipid membrane defects, Lipid membranes, Lipids, Nanocrystals, Nanomechanics, Optical and electronic properties, Quartz, Quartz crystal microbalance with dissipation, Quartz crystal microbalances, Quartz-crystal microbalance, Semiconductor nanoparticles, Semiconductor quantum dots, Supported lipid bilayers

Tozzi, C., Walani, N., Arroyo, M., (2019). Out-of-equilibrium mechanochemistry and self-organization of fluid membranes interacting with curved proteins New Journal of Physics 21, (9), 093004

The function of biological membranes is controlled by the interaction of the fluid lipid bilayer with various proteins, some of which induce or react to curvature. These proteins can preferentially bind or diffuse towards curved regions of the membrane, induce or stabilize membrane curvature and sequester membrane area into protein-rich curved domains. The resulting tight interplay between mechanics and chemistry is thought to control organelle morphogenesis and dynamics, including traffic, membrane mechanotransduction, or membrane area regulation and tension buffering. Despite all these processes are fundamentally dynamical, previous work has largely focused on equilibrium and a self-consistent theoretical treatment of the dynamics of curvature sensing and generation has been lacking. Here, we develop a general theoretical and computational framework based on a nonlinear Onsager's formalism of irreversible thermodynamics for the dynamics of curved proteins and membranes. We develop variants of the model, one of which accounts for membrane curving by asymmetric crowding of bulky off-membrane protein domains. As illustrated by a selection of test cases, the resulting governing equations and numerical simulations provide a foundation to understand the dynamics of curvature sensing, curvature generation, and more generally membrane curvature mechano-chemistry.

JTD Keywords: Curvature generation, Curvature sensing, Lipid bilayers, Membrane proteins

Gumí-Audenis, B., Giannotti, M. I., (2019). Structural and mechanical characterization of supported model membranes by AFM Biomimetic Lipid Membranes: Fundamentals, Applications, and Commercialization (ed. Kök, Fatma N., Arslan Yildiz, Ahu, Inci, Fatih), Springer International Publishing (Cham, Germany) , 1-27

Several cellular processes, including adhesion, signaling and transcription, endocytosis, and membrane resealing, among others, involve conformational changes such as bending, vesiculation, and tubulation. These mechanisms generally involve membrane separation from the cytoskeleton as well as strong bending, for which the membrane chemical composition and physicochemical properties, often highly localized and dynamic, are key players. The mechanical role of the lipid membrane in force triggered (or sensing) mechanisms in cells is important, and understanding the lipid bilayers’ physical and mechanical properties is essential to comprehend their contribution to the overall membrane. Atomic force microscopy (AFM)-based experimental approaches have been to date very valuable to deepen into these aspects. As a stand-alone, high-resolution imaging technique and force transducer with the possibility to operate in aqueous environment, it defies most other surface instrumentation in ease of use, sensitivity and versatility. In this chapter, we introduce the different AFM-based methods to assess topological and nanomechanical information on model membranes, specifically to supported lipid bilayers (SLBs), including several examples ranging from pure phospholipid homogeneous bilayers to multicomponent and phase-separated SLBs, increasing the bilayer complexity, in the direction of mimicking biological membranes.

JTD Keywords: Atomic force microscopy, Force spectroscopy, Model membranes, Nanomechanics, Supported lipid bilayers

Crespo-Villanueva, Adrián, Gumí-Audenis, Berta, Sanz, Fausto, Artzner, Franck, Mériadec, Cristelle, Rousseau, Florence, Lopez, Christelle, Giannotti, M. I., Guyomarc'h, Fanny, (2018). Casein interaction with lipid membranes: Are the phase state or charge density of the phospholipids affecting protein adsorption? Biochimica et Biophysica Acta (BBA) - Biomembranes 1860, (12), 2588-2598

Casein micelles are ~200 nm electronegative particles that constitute 80 wt% of the milk proteins. During synthesis in the lactating mammary cells, caseins are thought to interact in the form of ~20 nm assemblies, directly with the biological membranes of the endoplasmic reticulum and/or the Golgi apparatus. However, conditions that drive this interaction are not yet known. Atomic force microscopy imaging and force spectroscopy were used to directly observe the adsorption of casein particles on supported phospholipid bilayers with controlled compositions to vary their phase state and surface charge density, as verified by X-ray diffraction and zetametry. At pH 6.7, the casein particles adsorbed onto bilayer phases with zwitterionic and liquid-disordered phospholipid molecules, but not on phases with anionic or ordered phospholipids. Furthermore, the presence of adsorbed caseins altered the stability of the yet exposed bilayer. Considering their respective compositions and symmetry/asymmetry, these results cast light on the possible interactions of casein assemblies with the organelles’ membranes of the lactating mammary cells.

JTD Keywords: Casein proteins, Phospholipid membrane, Supported lipid bilayer, Atomic force microscopy

Gumí-Audenis, Berta, Costa, Luca, Carlá, Francesco, Comin, Fabio, Sanz, Fausto, Giannotti, M. I., (2016). Structure and nanomechanics of model membranes by atomic force microscopy and spectroscopy: Insights into the role of cholesterol and sphingolipids Membranes , 6, (4), 58

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

JTD Keywords: Atomic force microscopy, Force spectroscopy, Lipid membranes, Supported lipid bilayers, Nanomechanics, Cholesterol, Sphingolipids, Membrane structure, XR-AFM combination

Rajzer, I., Menaszek, E., Kwiatkowski, R., Planell, J. A., Castaño, O., (2014). Electrospun gelatin/poly(ε-caprolactone) fibrous scaffold modified with calcium phosphate for bone tissue engineering Materials Science and Engineering: C 44, 183-190

In this study gelatin (Gel) modified with calcium phosphate nanoparticles (SG5) and polycaprolactone (PCL) were used to prepare a 3D bi-layer scaffold by collecting electrospun PCL and gelatin/SG5 fibers separately in the same collector. The objective of this study was to combine the desired properties of PCL and Gel/SG5 in the same scaffold in order to enhance mineralization, thus improving the ability of the scaffold to bond to the bone tissue. The scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and the wide angle X-ray diffraction (WAXD) measurements confirmed that SG5 nanoparticles were successfully incorporated into the fibrous gelatin matrix. The composite Gel/SG5/PCL scaffold exhibited more enhanced mechanical properties than individual Gel and Gel/SG5 scaffolds. The presence of SG5 nanoparticles accelerated the nucleation and growth of apatite crystals on the surface of the composite Gel/SG5/PCL scaffold in simulated body fluid (SBF). The osteoblast response in vitro to developed electrospun scaffolds (PCL and Gel/SG5/PCL) was investigated by using normal human primary NHOst cell lines. NHOst cell culture studies showed that higher alkaline phosphatase (ALP) activity and better mineralization were obtained in the case of composite materials than in pure PCL scaffolds. The mechanically strong PCL scaffold served as a skeleton, while the Gel/SG5 fibers facilitated cell spreading and mineralization of the scaffold.

JTD Keywords: Bilayer fibrous scaffold, Ceramic nanoparticles, Electrospinning, Gelatin, Polycaprolactone, Biomechanics, Bone, Calcium phosphate, Cell culture, Electrospinning, Fourier transform infrared spectroscopy, Mechanical properties, Mineralogy, Nanoparticles, Phosphatases, Polycaprolactone, Scanning electron microscopy, X ray diffraction, Polycaprolactone, Alkaline phosphatase activity, Bone tissue engineering, Calcium phosphate nanoparticles, Ceramic nanoparticles, Fibrous scaffolds, Gelatin, Simulated body fluids, Wide-angle x-ray diffraction, Electrospuns, Scaffolds (biology), Electrospinning

Redondo-Morata, L., Giannotti, M. I., Sanz, F., (2014). Structural impact of cations on lipid bilayer models: Nanomechanical properties by AFM-force spectroscopy Molecular Membrane Biology , 31, (1), 17-28

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.

JTD Keywords: Atomic force microscopy, Cations, Force spectroscopy, Lipid bilayer, Mechanical stability

Hoyo, J., Guaus, E., Oncins, G., Torrent-Burgués, J., Sanz, F., (2013). Incorporation of Ubiquinone in supported lipid bilayers on ITO Journal of Physical Chemistry B , 117, (25), 7498-7506

Ubiquinone (UQ) is one of the main electron and proton shuttle molecules in biological systems, and dipalmitoylphosphatidylcholine (DPPC) is one of the most used model lipids. Supported planar bilayers (SPBs) are extensively accepted as biological model membranes. In this study, SPBs have been deposited on ITO, which is a semiconductor with good electrical and optical features. Specifically, topographic atomic force microscopy (AFM) images and force curves have been performed on SPBs with several DPPC:UQ ratios to study the location and the interaction of UQ in the SPB. Additionally, cyclic voltammetry has been used to understand the electrochemical behavior of DPPC:UQ SPBs. Obtained results show that, in our case, UQ is placed in two main different positions in SPBs. First, between the DPPC hydrophobic chains, fact that originates a decrease in the breakthrough force of the bilayer, and the second between the two leaflets that form the SPBs. This second position occurs when increasing the UQ content, fact that eventually forms UQ aggregates at high concentrations. The formation of aggregates produces an expansion of the SPB average height and a bimodal distribution of the breakthrough force. The voltammetric response of UQ depends on its position on the bilayer.

JTD Keywords: Bimodal distribution, Biological models, Dipalmitoyl phosphatidylcholine, Electrochemical behaviors, Hydrophobic chains, Supported lipid bilayers, Supported planar bilayers, Voltammetric response

Redondo-Morata, Lorena, Oncins, Gerard, Sanz, Fausto, (2012). Force spectroscopy reveals the effect of different ions in the nanomechanical behavior of phospholipid model membranes: The case of potassium cation Biophysical Journal , 102, (1), 66-74

How do metal cations affect the stability and structure of phospholipid bilayers? What role does ion binding play in the insertion of proteins and the overall mechanical stability of biological membranes? Investigators have used different theoretical and microscopic approaches to study the mechanical properties of lipid bilayers. Although they are crucial for such studies, molecular-dynamics simulations cannot yet span the complexity of biological membranes. In addition, there are still some experimental difficulties when it comes to testing the ion binding to lipid bilayers in an accurate way. Hence, there is a need to establish a new approach from the perspective of the nanometric scale, where most of the specific molecular phenomena take place. Atomic force microscopy has become an essential tool for examining the structure and behavior of lipid bilayers. In this work, we used force spectroscopy to quantitatively characterize nanomechanical resistance as a function of the electrolyte composition by means of a reliable molecular fingerprint that reveals itself as a repetitive jump in the approaching force curve. By systematically probing a set of bilayers of different composition immersed in electrolytes composed of a variety of monovalent and divalent metal cations, we were able to obtain a wealth of information showing that each ion makes an independent and important contribution to the gross mechanical resistance and its plastic properties. This work addresses the need to assess the effects of different ions on the structure of phospholipid membranes, and opens new avenues for characterizing the (nano)mechanical stability of membranes.

JTD Keywords: Molecular-dynamics simulation, Liquid expanded monolayers, Lipid-bilayers, Hofmeister series, Monovalent salt, Phosphatidylcholine, Microscopy, Binding, Surfaces, NaCl

Redondo, L., Giannotti, M. I., Sanz, F., (2012). Stability of lipid bilayers as model membranes: Atomic force microscopy and spectroscopy approach Atomic force microscopy in liquid (ed. Baró, A. M., Reifenberger, R. G.), Wiley-VCH Verlag GmbH & Co.KGaA (Weinheim, Germany) Part I: General Atomic Force Microscopy, 259-284

Garcia-Manyes, S., Redondo-Morata, L., Oncins, G., Sanz, F., (2010). Nanomechanics of lipid bilayers: Heads or tails? Journal of the American Chemical Society American Chemical Society 132, (37), 12874-12886

Understanding the effect of mechanical stress on membranes is of primary importance in biophysics. Here we use force spectroscopy AFM to quantitatively characterize the nanomechanical stability of supported lipid bilayers as a function of their chemical composition. The onset of plastic deformation reveals itself as a repetitive jump in the approaching force curve, which represents a molecular fingerprint for the bilayer mechanical stability. By systematically probing a set of chemically distinct supported lipid bilayers (SLBs), we first show that both the headgroup and tail have a decisive effect on their mechanical properties. While the mechanical stability of the probed SLBs linearly increases by 3.3 nN upon the introduction of each additional -CH2- in the chain, it exhibits a significant dependence on the phospholipid headgroup, ranging from 3 nN for DPPA to 66 nN for DPPG. Furthermore, we also quantify the reduction of the membrane mechanical stability as a function of the number of unsaturations and molecular branching in the chemical structure of the apolar tails. Finally, we demonstrate that, upon introduction of cholesterol and ergosterol, contrary to previous belief the mechanical stability of membranes not only increases linearly in the liquid phase (DLPC) but also for phospholipids present in the gel phase (DPPC). Our results are discussed in the framework of the continuum nucleation model. This work highlights the compelling effect of subtle variations in the chemical structure of phospholipid molecules on the membrane response when exposed to mechanical forces, a mechanism of common occurrence in nature.

JTD Keywords: Atomic-force microscopy, Molecular-dynamics simulation, Aqueous-electrolyte solutions, Supported planar membranes, Phospholipid-bilayers, Biological-membranes, Physical-properties, Fluid membranes, Model membranes, Chain-length

Garcia-Manyes, S., Sanz, F., (2010). Nanomechanics of lipid bilayers by force spectroscopy with AFM: A perspective Biochimica et Biophysica Acta - Biomembranes , 1798, (4), 741-749

Lipid bilayers determine the architecture of cell membranes and regulate a myriad of distinct processes that are highly dependent on the lateral organization of the phospholipid molecules that compose the membrane. Indeed, the mechanochemical properties of the membrane are strongly correlated with the function of several membrane proteins, which demand a very specific, highly localized physicochemical environment to perform their function. Several mesoscopic techniques have been used in the past to investigate the mechanical properties of lipid membranes. However, they were restricted to the study of the ensemble properties of giant bilayers. Force spectroscopy with AFM has emerged as a powerful technique able to provide valuable insights into the nanomechanical properties of supported lipid membranes at the nanometer/nanonewton scale in a wide variety of systems. In particular, these measurements have allowed direct measurement of the molecular interactions arising between neighboring phospholipid molecules and between the lipid molecules and the surrounding solvent environment. The goal of this review is to illustrate how these novel experiments have provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Here we report in detail the main discoveries achieved by force spectroscopy with AFM on supported lipid bilayers, and we also discuss on the exciting future perspectives offered by this growing research field.

JTD Keywords: Force spectroscopy, Atomic force microscopy, Lipid bilayer, Nanomechanics

Nussio, M. R., Oncins, G., Ridelis, I., Szili, E., Shapter, J. G., Sanz, F., Voelcker, N. H., (2009). Nanomechanical characterization of phospholipid bilayer islands on flat and porous substrates: A force spectroscopy study Journal of Physical Chemistry B , 113, (30), 10339-10347

In this study, we compare for the first time the nanomechanical properties of lipid bilayer islands on flat and porous surfaces. 1,2-Dimyzistoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayers were deposited on flat (silicon and mica) and porous silicon (pSi) substrate surfaces and examined using atomic force spectroscopy and force volume imaging. Force spectroscopy measurements revealed the effects of the underlying substrate and of the lipid phase on the nanomechanical properties of bilayers islands. For mica and silicon, significant differences in breakthrough force between the center and the edges of bilayer islands were observed for both phospolipids. These differences were more pronounced for DMPC than for DPPC, presumably due to melting effects at the edges of DMPC bilayers. In contrast, bilayer islands deposited on pSi yielded similar breakthrough forces in the central region and along the perimeter of the islands, and those values in turn were similar to those measured along the perimeter of bilayer islands deposited on the flat substrates. The study also demonstrates that pSi is suitable solid support for the formation of pore-spanning phospholipid bilayers with potential applications in transmembrane protein studies, drug delivery, and biosensing.

JTD Keywords: Black lipid-membranes, Gold surfaces, Supported bilayers, Channel activity, Micro-BLMS, Silicon, Proteins, Vesicles, AFM, Temperature measurement

Bravo, R., Arimon, M., Valle-Delgado, J. J., Garcia, R., Durany, N., Castel, S., Cruz, M., Ventura, S., Fernàndez-Busquets, X., (2008). Sulfated polysaccharides promote the assembly of amyloid beta(1-42) peptide into stable fibrils of reduced cytotoxicity Journal of Biological Chemistry , 283, (47), 32471-32483

The histopathological hallmarks of Alzheimer disease are the self-aggregation of the amyloid beta peptide (A beta) in extracellular amyloid fibrils and the formation of intraneuronal Tau filaments, but a convincing mechanism connecting both processes has yet to be provided. Here we show that the endogenous polysaccharide chondroitin sulfate B (CSB) promotes the formation of fibrillar structures of the 42-residue fragment, A beta(1-42). Atomic force microscopy visualization, thioflavin T fluorescence, CD measurements, and cell viability assays indicate that CSB-induced fibrils are highly stable entities with abundant beta-sheet structure that have little toxicity for neuroblastoma cells. We propose a wedged cylinder model for A beta(1-42) fibrils that is consistent with the majority of available data, it is an energetically favorable assembly that minimizes the exposure of hydrophobic areas, and it explains why fibrils do not grow in thickness. Fluorescence measurements of the effect of different A beta(1-42) species on Ca2+ homeostasis show that weakly structured nodular fibrils, but not CSB-induced smooth fibrils, trigger a rise in cytosolic Ca2+ that depends on the presence of both extracellular and intracellular stocks. In vitro assays indicate that such transient, local Ca2+ increases can have a direct effect in promoting the formation of Tau filaments similar to those isolated from Alzheimer disease brains.

JTD Keywords: AFM, Alzheimers-disease, Chondroitin sulfate, Heparan-sulfate, Lipid-bilayers, Beta-peptide, In-vitro, Neurodegenerative diseases, Extracellular-matrix, Prion protein

Oncins, G., Torrent-Burgues, J., Sanz, F., (2008). Nanomechanical properties of arachidic acid Langmuir-Blodgett films Journal of Physical Chemistry C 112, (6), 1967-1974

The nanomechanical properties of Langmuir-Blodgett monolayers of arachidic acid extracted at surface pressures of 1, 15, and 35 mN/m and deposited on mica were investigated by atomic force microscopy, force spectroscopy, and lateral force microscopy. It was experimentally demonstrated that the arachidic acid molecular orientation depends on the extraction pressure. According to this, tilting angles of 50, 34, and 22 degrees with respect to the surface perpendicular were detected and identified as conformations that maximize van der Waals interactions between the arachidic acid alkyl chains. The vertical force needed to puncture the monolayers with the AFM tip strongly depends on the molecular tilting angles attained at different monolayer extraction surface pressures, obtaining values that range from 13.07 +/- 3.24 nN for 50 degrees to 22.94 +/- 5.49 nN for 22 degrees tilting angles. The different molecular interactions involved in the monolayer cohesion are discussed and quantitatively related to the experimental monolayer breakthrough forces. The friction measurements performed from low vertical forces up to monolayer disruption reveal the existence of three well-defined regimes: first, a low friction response due to the elastic deformation of the monolayer, which is followed by a sharp increase in the friction force due to the onset of a sudden plastic deformation. The last regime corresponds to the monolayer rupture and the contact between tip and substrate. The friction coefficient of the substrate is seen to depend on the monolayer extraction pressure, a fact that is discussed in terms of the relationship between the sample compactness and its rupture mechanism.

JTD Keywords: AFM, SAM, Reflection-absortion spectroscopy, Lipid-bilayers, Frictional-properies, Molecular-structure, Thermal behavior, Nanometer-scale, Chain-length, LB films

Domènech, Ò., Morros, A., Cabañas, M. E., Teresa Montero, M., Hernéndez-Borrell, J., (2007). Supported planar bilayers from hexagonal phases Biochimica et Biophysica Acta - Biomembranes , 1768, (1), 100-106

In this work the presence of inverted hexagonal phases HII of 1-palmitoy-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and cardiolipin (CL) (0.8:0.2, mol/mol) in the presence of Ca2+ were observed via 31P-NMR spectroscopy. When suspensions of the same composition were extended onto mica, HII phases transformed into structures which features are those of supported planar bilayers (SPBs). When characterized by atomic force microscopy (AFM), the SPBs revealed the existence of two laterally segregated domains (the interdomain height being ∼ 1 nm). Cytochrome c (cyt c), which binds preferentially to acidic phospholipids like CL, was used to demonstrate the nature of the domains. We used 1-anilinonaphtalen-8-sulfonate (ANS) to demonstrate that in the presence of cyt c, the fluorescence of ANS decreased significantly in lamellar phases. Conversely, the ANS binding to HII phases was negligible. When cyt c was injected into AFM fluid imaging cells, where SPBs of POPE:CL had previously formed poorly defined structures, protein aggregates (∼ 100 nm diameter) were ostensibly observed only on the upper domains, which suggests not only that they are mainly formed by CL, but also provides evidence of bilayer formation from HII phases. Furthermore, a model for the nanostructure of the SPBs is herein proposed.

JTD Keywords: 31P-NMR, AFM, ANS fluorescence, Cytochrome c (cyt c), Hexagonal phase (HII), Liposome, Supported planar bilayers (SPBs)