Abeta42-peptide assembly on lipid bilayers

One of the major pathological features of Alzheimer's disease (AD) is the presence of extracellular amyloid plaques that are composed predominantly of the amyloid-beta peptide (Abeta). Diffuse plaques associated with AD are composed predominantly of Abeta42, whereas senile plaques contain both Abeta40 and Abeta42. Recently, it has been suggested that diffuse plaque formation is initiated as a plasma membrane-bound Abeta species and that Abeta42 is the critical component. In order to investigate this hypothesis, we have examined Abeta42-membrane interactions using in situ atomic force microscopy and fluorescence spectroscopy. Our studies demonstrate the association of Abeta42 with planar bilayers composed of total brain lipids, which results initially in peptide aggregation and then fibre formation. Modulation of the cholesterol content is correlated with the extent of Abeta42-assembly on the bilayer surface. Although Abeta42 was not visualized directly on cholesterol-depleted bilayers, fluorescence anisotropy and fluorimetry demonstrate Abeta42-induced membrane changes. Our results demonstrate that the composition of the lipid bilayer governs the outcome of Abeta interactions.     

Alternate aggregation pathways of the Alzheimer beta-amyloid peptide: Abeta association kinetics at endosomal pH

The deposition of beta-amyloid peptide (Abeta) fibrils around neurons is an invariable feature of Alzheimer's disease and there is increasing evidence that fibrillar deposits and/or prefibrillar intermediates play a central role in the observed neurodegeneration. One site of Abeta generation is the endosomes, and we have investigated the kinetics of Abeta association at endosomal pH over physiologically relevant time frames. We have identified three distinct Abeta association phases that occur at rates comparable to endosomal transit times. Rapid formation of burst phase aggregates, larger than 200nm, was observed within 15 seconds. Two slower association phases were detected by fluorescence resonance energy transfer and termed phase 1 and phase 2 aggregation reactions. At 20 microM Abeta, pH 6, the half lives of the phase 1 and phase 2 aggregation phases were 3.15 minutes and 17.66 minutes, respectively. Atomic force microscopy and dynamic light scattering studies indicate that the burst phase aggregate is large and amorphous, while phase 1 and 2 aggregates are spherical with hydrodynamic radii around 30 nm. There is an apparent equilibrium, potentially mediated through a soluble Abeta intermediate, between the large burst phase aggregates and phase 1 and 2 spherical particles. The large burst phase aggregates form quickly, however, they disappear as the equilibrium shifts toward the spherical aggregates. These aggregated species do not contain alpha-helical or beta-structure as determined by circular dichroism spectroscopy. However, after two weeks beta-structure is observed and is attributable to the insoluble portion of the sample. After two months, mature amyloid fibrils appear and the spherical aggregates are significantly diminished.     

Secondary structure of alpha-synuclein oligomers: characterization by raman and atomic force microscopy

Formation of alpha-synuclein aggregates is proposed to be a crucial event in the pathogenesis of Parkinson's disease. Large soluble oligomeric species are observed as probable intermediates during fibril formation and these, or related aggregates, may constitute the toxic element that triggers neurodegeneration. Unfortunately, there is a paucity of information regarding the structure and composition of these oligomers. Here, the morphology and the conformational characteristics of the oligomers and filaments are investigated by a combined atomic force microscopy (AFM) and Raman microscopic approach on a common mica surface. AFM showed that in vitro early stage oligomers were globular with variable heights, while prolonged incubation caused the oligomers to become elongated as protofilaments. The height of the subsequently formed alpha-synuclein filaments was similar to that of the protofilaments. Analysis of the Raman amide I band profiles of the different alpha-synuclein oligomers establishes that the spheroidal oligomers contain a significant amount of alpha-helical secondary structure (47%), which decreases to about 37% in protofilaments. At the same time, when protofilaments form, beta-sheet structure increases to about 54% from the approximately 29% observed in spheroidal oligomers. Upon filament formation, the major conformation is beta-sheet (66%), confirmed by narrowing of the amide I band and the profile maximum shifting to 1667 cm(-1). The accumulation of spheroidal oligomers of increasing size but unchanged vibrational spectra during the fibrillization process suggests that a cooperative conformational change may contribute to the kinetic control of fibrillization.     

Monitoring biomolecular interactions by time-lapse atomic force microscopy

The atomic force microscope (AFM) is a unique imaging tool that enables the tracking of single macromolecule events in response to physiological effectors and pharmacological stimuli. Direct correlation can therefore be made between structural and functional states of individual biomolecules in an aqueous environment. This review explores how time-lapse AFM has been used to learn more about normal and disease-associated biological processes. Three specific examples have been chosen to illustrate the capabilities of this technique. In the cell, actin polymerizes into filaments, depolymerizes, and undergoes interactions with numerous effector molecules (i.e., severing, capping, depolymerizing, bundling, and cross-linking proteins) in response to many different stimuli. Such events are critical for the function and maintenance of the molecular machinery of muscle contraction and the dynamic organization of the cytoskeleton. One goal is to use time-lapse AFM to examine and manipulate some of these events in vitro, in order to learn more about how these processes occur in the cell. Aberrant protein polymerization into amyloid fibrils occurs in a multitude of diseases, including Alzheimer's and type 2 diabetes. Local amyloid deposits may cause organ dysfunction and cell death; hence, it is of interest to learn how to interfere with fibril formation. One application of time-lapse AFM in this area has been the direct visualization of amyloid fibril growth in vitro. This experimental approach holds promise for the future testing of potential therapeutic drugs, for example, by directly visualizing at which level of fibril assembly (i.e., nucleation, elongation, branching, or lateral association of protofibrils) a given active compound will interfere. Nuclear pore complexes (NPCs) are large supramolecular assemblies embedded in the nuclear envelope. Transport of ions, small molecules, proteins, RNAs, and RNP particles in and out of the nucleus occurs via NPCs. Time-lapse AFM has been used to structurally visualize the response of individual NPC particles to various chemical and physical effectors known to interfere with nucleocytoplasmic transport. Taken together, such time-lapse AFM studies could provide novel insights into the molecular mechanisms of fundamental biological processes under both normal and pathological conditions at the single molecule level.     

High-resolution atomic force microscopy visualization of metalloproteins and their complexes

Background

Metalloproteins myeloperoxidase (MPO), ceruloplasmin (CP) and lactoferrin (LF) play an important role in regulation of inflammation and oxidative stress in vertebrates. It was previously shown that these proteins may work synergetically as antimicrobial and anti-inflammatory agents by forming complexes, such as MPO-CP and LF-CP. However, interaction of metalloprotein molecules with each other has never been characterized at a single-molecule level.

原子力显微技术在酶学研究中的应用

酶在生物体的生命活动中占有及其重要的地位,机体功能的和谐统一有赖于酶的作用。原子力显微技术(AFM)作为一门新发展起来的技术,为人们认识酶的结构与功能提供了又一新的窗口。AFM能够在生理条件下对生物样品进行三维成像,在分子水平上实时监测生理生化反应。AFM还能够在皮牛顿精度上测定分子间作用力。目前,AFM已用于单分子酶的化学性质及其作用原理的研究。本简述AFM在酶学中的应用情况。     

Force spectroscopy reveals the presence of structurally modified dimers in transthyretin amyloid annular oligomers

Toxicity in amyloidogenic protein misfolding disorders is thought to involve intermediate states of aggregation associated with the formation of amyloid fibrils. Despite their relevance, the heterogeneity and transience of these oligomers have placed great barriers in our understanding of their structural properties. Among amyloid intermediates, annular oligomers or annular protofibrils have raised considerable interest because they may contribute to a mechanism of cellular toxicity via membrane permeation. Here we investigated, by using AFM force spectroscopy, the structural detail of amyloid annular oligomers from transthyretin (TTR), a protein involved in systemic and neurodegenerative amyloidogenic disorders. Manipulation was performed in situ , in the absence of molecular handles and using persistence length‐fit values to select relevant curves. Force curves reveal the presence of dimers in TTR annular oligomers that unfold via a series of structural intermediates. This is in contrast with the manipulation of native TTR that was more often manipulated over length scales compatible with a TTR monomer and without unfolding intermediates. Imaging and force spectroscopy data suggest that dimers are formed by the assembly of monomers in a head‐to‐head orientation with a nonnative interface along their β‐strands. Furthermore, these dimers stack through nonnative contacts that may enhance the stability of the misfolded structure.     

High-speed atomic force microscopy: Imaging and force spectroscopy

Atomic force microscopy (AFM) is the type of scanning probe microscopy that is probably best adapted for imaging biological samples in physiological conditions with submolecular lateral and vertical resolution. In addition, AFM is a method of choice to study the mechanical unfolding of proteins or for cellular force spectroscopy. In spite of 28 years of successful use in biological sciences, AFM is far from enjoying the same popularity as electron and fluorescence microscopy. The advent of high-speed atomic force microscopy (HS-AFM), about 10 years ago, has provided unprecedented insights into the dynamics of membrane proteins and molecular machines from the single-molecule to the cellular level. HS-AFM imaging at nanometer-resolution and sub-second frame rate may open novel research fields depicting dynamic events at the single bio-molecule level. As such, HS-AFM is complementary to other structural and cellular biology techniques, and hopefully will gain acceptance from researchers from various fields. In this review we describe some of the most recent reports of dynamic bio-molecular imaging by HS-AFM, as well as the advent of high-speed force spectroscopy (HS-FS) for single protein unfolding.     

Ultrastable atomic force microscopy: Improved force and positional stability

Atomic force microscopy (AFM) is an exciting technique for biophysical studies of single molecules, but its usefulness is limited by instrumental drift. We dramatically reduced positional drift by adding two lasers to track and thereby actively stabilize the tip and the surface. These lasers also enabled label-free optical images that were spatially aligned to the tip position. Finally, sub-pN force stability over 100 s was achieved by removing the gold coating from soft cantilevers. These enhancements to AFM instrumentation can immediately benefit research in biophysics and nanoscience.     

原子力显微镜单分子力谱研究生物分子间相互作用

原子力显微镜单分子力谱是近年来发展起来的能在单分子水平研究生物分子相互作用的新工具。本文综述了单分子力谱的测定原理、方法及其在研究蛋白．蛋白、蛋白-DNA相互作用,蛋白质去折叠和活细胞上配体／受体结合中的应用进展。     

Functionalization of amino-modified probes for atomic force microscopy

The probes for atomic force microscopy (AFM) functionalized with bovine serum albumin (BSA) were obtained; they can be used for molecular recognition studies. The procedure of modification and functionalization of the AFM probe included three stages. First, amino probes were obtained by modification in vapors of an amino silane derivative. Then, a covalent bond was formed between the surface amino groups of the probe and a homobifunctional aminoreactive crosslinker. Finally, the probe with a covalently attached crosslinker was functionalized with BSA molecules. The AFM probes were characterized by force measurements at different stages of the modification; the adhesion force and the work of adhesion force were determined. The modification process was confirmed by visualization of BSA and supercoiled pGEMEX DNA molecules immobilized on the standard amino mica and on amino mica modified with a crosslinker.     

原子力显微技术成像在生物医学中的应用

原子力显微技术利用探针尖端与标本之间相互作用的力场对标本进行三维成像。这种成像可在生理条件下进行 ,可进行动态观察和标本容易制备是有别于其它成像技术如电子显微镜成像等的特点。对于细胞和生物大分子 ,能够在生理条件下成像具有重要意义。它意味着人们在认识生命本质的方法学方面 ,又向前迈出了新的一步。本文简要综述对细胞和生物大分子的成像在生物医学方面的应用。     

Quasi-linear viscoelastic properties of costal cartilage using atomic force microscopy

Costal cartilage (CC) is one of the load-bearing tissues of the rib cage. Literature on material characterisation of the CC is limited. Atomic force microscopy (AFM) has been extremely successful in characterising the elastic properties of soft biomaterials such as articular cartilage and hydrogels, which are often the material of choice for cartilage models. But AFM data on CC are absent in the literature. In this study, AFM indentations using spherical beaded tips were performed on human CC to isolate the mechanical properties. A novel method was developed for modelling the relaxation indentation experiments based on Fung's quasi-linear viscoelasticity and a continuous relaxation spectrum. This particular model has been popular for uniaxial compression test data analysis. Using the model, the mean Young's modulus of CC was found to be about 2.17, 4.11 and 5.49 MPa for three specimens. A large variation of modulus was observed over the tissue. Also, the modulus values decreased with distance from the costochondral junction.     

Unique physicochemical profile of beta-amyloid peptide variant Abeta1-40E22G protofibrils: conceivable neuropathogen in arctic mutant carriers

A new early-onset form of Alzheimer's disease (AD) was described recently where a point mutation was discovered in codon 693 of the beta-amyloid (Abeta) precursor protein gene, the Arctic mutation. The mutation translates into a single amino acid substitution, glutamic acid-->glycine, in position 22 of the Abeta peptide. The mutation carriers have lower plasma levels of Abeta than normal, while in vitro studies show that Abeta1-40E22G protofibril formation is significantly enhanced. We have explored the nature of the Abeta1-40E22G peptide in more detail, in particular the protofibrils. Using size-exclusion chromatography (SEC) and circular dichroism spectroscopy (CD) kinetic and secondary structural characteristics were compared with other Abeta1-40 peptides and the Abeta12-28 fragment, all having single amino acid substitutions in position 22. We have found that Abeta1-40E22G protofibrils are a group of comparatively stabile beta-sheet-containing oligomers with a heterogeneous size distribution, ranging from >100 kDa to >3000 kDa. Small Abeta1-40E22G protofibrils are generated about 400 times faster than large ones. Salt promotes their formation, which significantly exceeds all the other peptides studied here, including the Dutch mutation Abeta1-40E22Q. Position 22 substitutions had significant effects on aggregation kinetics of Abeta1-40 and in Abeta12-28, although the qualitative aspects of the effects differed between the native peptide and the fragment, as no protofibrils were formed by the fragments. The rank order of protofibril formation of Abeta1-40 and its variants was the same as the rank order of the length of the nucleation/lag phase of the Abeta12-28 fragments, E22V>E22A?E22G>E22Q?E22, and correlated with the degree of hydrophobicity of the position 22 substituent. The molecular mass of peptide monomers and protofibrils were estimated better in SEC studies using linear rather than globular calibration standards. The characteristics of the Abeta1-40E22G suggest an important role for the peptide in the neuropathogenesis in the Arctic form of AD.     

Polymorphism and ultrastructural organization of prion protein amyloid fibrils: an insight from high resolution atomic force microscopy

Amyloid fibrils were produced from the full-length mouse prion protein (PrP) under solvent conditions similar to those used for the generation of synthetic prions from PrP 89-230. Analysis of the ultrastructure by atomic force microscopy revealed extremely broad polymorphism in fibrils formed under a single growth condition. Fibrils varied with respect to the number of constitutive filaments and the manner in which the filaments were assembled. PrP polymerization was found to show several peculiar features: (i) the higher-order fibrils/ribbons were formed through a highly hierarchical mechanism of assembly of lower-order fibrils/ribbons; (ii) the lateral assembly proceeded stepwise; at each step, a semi-stable fibrillar species were generated, which were then able to enter the next level of assembly; (iii) the assembly of lower into higher-order fibrils occurred predominantly in a vertical dimension via stacking of ribbons on top of each other; (iv) alternative modes of lateral association co-existed under a single growth condition; (iv) the fibrillar morphology changed even within individual fibrils, illustrating that alternative modes of filament assembly are inter-convertible and thermodynamically equivalent. The most predominant fibrillar types were classified into five groups according to their height, each of which was divided in up to three subgroups according to their width. Detailed analysis of ultrastructure revealed that the fibrils of the major subtype (height 3.61(+/-0.28)nm, width 31.1(+/-2.0)nm) were composed of two ribbons, each of which was composed of two filaments. The molecular volume calculations indicated that a single PrP molecule occupied a distance of approximately 1.2 nm within a single filament. High polymorphism in fibrils generated in vitro is reminiscent of high morphological diversity of scrapie-associated fibrils isolated from scrapie brains, suggesting that polymorphism is peculiar for polymerization of PrP regardless of whether fibrils are formed in vitro or under pathological conditions in vivo.     

Tapping mode atomic force microscopy of scleroglucan networks

Tapping mode Atomic Force Microscopy (TmAFM) has been used to study the fungal polysaccharide scleroglucan deposited from aqueous solution and dimethyl sulfoxide (DMSO) onto a mica surface. The solutions from which the microscope samples were produced were prepared by first dissolving the solid scleroglucan in 0.1M NaOH, then neutralizing the solution with HCl, followed by dilution to the required concentration in either water or DMSO. It was found that from the aqueous solution described above, scleroglucan forms networks. Based on a comparison of the denatured-renatured and aqueous solution samples, network formation is due to the imperfect registration between the chains forming the triple helices. The relatively large stiffness of the scleroglucan triple helix is also assumed to contribute to the formation of the extended networks. The triple helix diameter was measured to be 0.92 ± 0.27 nm, which is in the same range as that obtained by other researchers using similar techniques. Denatured scleroglucan, deposited from DMSO onto mica, forms a web-like layer on top of which there are sphere-like structures. These morphologies are believed to be due to triple helix denaturation yielding highly flexible single chains in DMSO, which results in coiling and web-like dense packing of scleroglucan upon deposition onto mica. Most interestingly after addition of water to the samples deposited from DMSO, some of the chains can be renatured into short, stiff rod-like structures which are similar to the structures observed by other researchers. The imaging data for aqueous solution deposition can be analyzed by plotting maximum end-to-end distance versus the perimeter of the networks deposited onto mica. This yields a Flory-like exponent of 0.67, which is almost similar in value to that obtained by other researchers for linear structures of scleroglucan but less than that expected for a polymer chain following a self-avoiding walk (v = 0.75) model on a two-dimensional surface. The fractal dimension that can be used to characterize the networks was determined graphically to be 1.22 ± 0.06. © 1997 John Wiley & Sons, Inc. Biopoly 42: 89–100, 1997     

Amyloid beta-protein (Abeta)1-40 protects neurons from damage induced by Abeta1-42 in culture and in rat brain

Previously, we found that amyloid beta-protein (Abeta)1-42 exhibits neurotoxicity, while Abeta1-40 serves as an antioxidant molecule by quenching metal ions and inhibiting metal-mediated oxygen radical generation. Here, we show another neuroprotective action of nonamyloidogenic Abeta1-40 against Abeta1-42-induced neurotoxicity in culture and in vivo. Neuronal death was induced by Abeta1-42 at concentrations higher than 2 microm, which was prevented by concurrent treatment with Abeta1-40 in a dose-dependent manner. However, metal chelators did not prevent Abeta1-42-induced neuronal death. Circular dichroism spectroscopy showed that Abeta1-40 inhibited the beta-sheet transformation of Abeta1-42. Thioflavin-T assay and electron microscopy analysis revealed that Abeta1-40 inhibited the fibril formation of Abeta1-42. In contrast, Abeta1-16, Abeta25-35, and Abeta40-1 did not inhibit the fibril formation of Abeta1-42 nor prevent Abeta1-42-induced neuronal death. Abeta1-42 injection into the rat entorhinal cortex (EC) caused the hyperphosphorylation of tau on both sides of EC and hippocampus and increased the number of glial fibrillary acidic protein (GFAP)-positive astrocytes in the ipsilateral EC, which were prevented by the concurrent injection of Abeta1-40. These results indicate that Abeta1-40 protects neurons from Abeta1-42-induced neuronal damage in vitro and in vivo, not by sequestrating metals, but by inhibiting the beta-sheet transformation and fibril formation of Abeta1-42. Our data suggest a mechanism by which elevated Abeta1-42/Abeta1-40 ratio accelerates the development of Alzheimer's disease (AD) in familial AD.     

原子力显微镜在细胞弹性研究中的应用

细胞表面的力学性质会随着细胞所处环境的不同而发生改变,它的变化间接反映出胞内复杂的生理过程。原子力显微镜（atomic force microscope,AFM）能以高的灵敏度和分辨率检测活体细胞,通过利用赫兹模型分析力曲线可以获得细胞的弹性信息。本文简介了原子力显微镜的工作原理与工作模式,着重介绍利用AFM力曲线检测细胞弹性的方法及其在细胞运动、细胞骨架、细胞黏附、细胞病理等方面的应用成果,表明AFM已经成为细胞弹性研究中十分重要的显微技术。     

Chip-based protein-protein interaction studied by atomic force microscopy

In this article, a technique for accurate direct measurement of protein‐to‐protein interactions before and after the introduction of a drug candidate is developed using atomic force microscopy (AFM). The method is applied to known immunosuppressant drug candidate Echinacea purpurea derived cynarin. T‐cell/CD28 is on‐chip immobilized and B‐cell/CD80 is immobilized on an AFM tip. The difference in unbinding force between these two proteins before and after the introduction of cynarin is measured. The method is described in detail including determination of the loading rates, maximum probability of bindings, and average unbinding forces. At an AFM loading rate of 1.44 × 10⁴ pN/s, binding events were largely reduced from 61 ± 5% to 47 ± 6% after cynarin introduction. Similarly, maximum probability of bindings reduced from 70% to 35% with a blocking effect of about 35% for a fixed contact time of 0.5 s or greater. Furthermore, average unbinding forces were reduced from 61.4 to 38.9 pN with a blocking effect of ～37% as compared with ～9% by SPR. AFM, which can provide accurate quantitative measures, is shown to be a good method for drug screening. The method could be applied to a wider variety of drug candidates with advances in bio‐chip technology and a more comprehensive AFM database of protein‐to‐protein interactions. Biotechnol. Bioeng. 2012; 109: 2460–2467. © 2012 Wiley Periodicals, Inc.     

Assessing the flexibility of intermediate filaments by atomic force microscopy

Eukaryotic cells contain three cytoskeletal filament systems that exhibit very distinct assembly properties, supramolecular architectures, dynamic behaviour and mechanical properties. Microtubules and microfilaments are relatively stiff polar structures whose assembly is modulated by the state of hydrolysis of the bound nucleotide. In contrast, intermediate filaments (IFs) are more flexible apolar structures assembled from a approximately 45 nm long coiled-coil dimer as the elementary building block. The differences in flexibility that exist among the three filament systems have been described qualitatively by comparing electron micrographs of negatively stained dehydrated filaments and by directly measuring the persistence length of F-actin filaments (approximately 3-10 microm) and microtubules (approximately 1-8 mm) by various physical methods. However, quantitative data on the persistence length of IFs are still missing. Toward this goal, we have carried out atomic force microscopy (AFM) in physiological buffer to characterise the morphology of individual vimentin IFs adsorbed to different solid supports. In addition, we compared these images with those obtained by transmission electron microscopy (TEM) of negatively stained dehydrated filaments. For each support, we could accurately measure the apparent persistence length of the filaments, yielding values ranging between 0.3 microm and 1 microm. Making simple assumptions concerning the adsorption mechanism, we could estimate the persistence length of an IF in a dilute solution to be approximately 1 microm, indicating that the lower measured values reflect constraints induced by the adsorption process of the filaments on the corresponding support. Based on our knowledge of the structural organisation and mechanical properties of IFs, we reason that the lower persistence length of IFs compared to that of F-actin filaments is caused by the presence of flexible linker regions within the coiled-coil dimer and by postulating the occurrence of axial slipping between dimers within IFs.