Structural Evidence for α-Synuclein Fibrils Using in Situ Atomic Force Microscopy

Human α-synuclein is a presynaptic terminal protein and can form insoluble fibrils that are believed to play an important role in the pathogenesis of several neurodegenerative diseases such as Parkinson‘s disease, dementia with Lewy bodies and Lewy body variant of Alzheimer‘s disease. In this paper, in situ atomic force microscopy has been used to study the structural properties of α-synuclein fibrils in solution using two different atomic force microscopy imaging modes: tapping mode and contact mode. In the in situ contact mode atomic force microscopy experiments α-synuclein fibrils quickly broke into fragments, and a similar phenomenon was found using tapping mode atomic force microscopy in which α-synuclein fibrils were incubated with guanidine hydrochloride (0.6 M). The α-synuclein fibrils kept their original filamentous topography for over 1h in the in situ tapping mode atomic force microscopy experiments. The present results provide indirect evidence on how 13-sheets assemble into α-synuclein fibrils on a nanometer scale.     

Growth of Collagen Fibril Seeds from Embryonic Tendon: Fractured Fibril Ends Nucleate New Tip Growth

Collagen fibrils are the principal tensile element of vertebrate tissues where they occur in the extracellular matrix as spatially organised arrays. A major challenge is to understand how the mechanisms of nucleation, growth and remodelling yield fibrils of tissue-specific diameter and length. Here we have developed a seeding system whereby collagen fibrils were isolated from avian embryonic tendon and added to purified collagen solution, in order to characterise fibril surface nucleation and growth mechanisms. Fragmentation of tendon in liquid nitrogen followed by Dounce homogenisation generated fibril length fragments. Most (> 94%) of the fractured ends of fibrils, which show an abrupt square profile, were found to act as nucleation sites for further growth by molecular accretion. The mechanism of this nucleation and growth process was investigated by transmission electron microscopy, atomic force microscopy and scanning transmission electron microscopy mass mapping. Typically, a single growth spur occurred on the N-terminal end of seed fibrils whilst twin spurs frequently formed on the C-terminal end before merging into a single tip projection. The surface nucleation and growth process generated a smoothly tapered tip that achieved maximum diameter when the axial extension reached ∼ 13 μm. Lateral growth also occurred along the entire length of all seed fibrils that contained tip projections. The data support a model of collagen fibril growth in which the broken ends of fibrils are nucleation sites for propagation in opposite axial directions. The observed fibril growth behaviour has direct relevance to tendon matrix remodelling and repair processes that might involve rupture of collagen fibrils.     

Atomic force microscopy and modeling of natural elastic fibrillin polymers

A central issue in the understanding of Marfan syndrome deals with the functional architecture of fibrillin-containing microfibrils. Fibrillin-rich microfibrils are long extracellular matrix fibrillar components exhibiting a 50 nm periodic beaded-structure with a width of around 20–25 nm after rotary shadowing and a 10–12 nm diameter when observed in ultra-thin sections. They are composed of fibrillin monomers more or less associated with many other components which are, for the most part, poorly characterized up to date. They are known to be elastic but few data have been accumulated to understand their properties. Atomic force microscopy (AFM) allowed us to morphologically differentiate fibrillin-rich microfibrils from other fibrillar components and to investigate the thin structure of these beaded filaments in their native state. They showed, in AFM, a periodic beaded structure ranging from 50 to 60 nm and a width of about 40 nm. The different sizes of fibrillin-containing microfibrils previously observed after rotary shadowing and in ultra-thin sections was resolved with our technique and is revealed to be 10 nm in diameter. Each beaded microfibril appears to be composed of heterogeneous beads connected by 2–3 arms. An orientation of the microfibrils has been shown, and allows us to propose a complementary model of microfibrillar monomer association.     

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。     

Nanostructure and mechanics of mummified type I collagen from the 5300-year-old Tyrolean Iceman

Skin protects the body from pathogens and degradation. Mummified skin in particular is extremely resistant to decomposition. External influences or the action of micro-organisms, however, can degrade the connective tissue and lay the subjacent tissue open. To determine the degree of tissue preservation in mummified human skin and, in particular, the reason for its durability, we investigated the structural integrity of its main protein, type I collagen. We extracted samples from the Neolithic glacier mummy known as ‘the Iceman’. Atomic force microscopy (AFM) revealed collagen fibrils that had characteristic banding patterns of 69 ± 5 nm periodicity. Both the microstructure and the ultrastructure of dermal collagen bundles and fibrils were largely unaltered and extremely well preserved by the natural conservation process. Raman spectra of the ancient collagen indicated that there were no significant modifications in the molecular structure. However, AFM nanoindentation measurements showed slight changes in the mechanical behaviour of the fibrils. Young''s modulus of single mummified fibrils was 4.1 ± 1.1 GPa, whereas the elasticity of recent collagen averages 3.2 ± 1.0 GPa. The excellent preservation of the collagen indicates that dehydration owing to freeze-drying of the collagen is the main process in mummification and that the influence of the degradation processes can be addressed, even after 5300 years.     

原子力显微镜在双微体形态学研究中的应用

原子力显微术(atomic force microscopy,AFM)是一种新型的纳米显微技术,由于其拥有标本制备简单、分辨率高等优点,因此常用于细胞超微结构的观察。双微体(double minute chromosomes,DMs)是基因扩增的主要表现形式,经常出现在肿瘤细胞及耐药细胞中,可使肿瘤细胞获得生存优势或产生耐药性,因此对双微体进行研究可使人类了解肿瘤的生长特性及其抗药性的产生机理。为寻找一种研究双微体的有效方法,本实验利用原子力显微镜对小鼠耐氨甲喋呤细胞3T3R500中的双微体进行观察,在获得双微体高分辨AFM形态图的同时,还对双微体的大小进行了测量,发现细胞中双微体大小存在差异。此外,就原子力显微镜在双微体研究中的一些技术细节进行了探讨。实验结果表明原子力显微术是研究双微体的一种有效手段。     

原子力显微镜观察丹皮多糖分子的形貌结构及自组装行为

运用原子力显微镜(Atomic Force Microscopy,AFM)技术对丹皮多糖的形貌结构进行研究。采用多糖水溶液液珠滴降法制样,AFM非接触模式(non-contact mode)扫描。AFM图像显示,丹皮多糖分子呈现近似球形的结构形貌,小球形颗粒直径为50 nm~80 nm左右,推测是丹皮多糖的基本结构单位。在一定浓度和条件下,丹皮多糖分子小球形颗粒可发生聚集,其中以直径约170 nm~220 nm的圈状和中空球聚集体结构较有代表性。将多糖稀溶液80℃加热,可促进多糖分子发生自组装,缔合形成长链超分子结构。超分子结构中的每一亚单位亦由小球形颗粒聚集紧缩而成。AFM图像研究表明丹皮多糖分子在水溶液中倾向采取球状或线团构象并通过链间氢键作用形成分子缔合组装。     

Changes in Structural-Mechanical Properties and Degradability of Collagen during Aging-associated Modifications

During aging, changes occur in the collagen network that contribute to various pathological phenotypes in the skeletal, vascular, and pulmonary systems. The aim of this study was to investigate the consequences of age-related modifications on the mechanical stability and in vitro proteolytic degradation of type I collagen. Analyzing mouse tail and bovine bone collagen, we found that collagen at both fibril and fiber levels varies in rigidity and Young''s modulus due to different physiological changes, which correlate with changes in cathepsin K (CatK)-mediated degradation. A decreased susceptibility to CatK-mediated hydrolysis of fibrillar collagen was observed following mineralization and advanced glycation end product-associated modification. However, aging of bone increased CatK-mediated osteoclastic resorption by ∼27%, and negligible resorption was observed when osteoclasts were cultured on mineral-deficient bone. We observed significant differences in the excavations generated by osteoclasts and C-terminal telopeptide release during bone resorption under distinct conditions. Our data indicate that modification of collagen compromises its biomechanical integrity and affects CatK-mediated degradation both in bone and tissue, thus contributing to our understanding of extracellular matrix aging.     

Protein composition and biomechanical properties of in vivo-derived basement membranes

Basement membranes (BMs) evolved together with the first metazoan species approximately 500 million years ago. Main functions of BMs are stabilizing epithelial cell layers and connecting different types of tissues to functional, multicellular organisms. Mutations of BM proteins from worms to humans are either embryonic lethal or result in severe diseases, including muscular dystrophy, blindness, deafness, kidney defects, cardio-vascular abnormalities or retinal and cortical malformations. In vivo-derived BMs are difficult to come by; they are very thin and sticky and, therefore, difficult to handle and probe. In addition, BMs are difficult to solubilize complicating their biochemical analysis. For these reasons, most of our knowledge of BM biology is based on studies of the BM-like extracellular matrix (ECM) of mouse yolk sac tumors or from studies of the lens capsule, an unusually thick BM. Recently, isolation procedures for a variety of BMs have been described, and new techniques have been developed to directly analyze the protein compositions, the biomechanical properties and the biological functions of BMs. New findings show that native BMs consist of approximately 20 proteins. BMs are four times thicker than previously recorded, and proteoglycans are mainly responsible to determine the thickness of BMs by binding large quantities of water to the matrix. The mechanical stiffness of BMs is similar to that of articular cartilage. In mice with mutation of BM proteins, the stiffness of BMs is often reduced. As a consequence, these BMs rupture due to mechanical instability explaining many of the pathological phenotypes. Finally, the morphology and protein composition of human BMs changes with age, thus BMs are dynamic in their structure, composition and biomechanical properties.     

用原子力显微镜观察人类精子的表面结构

目的:探讨用原子力显微镜观察精子表面结构的方法。方法:经常规洗涤正常人精液后进一步除去精子表面和生理溶液中的蛋白质,直接用原子力显微镜观察人类精子的表面形态。结果:获得了人类精子表面的细微结构和精子头的三维数据。精子全长约47μm,精子头约4.6μm,顶体位于精子头前端1/2~2/3,顶体前端扁平。精子赤道区有两圈环形凸起。结论:不需特殊处理,用原子力显微镜能直接观察精子表面的超微结构,并获得量化的三维数据。     

Loading a ring: structure of the Bacillus subtilis DnaB protein, a co-loader of the replicative helicase

Loading of the ring-shaped replicative helicase is a critical step in the initiation of DNA replication. Bacillus subtilis has adopted a two-protein strategy to load its hexameric replicative helicase: DnaB and DnaI interact with the helicase and mediate its delivery onto DNA. We present here the 3D electron microscopy structure of the DnaB protein, along with a detailed analysis of both its oligomeric state and its domain organization. DnaB is organized as an asymmetric tetramer that is comprised of two stacked components, one arranged as a closed collar and the other as an open sigma shape. Intriguingly, the 3D map of DnaB exhibits an overall architecture similar to the structure of the Escherichia coli gamma-complex, the loader of the ring-shaped processivity factor. We propose a model whereby each DnaB monomer participates in both stacked components of the tetramer and displays a different overall shape. This asymmetric quaternary organization could be a general feature of ring loaders.     

The supramolecular organization of collagen VI microfibrils

Collagen VI has a ubiquitous distribution throughout connective tissues, and has key roles in linking cells and matrix macromolecules. We have generated three-dimensional reconstructions of collagen VI microfibrils using automated electron tomography (AET) in order to obtain new insights into the organisation of collagen VI in assembled microfibrils. Analysis of the reconstruction data has allowed the resolution of the double-beaded structure into smaller subunits. Volume calculations from the tomography data indicate that ten and six A-domains could be packed into the N and C-terminal regions from each monomer, respectively. A putative location for the globular N-terminal regions of the alpha3 chain, important for microfibril assembly and function, has been identified. Some surfaces of the alpha3 chain N-terminal domains appear to be exposed on the surface of a microfibril, where they may provide an interactive surface for molecules. Analysis of the interbead region provides evidence for complex triple helical supercoiling in microfibrils. Frequently, two strands were visualised emerging from the beaded region and merging into a single interbead region. Measurements taken from the AET data show that there is a decrease in periodicity from dimer/tetramer to microfibrils. Molecular combing reverses this effect by mechanically increasing periodicity to give measurements similar to the component dimers/tetramers. Together, these data have provided important new insights into the organisation and function of these large macromolecular assemblies.