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.     

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.     

The supramolecular organization of fibrillin-rich microfibrils

We propose a new model for the alignment of fibrillin molecules within fibrillin microfibrils. Automated electron tomography was used to generate three-dimensional microfibril reconstructions to 18.6-A resolution, which revealed many new organizational details of untensioned microfibrils, including heart-shaped beads from which two arms emerge, and interbead diameter variation. Antibody epitope mapping of untensioned microfibrils revealed the juxtaposition of epitopes at the COOH terminus and near the proline-rich region, and of two internal epitopes that would be 42-nm apart in unfolded molecules, which infers intramolecular folding. Colloidal gold binds microfibrils in the absence of antibody. Comparison of colloidal gold and antibody binding sites in untensioned microfibrils and those extended in vitro, and immunofluorescence studies of fibrillin deposition in cell layers, indicate conformation changes and intramolecular folding. Mass mapping shows that, in solution, microfibrils with periodicities of <70 and >140 nm are stable, but periodicities of approximately 100 nm are rare. Microfibrils comprise two in-register filaments with a longitudinal symmetry axis, with eight fibrillin molecules in cross section. We present a model of fibrillin alignment that fits all the data and indicates that microfibril extensibility follows conformation-dependent maturation from an initial head-to-tail alignment to a stable approximately one-third staggered arrangement.     

Spatial organization of cellulose microfibrils and matrix polysaccharides in primary plant cell walls as imaged by multichannel atomic force microscopy

We used atomic force microscopy (AFM), complemented with electron microscopy, to characterize the nanoscale and mesoscale structure of the outer (periclinal) cell wall of onion scale epidermis – a model system for relating wall structure to cell wall mechanics. The epidermal wall contains ~100 lamellae, each ~40 nm thick, containing 3.5‐nm wide cellulose microfibrils oriented in a common direction within a lamella but varying by ~30 to 90° between adjacent lamellae. The wall thus has a crossed polylamellate, not helicoidal, wall structure. Montages of high‐resolution AFM images of the newly deposited wall surface showed that single microfibrils merge into and out of short regions of microfibril bundles, thereby forming a reticulated network. Microfibril direction within a lamella did not change gradually or abruptly across the whole face of the cell, indicating continuity of the lamella across the outer wall. A layer of pectin at the wall surface obscured the underlying cellulose microfibrils when imaged by FESEM, but not by AFM. The AFM thus preferentially detects cellulose microfibrils by probing through the soft matrix in these hydrated walls. AFM‐based nanomechanical maps revealed significant heterogeneity in cell wall stiffness and adhesiveness at the nm scale. By color coding and merging these maps, the spatial distribution of soft and rigid matrix polymers could be visualized in the context of the stiffer microfibrils. Without chemical extraction and dehydration, our results provide multiscale structural details of the primary cell wall in its near‐native state, with implications for microfibrils motions in different lamellae during uniaxial and biaxial extensions.     

基于AFM的细胞表面超微形貌成像与机械特性测量研究进展

原子力显微镜(AFM)以其独特的优势(纳米级空间分辨率、皮牛级力灵敏度、免标记、可在溶液下工作)成为细胞生物学的重要研究手段.AFM不仅可以对活细胞表面超微形貌进行可视化表征,同时还可通过压痕技术对细胞机械特性(如杨氏模量)进行定量测量,为原位探索纳米尺度下单个活细胞动态生理活动及力学行为提供了可行性.过去的数十年中,研究人员利用AFM在细胞超微形貌成像和机械特性测量方面开展了广泛的应用研究,展示了有关细胞生理活动的大量新认识,为生命医药学领域相关问题的解决提供了新的思路;同时AFM自身的性能也在不断得到改进和提升,进一步促进了其在生命科学领域的应用.本文结合作者在应用AFM观测纳米尺度下癌症靶向药物作用效能方面的研究工作,介绍了AFM成像与细胞机械特性测量的原理,总结了近年来AFM用于细胞表面超微形貌成像与机械特性测量所取得的进展,讨论了AFM表征与检测细胞生理特性存在的问题,并对其未来发展方向进行了展望.     

Charting the surfaces of the purple membrane

The preponderance of structural data of the purple membrane from X-ray diffraction (XRD), electron crystallography (EC), and atomic force microscopy (AFM) allows us to ask questions about the structure of bacteriorhodopsin itself, as well as about the information derived from the different techniques. The transmembrane helices of bacteriorhodopsin are quite similar in both EC and XRD models. In contrast, the loops at the surfaces of the purple membrane show the highest variability between the atomic models, comparable to the height variance measured by AFM. The excellent agreement of the AFM topographs with the atomic models from XRD builds confidence in the results. Small technical difficulties in EC lead to poorer resolution of the loop structures, although the combination of atomic models with AFM surfaces allows clear interpretation of the extent and flexibility of the loop structures. While XRD remains the premier technique to determine very-high-resolution structures, EC offers a method to determine loop structures unhindered by three-dimensional crystal contacts, and AFM provides information about surface structures and their flexibility under physiological conditions.     

Screening for Antioxidants of Microbial Origin

Asymmetric hydrolysis of (dl)-1-acyloxy-2-halo-1-phenylethanes by lipoprotein lipase Amano P from Pseudomonas fluorescens and the lipase from Chromobacterium viscosum afforded the optically active (R) residual substrates and (S)-2-halo-1-hydroxy-1-phenylethanes in 100% enantiomeric excess (e.e.). The length of acyl residues from acetyl to octanoyl in the substrates did not influence the enantioselectivity.

Both enantiomers of optically active styrene oxides were synthesized from the enzymatic products.     

Force generation and shift of mass between myosin and actin in skinned striated muscle fibres at low calcium concentrations

Skinned muscle fibres from the gracilis muscle of the rabbit were used to record small angle X-ray diffraction spectra under various contractile conditions. The intracellular calcium concentration, expressed as pCa, was varied between 8.0 and 5.74. Equatorial diffraction spectra were fitted by a function consisting of five Gaussian curves and a hyperbola to separate the (1.0), (1.1), (2.0), (2.1) and Z-line diffraction peaks. The hyperbola was used to correct for residual scattering in the preparation. The ratio between the intensities of the (1.1) and (1.0) peaks was defined as the relative transfer of mass between myosin and actin, due to crossbridge formation after activation by calcium. The relation between the ratio and the relative force of the fibre (normalized to the force at pCa 5.74 and sarcomere length 2.0 μm) was linear. At high pCa (from pCa 6.34 to 8.0) no active force was observed, while the ratio still decreased. Sarcomere length was recorded by laser diffraction. The laser diffraction patterns did not show changes in sarcomere length due to activation in the high pCa range (between 8.0 and 6.34). From these results the conclusion is drawn that crossbridge movement occurs even at subthreshold calcium concentrations in the cell, when no active force is exerted. Since no force is generated this movement may be related to crossbridges in the weakly bound state. Received: 20 June 1996 / Revised version: 12 January 1998 / Accepted: 18 March 1998     

结合AFM探测副伤寒沙门氏茵B感染红细胞膜的生物力学特性

采用原子力显微镜和衍射显微术,在纳米精确尺度探测副伤寒沙门氏菌B（Sp B）感染宿主红细胞（RBC）膜微观结构和力学特性,涉及细胞的形变、膜面内剪切模量和弯曲模量。结合这两种单分子测量技术,利用相关的数学模型表述RBC膜对菌体印B的入侵非常敏感。实验结果显示,不同感染期间的SpB寄生菌体,能够引起宿主RBC膜结构改变,形变能力降低,膜剪切模量和弯曲模量显著增加。这些力学特性的变化影响RBC的输氧和循环功能。实验结果表明,印B具有独特的鞭毛调控系统,入侵的毒性菌体寄生蛋白与血影蛋白网络中的运输蛋白有特异结合位点,导致RBC膜骨架网络、波动力学和细胞内、外基质都产生应激反应,这有可能为理解勋曰感染RBC的发病机理和寄生途径提供一些新的实验思路和分析依据。     

A Structural Model for Apolipoprotein C-II Amyloid Fibrils: Experimental Characterization and Molecular Dynamics Simulations

The self-assembly of specific proteins to form insoluble amyloid fibrils is a characteristic feature of a number of age-related and debilitating diseases. Lipid-free human apolipoprotein C-II (apoC-II) forms characteristic amyloid fibrils and is one of several apolipoproteins that accumulate in amyloid deposits located within atherosclerotic plaques. X-ray diffraction analysis of aligned apoC-II fibrils indicated a simple cross-β-structure composed of two parallel β-sheets. Examination of apoC-II fibrils using transmission electron microscopy, scanning transmission electron microscopy, and atomic force microscopy indicated that the fibrils are flat ribbons composed of one apoC-II molecule per 4.7-Å rise of the cross-β-structure. Cross-linking results using single-cysteine substitution mutants are consistent with a parallel in-register structural model for apoC-II fibrils. Fluorescence resonance energy transfer analysis of apoC-II fibrils labeled with specific fluorophores provided distance constraints for selected donor-acceptor pairs located within the fibrils. These findings were used to develop a simple ‘letter-G-like’ β-strand-loop-β-strand model for apoC-II fibrils. Fully solvated all-atom molecular dynamics (MD) simulations showed that the model contained a stable cross-β-core with a flexible connecting loop devoid of persistent secondary structure. The time course of the MD simulations revealed that charge clusters in the fibril rearrange to minimize the effects of same-charge interactions inherent in parallel in-register models. Our structural model for apoC-II fibrils suggests that apoC-II monomers fold and self-assemble to form a stable cross-β-scaffold containing relatively unstructured connecting loops.     

Structure and orientation of collagen fibres in human mitral valve

The structure and distribution of collagen fibres in chordae tendineae, anterior leaflet and annulus fibrous of human mitral valve has been investigated using high and small angle X-ray diffraction. The molecular packing of collagen in native mitral valve components is very similar to that in native rat tail tendon. The distribution and orientation of collagen fibres in unstretched and stretched specimens has been deduced by the arcing of the high and small angle meridional reflections. Collagen fibres, which are aligned along the chordae tendineae, are preferentially distributed along the branchings of the chordae into the anterior leaflet and then course towards the annulus fibrous. However, in the anterior leaflet a considerable amount of collagen fibres are organized in a tridimensional isotropic network even after high deformation of the tissue.     

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。     

Nanoscale Elastic Changes in 2D Ti3C2Tx (MXene) Pseudocapacitive Electrodes

Designing sustainable electrodes for next generation energy storage devices relies on the understanding of their fundamental properties at the nanoscale, including the comprehension of ions insertion into the electrode and their interactions with the active material. One consequence of ion storage is the change in the electrode volume resulting in mechanical strain and stress that can strongly affect the cycle life. Therefore, it is important to understand the changes of dimensions and mechanical properties occurring during electrochemical reactions. While the characterization of mechanical properties via macroscopic measurements is well documented, in situ characterization of their evolution has never been achieved at the nanoscale. It is reported here with in situ imaging, combined with density functional theory of the elastic changes of a 2D titanium carbide (Ti₃C₂T_x) based electrode in direction normal to the basal plane (electrode surface) during alkaline cation intercalation/extraction. 2D carbides, known as MXenes, are promising new materials for supercapacitors and various kinds of batteries, and understanding the coupling between their mechanical and electrochemical properties is therefore necessary. The results show a strong correlation between the cations content and the out‐of‐plane elastic modulus. This strategy enables identifying the preferential intercalation pathways within a single particle, which is important for understanding ionic transport in these materials.     

Researching into the cellular shape, volume and elasticity of mesenchymal stem cells, osteoblasts and osteosarcoma cells by atomic force microscopy

Within the bone lie several different cell types, including osteoblasts (OBs) and mesenchymal stem cells (MSCs). The MSCs are ideal targets for regenerative medicine of bone due to their differentiation potential towards OBs. Human MSCs exhibit two distinct morphologies: rapidly self-renewing cells (RS) and flat cells (FC) with very low proliferation rates. Another cell type found in pathological bone conditions is osteosarcoma. In this study, we compared the topographic and morphometric features of RS and FC cells, human OBs and MG63 osteosarcoma cells by atomic force microscopy (AFM). The results demonstrated clear differences: FC and hOB cells showed similar ruffled topography, whereas RS and MG63 cells exhibited smoother surfaces. Furthermore, we investigated how selected substrates influence cell morphometry. We found that RS and MG63 cells were flatter on fibrous substrates such as polystyrene and collagen I, but much more rounded on glass, the smoothest surface. In contrast, cells with large area, namely FC and hOB cells, did not exhibit pronounced changes in flatness with regards to the different substrates. They were, however, remarkably flatter in comparison to RS and MG63 cells. We could explain the differences in flatness by the extent of adhesion. Indeed, FC and hOB cells showed much higher content of focal adhesions. Finally, we used the AFM to determine the cellular Young's modulus. RS, FC and hOB cells showed comparable stiffness on the three different substrates, while MG63 cells demonstrated the unique feature of increased elasticity on collagen I. In summary, our results show, for the first time, a direct comparison between the morphometric and biophysical features of different human cell types derived from normal and pathological bone. Our study manifests the opinion that along with RNA, proteomic and functional research, morphological and biomechanical characterization of cells also reveals novel cell features and interrelationships.     

Some Recent Developments in Computational Chemistry

Abstract

Some recent developments in the use of computational methods to predict the properties of condensed phases are discussed; the use of Gibbs ensemble Monte Carlo to predict the phase equilibria of bulk phases, the use of molecular dynamics to elucidate Atomic Force Microscopy experiments on organic films, and the use of combined Monte Carlo/molecular dynamics techniques to enable the direct prediction of particle fluxes along pressure gradients in model microporous materials.     

Extracellular silk fibres in Stannophyllum (Rhizopodea: Protozoa)

Summary The agglutinated shell or capsule of Stannophyllum zonarium, a deep sea rhizopod, contains a mass of extracellular proteinaceous fibres called linellae. They are 2–3 m diameter, weakly positively birefringent and on examination by X-ray diffraction give an amorphous type of diagram. An amino acid analysis is notable for its very high glycine content and indicates that the molecule may be related to those found in arthropod silk proteins.We wish to thank Mr. G. S. Bell and Mr. C. G. Ogden for their help with the amino acid analysis and electron microscopy respectively.     

Folding and stretching in a Go-like model of titin

Mechanical stretching of the I27 domain of titin and of its double and triple repeats are studied through molecular dynamics simulations of a Go-like model with Lennard-Jones contact interactions. We provide a thorough characterization of the system and correlate the sequencing of the folding and unraveling events with each other and with the contact order. The roles of cantilever stiffness and pulling rate are studied. Unraveling of tandem titin structures has a serial nature. The force-displacement curves in this coarse-grained model are similar to those obtained through all atom calculations.     

Formulation matters! A spectroscopic and molecular dynamics investigation on the peptide CIGB552 as itself and in its therapeutical formulation

Synthetic therapeutic peptides (STP) are intensively studied as new-generation drugs, characterized by high purity, biocompatibility, selectivity and stereochemical control. However, most of the studies are focussed on the bioactivity of STP without considering how the formulation actually used for therapy administration could alter the physico-chemical properties of the active principle. The aggregation properties of a 20-mer STP (Ac-His-Ala-Arg-Ile-Lys-D-Pro-Thr-Phe-Arg-Arg-D-Leu-Lys-Trp-Lys-Tyr-Lys-Gly-Lys-Phe-Trp-NH₂), showing antitumor activity, were investigated by optical spectroscopy and atomic force microscopy imaging, as itself (CIGB552) and in its therapeutic formulation (CIGB552TF). It has found that the therapeutic formulation deeply affects the aggregation properties of the investigated peptide and the morphology of the aggregates formed on mica by deposition of CIGB552 and CIGB552TF millimolar solutions. Molecular dynamics simulations studied the first steps of CIGB552 aggregation under physiological ionic strength conditions (NaCl 150 mM), showing that peptide oligomers, from dimers to tetramers, are preferentially formed in this environment. Interestingly, cell viability assays performed on H-460 cell lines indicate a major antiproliferative activity of the peptide in its therapeutic formulation with respect to the peptide aqueous solution.     

Structural Insights into the Substrate Specificity Switch Mechanism of the Type III Protein Export Apparatus

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Softness, strength and self-repair in intermediate filament networks

One cellular function of intermediate filaments is to provide cells with compliance to small deformations while strengthening them when large stresses are applied. How IFs accomplish this mechanical role is revealed by recent studies of the elastic properties of single IF protein polymers and by viscoelastic characterization of the networks they form. IFs are unique among cytoskeletal filaments in withstanding large deformations. Single filaments can stretch to more than 3 times their initial length before breaking, and gels of IF withstand strains greater than 100% without damage. Even after mechanical disruption of gels formed by crossbridged neurofilaments, the elastic modulus of these gels rapidly recovers under conditions where gels formed by actin filaments are irreversibly ruptured. The polyelectrolyte properties of IFs may enable crossbridging by multivalent counterions, but identifying the mechanisms by which IFs link into bundles and networks in vivo remains a challenge.