Fine mapping of inherent flexibility variation along DNA molecules. Validation by atomic force microscopy (AFM) in buffer

Curvature and flexibility are structural properties of central importance to genome function. However, due to the difficulties in finding suitable experimental conditions, methods for studying one without the interference of the other have proven to be difficult. We propose a new approach that provides a measure of inherent flexibility of DNA by taking advantage of two powerful techniques, X-ray crystallography and nuclear magnetic resonance. Both techniques are able to detect local curvature on DNA fragments but, while the first analyzes DNA in the solid state, the second works on DNA in solution. Comparison of the two data sets allowed us to calculate the relative contribution to flexibility of the three rotations and three translations, which relate successive base pair planes for the ten different dinucleotide steps. These values were then used to compute the variation of flexibility along a given nucleotide sequence. This allowed us to validate the method experimentally through comparisons with maps of local fluctuations in DNA molecule trajectory constructed from atomic force microscopy imaging in solution. We conclude that the six dinucleotide-step parameters defined here provide a powerful tool for the exploration of DNA structure and, consequently will make an important contribution to our understanding of DNA-sequence-dependent biological processes.     

Quantitative characterization of single‐cell adhesion properties by atomic force microscopy using protein‐functionalized microbeads

A method was developed to characterize the adhesion properties of single cells by using protein‐functionalized atomic force microscopy (AFM) probes. The quantification by force spectroscopy of the mean detachment force between cells and a gelatin‐functionalized colloidal tip reveals differences in cell adhesion properties that are not within reach of a traditional bulk technique, the washing assay. In this latter method, experiments yield semiquantitative and average adhesion properties of a large population of cells. They are also limited to stringent conditions and cannot highlight disparities in adhesion in the subset of adherent cells. In contrast, this AFM‐based method allows for a reproducible and quantitative investigation of the adhesive properties of individual cells in common cell culture conditions and allows for the detection of adhesive subpopulations of cells. These characteristics meet the critical requirements of many fields, such as the study of cancer cell migratory abilities.     

Observation of single-stranded DNA on mica and highly oriented pyrolytic graphite by atomic force microscopy

Atomic force microscopy was used to image single-stranded DNA (ssDNA) adsorbed on mica modified by Mg(2+), by 3-aminopropyltriethoxysilane or on modified highly oriented pyrolytic graphite (HOPG). ssDNA molecules on mica have compact structures with lumps, loops and super twisting, while on modified HOPG graphite ssDNA molecules adopt a conformation without secondary structures. We have shown that the immobilization of ssDNA under standard conditions on modified HOPG eliminates intramolecular base-pairing, thus this method could be important for studying certain processes involving ssDNA in more details.     

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。     

原子力显微镜 (AFM) 在细菌生物被膜研究中的应用

原子力显微镜(AFM)作为一项重要的表面可视化技术,以其独特的优势(纳米级的空间分辨率、皮牛级力灵敏度、免标记、可在溶液环境下工作)被广泛应用于生物被膜的研究。AFM不仅可以在近生理环境下对生物被膜表面超微形貌进行可视化表征,同时还可以通过纳米压痕对生物被膜的机械特性(弹性和粘性)进行定量测量,利用AFM单细胞和单分子力谱技术可以获得生物被膜形成过程中细胞-基底以及细胞-细胞之间的相互作用力,为生物被膜的实时原位系统研究提供了可行性。本文简述了AFM的基本操作原理,综述了近年来AFM用于生物被膜表面超微结构成像、机械特性测量以及相互作用力研究方面的进展,并对AFM在生物被膜研究中面临的问题和未来的发展方向进行了讨论。     

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.     

Direct visualization of KirBac3.1 potassium channel gating by atomic force microscopy

KirBac3.1 belongs to a family of transmembrane potassium (K⁺) channels that permit the selective flow of K-ions across biological membranes and thereby regulate cell excitability. They are crucial for a wide range of biological processes and mutations in their genes cause multiple human diseases. Opening and closing (gating) of Kir channels may occur spontaneously but is modulated by numerous intracellular ligands that bind to the channel itself. These include lipids (such as PIP₂), G-proteins, nucleotides (such as ATP) and ions (e.g. H⁺, Mg²⁺, Ca²⁺). We have used high-resolution atomic force microscopy (AFM) to examine KirBac3.1 in two different configurations. AFM imaging of the cytoplasmic surface of KirBac3.1 embedded in a lipid bilayer has allowed visualization of the tetrameric assembly of the ligand-binding domain. In the absence of Mg²⁺, the four subunits appeared as four protrusions surrounding a central depression corresponding to the cytoplasmic pore. They did not display 4-fold symmetry, but formed a dimer-of-dimers with 2-fold symmetry. Upon addition of Mg²⁺, a marked rearrangement of the intracellular ligand-binding domains was observed: the four protrusions condensed into a single protrusion per tetramer, and there was an accompanying increase in protrusion height. The central cavity within the four intracellular domains also disappeared on addition of Mg²⁺, indicating constriction of the cytoplasmic pore. These structural changes are likely transduced to the transmembrane helices, which gate the K⁺ channel. This is the first time AFM has been used as an interactive tool to study K⁺ channels. It has enabled us to directly measure the conformational changes in the protein surface produced by ligand binding.     

Direct monitoring of drug‐induced mechanical response of individual cells by atomic force microscopy

Mechanical characteristics of individual cells play a vital role in many biological processes and are considered as indicators of the cells’ states. Disturbances including methyl‐β‐cyclodextrin (MβCD) and cytochalasin D (cytoD) are known to significantly affect the state of cells, but little is known about the real‐time response of single cells to these drugs in their physiological condition. Here, nanoindentation‐based atomic force microscopy (AFM) was used to measure the elasticity of human embryonic kidney cells in the presence and absence of these pharmaceuticals. The results showed that depletion of cholesterol in the plasma membrane with MβCD resulted in cell stiffening whereas depolymerization of the actin cytoskeleton by cytoD resulted in cell softening. Using AFM for real‐time measurements, we observed that cells mechanically responded right after these drugs were added. In more detail, the cell´s elasticity suddenly increased with increasing instability upon cholesterol extraction while it is rapidly decreased without changing cellular stability upon depolymerizing actin cytoskeleton. These results demonstrated that actin cytoskeleton and cholesterol contributed differently to the cell mechanical characteristics.     

Exploring the mechanical properties of single vimentin intermediate filaments by atomic force microscopy

Intermediate filaments (IFs), together with actin filaments and microtubules, compose the cytoskeleton. Among other functions, IFs impart mechanical stability to cells when exposed to mechanical stress and act as a support when the other cytoskeletal filaments cannot keep the structural integrity of the cells. Here we present a study on the bending properties of single vimentin IFs in which we used an atomic force microscopy (AFM) tip to elastically deform single filaments hanging over a porous membrane. We obtained a value for the bending modulus of non-stabilized IFs between 300 MPa and 400 MPa. Our results together with previous ones suggest that IFs present axial sliding between their constitutive building blocks and therefore have a bending modulus that depends on the filament length. Measurements of glutaraldehyde-stabilized filaments were also performed to reduce the axial sliding between subunits and therefore provide a lower limit estimate of the Young's modulus of the filaments. The results show an increment of two to three times in the bending modulus for the stabilized IFs with respect to the non-stabilized ones, suggesting that the Young's modulus of vimentin IFs should be around 900 MPa or higher.     

Atomic force microscopy study of the collagen fibre structure

Observations of intact reconstituted and native collagen fibres were performed with the atomic force microscope. The results are compared between the two types of fibres and with those obtained previously with the electron microscope on freeze-etched or negative stained samples. Some of the findings presented here indicate that the specimens observed in air with the atomic force microscope were still in a hydrated state.     

原子力显微镜研究小鼠免疫球蛋白G自组装

应用原子力显微镜观察小鼠免疫球蛋白G样品,结果不管是IgG1还是IgG1与IgG2的混合样品,在制样2-3d后,均发现有圆环状聚集区,也有部分圆面状聚集区域,一般环中心有核,核为IgG分子多聚体的再聚集,多聚体中亚基（单体）数目因环而异,在圆环和贺湎状区域外,也有零星分散的聚集体,不参与成环,另外,IgG1和IgG1 IgG2聚集情况有所不同,后者更倾向于形成圆面状聚集,另外,在聚集发生前,亚基已自组装形成各种形状的多聚体,然后多聚体进一步聚集形成各种圆环或圆面状,对免疫球蛋白G先自组装而后聚集的过程,从生物学,物理学角度作了初步解释。     

Visualization of alkali-denatured supercoiled plasmid DNA by atomic force microscopy

To study the alkali denaturation of supercoiled DNA, plasmid pBR322 was treated with gradient concentrations of NaOH solution. The results of gel electrophoresis showed that the alkali denaturation of the supercoiled DNA occurred in a narrow range of pH value (12.88-12.90). The alkali-denatured supercoiled DNA ran, as a sharp band, faster than the supercoiled DNA. The supercoiled plasmid DNA of pBR322, pACYC184 and pJGX15A were denatured by NaOH, and then visualized by atomic force microscopy. Compared with the supercoiled DNA, the atomic force microscopy images of the alkali-denatured supercoiled DNA showed rough surface with many kinks, bulges on double strands with inhomogeneous diameters. The apparent contour lengths of the denatured DNA were shortened by 16%, 16% and 50% for pBR322, pACYC184 and pJGX15A, respectively. All evidence suggested that the alkali-denatured supercoiled DNA had a stable conformation with unregistered, topologically constrained double strands and intrastrand secondary structure.     

Ultra-high resolution imaging of DNA and nucleosomes using non-contact atomic force microscopy

Visualisation of nano-scale biomolecules aids understanding and development in molecular biology and nanotechnology. Detailed structure of nucleosomes adsorbed to mica has been captured in the absence of chemical-anchoring techniques, demonstrating the usefulness of non-contact atomic force microscopy (NC-AFM) for ultra-high resolution biomolecular imaging. NC-AFM offers significant advantages in terms of resolution, speed and ease of sample preparation when compared to techniques such as cryo-electron microscopy and X-ray crystallography. In the absence of chemical modification, detailed structure of DNA deposited on a gold substrate was observed for the first time using NC-AFM, opening up possibilities for investigating the electrical properties of unmodified DNA.     

Atomic force microscopy comes of age

AFM (atomic force microscopy) analysis, both of fixed cells, and live cells in physiological environments, is set to offer a step change in the research of cellular function. With the ability to map cell topography and morphology, provide structural details of surface proteins and their expression patterns and to detect pico‐Newton force interactions, AFM represents an exciting addition to the arsenal of the cell biologist. With the explosion of new applications, and the advent of combined instrumentation such as AFM—confocal systems, the biological application of AFM has come of age. The use of AFM in the area of biomedical research has been proposed for some time, and is one where a significant impact could be made. Fixed cell analysis provides qualitative and quantitative subcellular and surface data capable of revealing new biomarkers in medical pathologies. Image height and contrast, surface roughness, fractal, volume and force analysis provide a platform for the multiparameter analysis of cell and protein functions. Here, we review the current status of AFM in the field and discuss the important contribution AFM is poised to make in the understanding of biological systems.     

Compaction of single supercoiled DNA molecules adsorbed onto amino mica

A model of possible conformational transitions of supercoiled DNA in vitro in the absence of proteins under the conditions of increasing degree of compaction was developed. A 3993-bp pGEMEX supercoiled DNA immobilized on various substrates (freshly cleaved mica, standard amino mica, and modified amino mica with a hydrophobicity higher than that of standard amino mica) was visualized by atomic force microscopy in air. On the modified amino mica, which has an increased density of surface positive charges, single molecules with an extremely high degree of compaction were visualized in addition to plectonemic DNA molecules. As the degree of DNA supercoiling increased, the length of the first-order superhelical axis of molecules decreased from 570 to 370 nm, followed by the formation of second-and third-order superhelical axes about 280 and 140 nm long, respectively. The compaction of molecules ends with the formation of minitoroids about 50 nm in diameter and molecules of spherical shape. It was shown that the compaction of single supercoiled DNA molecules immobilized on amino mica to the level of minitoroids and spheroids is due to the shielding of mutually repulsing negatively charged phosphate groups of DNA by positively charged amino groups of the amino mica, which has a high charge density of its surface.     

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。     

颗石藻颗石粒形态的原子力显微观测方法: 以赫氏艾密里藻为例

颗石藻(coccolithophore)作为一种模式生物, 在重建古海洋气候和环境以及预测未来全球气候变化中起着很重要的作用, 赫氏艾密里藻(Emiliania huxleyi)是颗石藻最为典型的代表种。钙质颗石粒(coccolith)是颗石藻形态分类的主要依据, 有着非常精细和复杂的结构, 在样品收集过程中很容易遭到破坏, 这是颗石藻鉴定中经常遇到的一个技术问题。国际上还没有统一的颗石藻定量采样和样品分析方法。本文采用原子力显微方法(atomic force microscopy, AFM)对赫氏艾密里藻的颗石粒形态进行了超显微观察研究, 获取不同扫描范围的高度图(height image)和形貌图(deflection image)以观测其形态结构, 并建立了针对颗石藻的原子力显微样品制备方法。通过离心与膜过滤两种方法收集赫氏艾密里藻, 比较后得出了一种简单、快速的适合于观测颗石藻在大气环境成像的样品处理、制备和图像采集方法: 3,000-4,000 rpm, 20℃离心5 min, 收集颗石藻, 去除有机杂质后取白色沉淀, 将沉淀物悬浮于0.05 M NH₄HCO₃溶液中, 悬浮液滴加于盖玻片表面, 20℃晾干后于样品台在AFM接触模式(contact mode)下原子级扫描, 扫描范围50 µm, 频率1 Hz, 可以得到优质的颗石粒形态图像, 有助于颗石藻的分类鉴别。该方法可用于室内不同环境梯度或参数下的颗石粒形态结构及颗石藻藻华的检测与研究。     

Oligomerization states of Bowman-Birk inhibitor by atomic force microscopy and computational approaches

Several methods have been applied to study protein-protein interaction from structural and thermodynamic point of view. The present study reveals that atomic force microscopy (AFM), molecular modeling, and docking approaches represent alternative methods offering new strategy to investigate structural aspects in oligomerization process of proteinase inhibitors. The topography of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) was recorded by AFM and compared with computational rigid-bodies docking approaches. Multimeric states of BTCI identified from AFM analysis showed globular-ellipsoidal shapes. Monomers, dimers, trimers, and hexamers were the most prominent molecular arrays observed in AFM images as evaluated by molecular volume calculations and corroborated by in silico docking and theoretical approaches. We therefore propose that BTCI adopts stable and well-packed self-assembled states in monomer-dimer-trimer-hexamer equilibrium. Although there are no correlation between specificity and packing efficiency among proteinases and proteinase inhibitors, the AFM and docked BTCI analyses suggest that these assemblies may exist in situ to play their potential function in oligomerization process.     

应用原子力显微镜分析正常淋巴细胞和Jurkat细胞的形态和机械性质

淋巴细胞形态和机械性质的变化与人的健康、疾病的治疗和诊断有着密切关系。本研究利用原子力显微镜研究淋巴细胞和Jurkat细胞形态和机械性质。结果显示,这2种细胞的形态较为相似,但通过对力曲线的分析得出这2种细胞的机械性质明显不同。正常淋巴细胞粘弹力范围大致为(796.7±248.5)pN,而Jurkat细胞分布于(158.5±37.5)pN;正常淋巴细胞的杨氏模量(0.471kPa±0.081kPa)近4倍于Jurkat细胞(0.0964kPa±0.0229kPa);而Jurkat细胞(4.322mN/m±0.382mN/m)的硬度近2倍于正常淋巴细胞(2.278mN/m±0.488mN/m)。结果表明原子力显微镜能可在临床诊断上区分正常细胞与肿瘤细胞,即使两者形态区别不明显。