Fungal surface remodelling visualized by atomic force microscopy

Most fungal growth is localized to the tips of hyphae, however, early stages of spore germination and the growth of certain morphological mutant strains exhibit non-polarized expansion. We used atomic force microscopy (AFM) to document changes in Aspergillus nidulans wall surfaces during non-polarized growth: spore germination, and growth in a strain containing the hypA1 temperature sensitive morphogenesis defect. We compared wall surface structures of both wild-type and mutant A. nidulans following growth at 28 ° and 42 °C, the latter being the restrictive temperature for hypA1. There was no appreciable difference in surface ultrastructure between wild-type and hypA1 spores, or hyphal walls grown at 28 °C. When dry mature A. nidulans conidia were wetted they lost their hydrophobin coat, indicating an intermediate stage between dormancy and swelling. The surface structure of hypA1 germlings grown at 42 °C was less organized than wild-type hyphae grown under the same conditions, and had a larger range of subunit sizes. AFM images of hyphal wall surface changes following a shift in growth temperature from restrictive (42 °C) to permissive (28 °C), showed a gradient of sizes for wall surface features similar to the trend observed for wild-type cells at branch points. Changes associated with the hyphal wall structure for A. nidulans hypA1 offer insight into the events associated with fungal germination, and wall remodelling.     

Imaging surface of gold-immunolabeled thin sections by atomic force microscopy

Atomic force microscopy (AFM) has been used to image the surface of thin sections of fungal infected plant tissue, with or without post-embedding immunocytochemical labeling with gold conjugates. Plant and fungal cells are easily identified from their size, shape and roughness. The cellular shape is similar to that observed by light or electron microscopy (LM or EM) and some internal organelles can even be individualized. The gold beads are easily observed and counted. Their dimensions varied according to the roughness of the surface, but fit with the expected sizes.     

Topography of cell traces studied by atomic force microscopy

Migrating adherent cells release material onto artificial substrates like glass and silicon while moving. Traces of mouse fibroblasts (L929) have been visualised by atomic force microscopy (AFM). “Non-contact” mode AFM in a liquid environment can extract topographic information from these traces. This dynamic mode allows the study of these soft structures without damage or compression. The AFM images show crossing and branching networks (with specific angles of branching), structured patches, nodular elements, linear elements with irregular height and other features. Fourier analysis of segment spacing in the strands is presented. These spatial features of fibroblast traces are strong indications that actin linked to structural proteins is involved in the formation of cell traces. We also give methods for trace preparation and undistorted imaging and discuss further perspectives. Received: 11 January 1999 / Revised version: 1 April 1999 / Accepted: 8 April 1999     

Visualization of plant cell walls by atomic force microscopy.

Atomic force microscopy has been used to visualize the ultrastructure of hydrated plant cell wall material from prepared apple (Malus pumila MILL; Cox orange pippin), water chestnut (Eleocharis dulcis L.), potato (Solanum tuberosum L.; Bintje), and carrot (Daucus carota L.; Amsterdamse bak) parenchyma. Samples of cell wall material in aqueous suspension were deposited onto freshly cleaved mica. Excess water was blotted away and the moist samples were imaged in air at ambient temperature and humidity. The three-dimensional images obtained highlighted the layered structure of the plant cell walls and revealed features interpreted as individual cellulose microfibrils and plasmodesmata.     

Characterization of cell elasticity correlated with cell morphology by atomic force microscope

Biomechanical properties of cells have been identified as an important factor in a broad range of biological processes. Based on measurements of mechanical properties by atomic force microscopy (AFM) particularly cell elasticity has been linked with human diseases, such as cancer. AFM has been widely used as a nanomechanical tool to probe the elasticity of living cells, however, standard methods for characterizing cell elasticity are still lacking. The local elasticity of a cell is conventionally used to represent the mechanical property of the cell. However, since cells have highly heterogeneous regions, elasticity mapping over the entire cell, rather than at a few points of measurement, is required. Using human aortic endothelial cells (HAECs) as a model, we have developed in this study a new method to evaluate cell elasticity more quantitatively. Based on the height information of the cell, a new characterization method was proposed to evaluate the elasticity of a cell. Using this method, elasticities of cells on different substrates were compared. Results showed that the elasticity of HAECs on softer substrate also has higher value compared to those on harder substrate given a certain height where the statistical distribution analysis confirmed that higher actin filaments density was located. Thus, the elasticity of small portions of a cell could not represent the entire cell property and may lead to invalid characterization. In order to gain a more comprehensive and detailed understanding of biomechanical properties for future clinical use, elasticity and cell morphology should therefore be correlated with discussion.     

High-resolution analysis of neuronal growth cone morphology by comparative atomic force and optical microscopy

Neuronal growth cones are motile sensory structures at the tip of axons, transducing guidance information into directional movements towards target cells. The morphology and dynamics of neuronal growth cones have been well characterized with optical techniques; however, very little quantitative information is available on the three-dimensional structure and mechanical properties of distinct subregions. In the present study, we imaged the large Aplysia growth cones after chemical fixation with the atomic force microscope (AFM) and directly compared our data with images acquired by light microscopy methods. Constant force imaging in contact mode in combination with force-distant measurements revealed an average height of 200 nm for the peripheral (P) domain, 800 nm for the transition (T) zone, and 1200 nm for the central (C) domain, respectively. The AFM images show that the filopodial F-actin bundles are stiffer than surrounding F-actin networks. Enlarged filopodia tips are 60 nm higher than the corresponding shafts. Measurements of the mechanical properties of the specific growth cone regions with the AFM revealed that the T zone is stiffer than the P and the C domain. Direct comparison of AFM and optical data acquired by differential interference contrast and fluorescence microscopy revealed a good correlation between these imaging methods. However, the AFM provides height and volume information at higher resolution than fluorescence methods frequently used to estimate the volume of cellular compartments. These findings suggest that AFM measurements on live growth cones will provide a quantitative understanding of how proteins can move between different growth cone regions.     

Study of phototrophic purple bacterium Rhodobacter sphaeroides cell morphology of wild-type and ipt-transformant by atomic force and electron microscopy

A comparative study of phototrophic purple bacterium Rhodobacter sphaeroides cell morphology of wild-type and ipt-transformant was done by atomic force and electron microscopy. It was shown that transformation led to a decrease in the number or total disappearance of the flagella, as well as to changes in the structure of the outer membrane of the bacteria cell wall. On the wild-type cell surface phage-like structures were found, and in transformed cells at their places hollows were identified. This study significantly extends an understanding of the changes occurring in the ipt-transformants of phototrophic purple bacterium Rhodobacter sphaeroides. This investigation not only confirmed earlier obtained data about the differences in the wild-type and ipt-transformant phototrophic purple bacteria cell wall, but also showed fine changes in the structure of its outer membrane.     

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。     

Imaging polysaccharides by atomic force microscopy

Techniques have been developed for the routine reliable imaging of polysaccharides by atomic force microscopy (AFM). The polysaccharides are deposited from aqueous solution onto the surface of freshly cleaved mica, air dried, and then imaged under alcohols. The rationale behind the development of the methodology is described and data is presented for the bacterial polysaccharides xanthan, acetan, and the plant polysaccharides 1-carrageenan and pectin. Studies on uncoated polysaccharides have demonstrated the improved resolution achievable when compared to more traditional metal-coated samples or replicas. For acetan the present methodology has permitted imaging of the helical structure. Finally, in addition to data obtained on individual polysaccharides, AFM images have also been obtained of the network structures formed by κ-carrageenan and gellan gum. © 1996 John Wiley & Sons, Inc.     

Direct observation of positive supercoils introduced by reverse gyrase through atomic force microscopy

Reverse gyrase is a hyperthermophilic enzyme that can introduce positive supercoiling in substrate DNA. It is showed in our studies that positive DNA supercoils were induced in both pBR322 vector and an artificially synthesized mini-plasmid DNA by reverse gyrase. The left-handed structures adopted by positively supercoiled DNA molecules could be identified from their right-handed topoisomers through atomic force microscopic examination. Additional structural comparisons revealed that positively supercoiled DNA molecule AFM images exhibited increased contour lengths. Moreover, enzymatic assays showed that the positively supercoiled DNA could not be cleaved by T7 endonuclease. Together, this suggests that the overwound structure of positive supercoils could prevent genomic duplex DNA from randomly forming single-stranded DNA regions and intra-stranded secondary structures.     

Observations on the internal and surface morphology of malaria infected blood cells using optical and atomic force microscopy

We describe a simple and fast method to probe the morphological changes on the exterior and interior of a malaria infected erythrocyte at different stages of parasite development. This involves the imaging and scanning of Giemsa stained malaria infected erythrocytes using optical microscopy and atomic force microscopy, respectively.     

Preparation of DNA catenanes and observation of their topological structures by atomic force microscopy.

DNA catenanes have been prepared by the reaction of T4 DNA ligase with linear DNA in the presence of nicked DNA. Single molecular images of DNA catenanes and large circular DNAs have been clearly observed by AFM using a tapping mode at room temperature and in an ambient atmosphere.     

Direct observation of the assembly of RecA/DNA complexes by atomic force microscopy

The formation of the RecA/DNA nucleofilament on nicked circular double stranded (ds) DNA in the presence of ATPgammaS was studied using the atomic force microscope (AFM) at nanometer resolution. The AFM allowed simultaneous observation of both dsDNA substrate and RecA protein-coated sections such that they are highly distinguishable. Using a time series of images, the complex formation was monitored. AFM imaging provided direct evidence that assembly of the nucleofilaments occurs via a nucleation and growth mechanism. The nucleation step is much slower than the growth phase, as demonstrated by the predominance of naked dsDNA at early and middle time points, followed by the rapid appearance of partially then fully formed complexes. Observation of the formation of nucleation sites without accompanying growth on unnicked dsDNA enabled an estimate of the nucleation rate, of 5 x 10(-5) RecA min(-1) bp(-1). The published model for the analysis of RecA assembly on dsDNA deduces a single kinetic parameter that prevents the separate determination of nucleation rate and growth rate. By directly measuring the nucleation rate with the AFM, this model is employed to determine a growth rate of 202 min(-1). These AFM results provide the first direct evidence of previous results on complex formation obtained only by indirect means.     

An atomic force microscopy investigation of protein crystal surface topography

Tapping mode atomic force microscopy was employed to study the surface structure of different protein crystals in a liquid environment. The (101) face of hen egg-white lysozyme crystals and the (111) face of horse spleen ferritin crystals were studied. On the (101) face of lysozyme crystals we observed islands delimitated by micro-steps and elongated in the [010] direction. The elongation direction coincides with the preferential growth direction predicted by a growth model reported in the literature. The islands observed on the ferritin (111) face are also delimitated by micro-steps but have circular symmetry. Sectioning of the images allowed us to measure the step heights. The surface free energy was estimated from the growth step morphology. Molecular resolution was achieved for ferritin crystals, showing a hexagonal surface packing, as expected for the molecular lattice of a (111) face in a fcc crystal.     

Imaging of the membrane surface of MDCK cells by atomic force microscopy.

The membrane surface of polarized renal epithelial cells (MDCK cells) grown as a monolayer was imaged with the atomic force microscope. The surface topography of dried cells determined by this approach was consistent with electron microscopy images previously reported. Fixed and living cells in aqueous medium gave more fuzzy images, likely because of the presence of the cell glycocalix. Treatment of living cells with neuraminidase, an enzyme that partly degrades the glycocalix, allowed sub-micrometer imaging. Protruding particles, 10 to 60 nm xy size, occupy most of the membrane surface. Protease treatment markedly reduced the size of these particles, indicating that they corresponded to proteins. Tip structure effects were probably involved in the exaggerated size of imaged membrane proteins. Although further improvements in the imaging conditions, including tip sharpness, are required, atomic force microscope already offers the unique possibility to image proteins at the membrane surface of living cells.     

Reproducible acquisition of Escherichia coli porin surface topographs by atomic force microscopy.

Crystalline membranes reconstituted from Escherichia coli OmpF porin and phospholipids were adsorbed to freshly cleaved mica and imaged in solution by the atomic force microscope. The extracellular as well as the periplasmic side of the porin trimers could be identified and the conditions to record topographs at 1-nm lateral and 0.1-nm vertical resolution were established.     

Direct observation of protein secondary structure in gas vesicles by atomic force microscopy.

The protein that forms the gas vesicle in the cyanobacterium Anabaena flos-aquae has been imaged by atomic force microscopy (AFM) under liquid at room temperature. The protein constitutes "ribs" which, stacked together, form the hollow cylindrical tube and conical end caps of the gas vesicle. By operating the microscope in deflection mode, it has been possible to achieve sub-nanometer resolution of the rib structure. The lateral spacing of the ribs was found to be 4.6 +/- 0.1 nm. At higher resolution the ribs are observed to consist of pairs of lines at an angle of approximately 55 degrees to the rib axis, with a repeat distance between each line of 0.57 +/- 0.05 nm along the rib axis. These observed dimensions and periodicities are consistent with those determined from previous x-ray diffraction studies, indicating that the protein is arranged in beta-chains crossing the rib at an angle of 55 degrees to the rib axis. The AFM results confirm the x-ray data and represent the first direct images of a beta-sheet protein secondary structure using this technique. The orientation of the GvpA protein component of the structure and the extent of this protein across the ribs have been established for the first time.     

Hyaluronic acid by atomic force microscopy.

Hyaluronic acid (HA) of different molecular weights has been examined by atomic force microscopy (AFM) in air. This technique allows 3-D surface images of soft samples without any pretreatment, such as shadowing or staining. In the present study we examined the supermolecular organization of HA chains when deposited on mica and graphite, to better understand the interchain and intrachain interactions of HA molecules in solution. The concentration of the solution deposited varied from 0.001 to 1 mg/ml. On both substrates, and independent of the concentration, high-molecular-mass HA formed networks in which molecules ran parallel for hundreds of nanometers, giving rise to flat sheets and tubular structures that separate and rejoin into similar neighboring aggregates. Accurate measurements of the thickness of the thinnest sheets were consistent with a monolayer of HA molecules, 0.3 nm thick, strongly indicating lateral aggregation forces between chains as well as rather strong hydrophilic interactions between mica and HA. The results agree with an existing model of HA tertiary structure in solution in which the network is stabilized by both hydrophilic and hydrophobic interactions. Our images support this model and indicate that hydrophobic interactions between chains may exert a pivotal role in aqueous solution.     

Biomolecular interactions measured by atomic force microscopy

Atomic force microscopy (AFM) is nowadays frequently applied to determine interaction forces between biological molecules. Starting with the detection of the first discrete unbinding forces between ligands and receptors by AFM only several years ago, measurements have become more and more quantitative. At the same time, theories have been developed to describe and understand the dynamics of the unbinding process and experimental techniques have been refined to verify this theory. In addition, the detection of molecular recognition forces has been exploited to map and image the location of binding sites. In this review we discuss the important contributions that have led to the development of this field. In addition, we emphasize the potential of chemically well-defined surface modification techniques to further improve reproducible measurements by AFM. This increased reproducibility will pave the way for a better understanding of molecular interactions in cell biology.