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.     

Histamine effects on endothelial cell fibronectin interaction studied by atomic force microscopy

Atomic force microscopy was used to investigate the cellular response to histamine, one of the major inflammatory mediators that cause endothelial hyperpermeability and vascular leakage. AFM probes were labeled with fibronectin and used to measure binding strength between alpha5beta1 integrin and fibronectin by quantifying the force required to break single fibronectin-integrin bonds. The cytoskeletal changes, binding probability, and adhesion force before and after histamine treatment on endothelial cells were monitored. Cell topography measurements indicated that histamine induces cell shrinkage. Local cell stiffness and binding probability increased twofold after histamine treatment. The force necessary to rupture single alpha5beta1-fibronectin bond increased from 34.0 +/- 0.5 pN in control cells to 39 +/- 1 pN after histamine treatment. Experiments were also conducted to confirm the specificity of the alpha5beta1-fibronectin interaction. In the presence of soluble GRGDdSP the probability of adhesion events decreased >50% whereas the adhesion force between alpha5beta1 and fibronectin remained unchanged. These data indicate that extracellular matrix-integrin interactions play an important role in the endothelial cell response to changes of external chemical mediators. These changes can be recorded as direct measurements on live endothelial cells by using atomic force microscopy.     

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     

Structure of human chromosomes studied by atomic force microscopy

In this work human chromosomes have been treated with RNase and pepsin to remove the layer of cellular material that covers the standard preparations on glass slides. This allows characterization of the topography of chromosomes at nanometer scale in air and in physiological solution by atomic force microscopy. Imaging of the dehydrated structure in air indicates radial arrangement of chromatin loops as the last level of DNA packing. However, imaging in liquid reveals a last level of organization consisting of a hierarchy of bands and coils. Additionally force curves between the tip and the chromosome in liquid are consistent with radial chromatin loops. These results and previous electron microscopy studies are analyzed, and a model is proposed for the chromosome structure in which radial loops and helical coils coexist.     

Coccolith biomineralisation studied with atomic force microscopy

Biomineralisation can only be understood as an interplay between organic and mineral phases. With this objective, we conducted an investigation of coccoliths using atomic force microscopy (AFM), an ultra-high resolution technique that requires no surface coating and can be used in air or under solution at ambient conditions of temperature and pressure. The detailed morphology, crystal structure, organic scales and organic coating of the coccolith species Coccolithus pelagicus , Helicosphaera carteri and Oolithotus fragilis were investigated. The fine structure of coccoliths is very complex, with the calcite either being smooth, dominated by steps or tuberculate; organic cover can be either granular or fibrous. Behaviour of coccolith surfaces during dissolution is influenced both by mineral and organic material and different surface types show variable resistance to dissolution. The organic coating protects element faces against etching. Through atomic resolution AFM, it is possible to establish the crystallographic structure of the distal shields of C. pelagicus and O. fragilis . Though elements of both species are dominated by stable crystal faces, there are important differences between them, with the external edge of elements being parallel to a cleavage direction in C. pelagicus but parallel to the atomic rows in O. fragilis . Thus, there is evidence that the biomineralisation of each species, and also of select areas of coccoliths of the same species, is markedly different.     

RecA-double stranded DNA complexes studied by atomic force microscopy.

RecA-double stranded (ds) DNA complexes have been studied by atomic force microscopy (AFM). When the complexes were prepared in the presence of ATP gamma S, fully covered RecA-dsDNA filaments were observed by AFM. When the concentration of RecA proteins was lower, various lengths of filaments were found. The variation of the observed structures may directly reflect the real distribution of the intermediate complexes in the reaction mixture, as the mixture was simply deposited on a mica surface for AFM observation without special fixation or staining. The use of a carbon nanotube (CNT) AFM tip enabled high resolution to reveal the periodicity of RecA-dsDNA filaments. Our observations demonstrated the potential of the AFM method for the structural studies of the RecA-dsDNA complexes, especially their intermediate states.     

Early events in insulin fibrillization studied by time-lapse atomic force microscopy

The importance of understanding the mechanism of protein aggregation into insoluble amyloid fibrils lies not only in its medical consequences, but also in its more basic properties of self-organization. The discovery that a large number of uncorrelated proteins can form, under proper conditions, structurally similar fibrils has suggested that the underlying mechanism is a general feature of polypeptide chains. In this work, we address the early events preceding amyloid fibril formation in solutions of zinc-free human insulin incubated at low pH and high temperature. Here, we show by time-lapse atomic force microscopy that a steady-state distribution of protein oligomers with a quasiexponential tail is reached within a few minutes after heating. This metastable phase lasts for a few hours, until fibrillar aggregates are observable. Although for such complex systems different aggregation mechanisms can occur simultaneously, our results indicate that the prefibrillar phase is mainly controlled by a simple coagulation-evaporation kinetic mechanism, in which concentration acts as a critical parameter. These experimental facts, along with the kinetic model used, suggest a critical role for thermal concentration fluctuations in the process of fibril nucleation.     

Enzyme-catalyzed hydrolysis of the supported phospholipid bilayers studied by atomic force microscopy

Atomic force microscopy (AFM) is employed to reveal the morphological changes of the supported phospholipid bilayers hydrolyzed by a phospholipase A₂ (PLA₂) enzyme in a buffer solution at room temperature. Based on the high catalytic selectivity of PLA₂ toward l-enantiomer phospholipids, five kinds of supported bilayers made of l- and d-dipalmitoylphosphatidylcholines (DPPC), including l-DPPC (upper leaflet adjacent to solution)/l-DPPC (bottom leaflet) (or l/l in short), l/d, d/l, d/d, and racemic ld/ld, were prepared on a mica surface in gel-phase, to explicate the kinetics and mechanism of the enzyme-induced hydrolysis reaction in detail. AFM observations for the l/l bilayer show that the hydrolysis rate for l-DPPC is significantly increased by PLA₂ and most of the hydrolysis products desorb from substrate surface in 40 min. As d-enantiomers are included in the bilayer, the hydrolysis rate is largely decreased in comparison with the l/l bilayer. The time used to hydrolyze the as-prepared bilayers by PLA₂ increases in the sequence of l/l, l/d, ld/ld, and d/l (d/d is inert to the enzyme action). d-enantiomers in the enantiomer hybrid bilayers remain on the mica surface at the end of the hydrolysis reaction. It was confirmed that the hydrolysis reaction catalyzed by PLA₂ preferentially occurs at the edges of pits or defects on the bilayer surface. The bilayer structures are preserved during the hydrolysis process. Based on these observations, a novel kinetics model is proposed to quantitatively account for the PLA₂-catalyzed hydrolysis of the supported phospholipid bilayers. The model simulation demonstrates that PLA₂ mainly binds with lipids at the perimeter of defects in the upper leaflet and leads to a hydrolysis reaction, yielding species soluble to the solution phase. The lipid molecules underneath subsequently flip up to the upper leaflet to maintain the hydrophilicity of the bilayer structure. Our analysis shows that d-enantiomers in the hybrid bilayers considerably reduce the hydrolysis rate by its ineffective binding with PLA₂.     

Thermal response of Langmuir-Blodgett films of dipalmitoylphosphatidylcholine studied by atomic force microscopy and force spectroscopy

The topographic evolution of supported dipalmitoylphosphatidylcholine (DPPC) monolayers with temperature has been followed by atomic force microscopy in liquid environment, revealing the presence of only one phase transition event at approximately 46 degrees C. This finding is a direct experimental proof that the two phase transitions observed in the corresponding bilayers correspond to the individual phase transition of the two leaflets composing the bilayer. The transition temperature and its dependency on the measuring medium (liquid saline solution or air) is discussed in terms of changes in van der Waals, hydration, and hydrophobic/hydrophilic interactions, and it is directly compared with the transition temperatures observed in the related bilayers under the same experimental conditions. Force spectroscopy allows us to probe the nanomechanical properties of such monolayers as a function of temperature. These measurements show that the force needed to puncture the monolayers is highly dependent on the temperature and on the phospholipid phase, ranging from 120+/-4 pN at room temperature (liquid condensed phase) to 49+/-2 pN at 65 degrees C (liquid expanded phase), which represents a two orders-of-magnitude decrease respective to the forces needed to puncture DPPC bilayers. The topographic study of the monolayers in air around the transition temperature revealed the presence of boundary domains in the monolayer surface forming 120 degrees angles between them, thus suggesting that the cooling process from the liquid-expanded to the liquid-condensed phase follows a nucleation and growth mechanism.     

Elastic properties of the cell wall of Aspergillus nidulans studied with atomic force microscopy

Currently, little is known about the mechanical properties of filamentous fungal hyphae. To study this topic, atomic force microscopy (AFM) was used to measure cell wall mechanical properties of the model fungus Aspergillus nidulans. Wild type and a mutant strain (deltacsmA), lacking one of the chitin synthase genes, were grown in shake flasks. Hyphae were immobilized on polylysine-coated coverslips and AFM force--displacement curves were collected. When grown in complete medium, wild-type hyphae had a cell wall spring constant of 0.29 +/- 0.02 N/m. When wild-type and mutant hyphae were grown in the same medium with added KCl (0.6 M), hyphae were significantly less rigid with spring constants of 0.17 +/- 0.01 and 0.18 +/- 0.02 N/m, respectively. Electron microscopy was used to measure the cell wall thickness and hyphal radius. By use of finite element analysis (FEMLAB v 3.0, Burlington, MA) to simulate AFM indentation, the elastic modulus of wild-type hyphae grown in complete medium was determined to be 110 +/- 10 MPa. This decreased to 64 +/- 4 MPa for hyphae grown in 0.6 M KCl, implying growth medium osmotic conditions have significant effects on cell wall elasticity. Mutant hyphae grown in KCl-supplemented medium were found to have an elastic modulus of 67 +/- 6 MPa. These values are comparable with other microbial systems (e.g., yeast and bacteria). It was also found that under these growth conditions axial variation in elastic modulus along fungal hyphae was small. To determine the relationship between composition and mechanical properties, cell wall composition was measured by anion-exchange liquid chromatography and pulsed electrochemical detection. Results show similar composition between wild-type and mutant strains. Together, these data imply differences in mechanical properties may be dependent on varying molecular structure of hyphal cell walls as opposed to wall composition.     

Double minute chromosomes in mouse methotrexate-resistant cells studied by atomic force microscopy

Double minute chromosomes (DMs) are acentric, autonomously replicating extra-chromosomes and frequently mediate gene amplification in tumor and drug resistant cells. Atomic force microscopy (AFM) is a powerful tool in microbiology. We used AFM to explore the ultrastructure of DMs in mouse fibroblasts 3T3R500. DMs in various phases of cell cycle were also studied in order to elucidate the mechanisms of their duplication and separation. Metaphase spread and induced premature condensed chromosomes (PCCs) were observed under the AFM. DMs were detected to be composed of two compact spheres linked by fibers. The fibers of DMs directly connected with metaphase chromosomes were observed. Many single-minutes and few DMs were detected in G1 PCCs, while more DMs were detected in S PCCs than in G1 PCCs. Besides, all of the DMs in G2 PCCs were coupled. Our present results suggested that DMs might divide into single-minutes during or before G1-phase, followed by duplication of the single-minutes in S-phase. Moreover, we introduced a new powerful tool to study DMs and got some ideal results.     

Stiffness of normal and pathological erythrocytes studied by means of atomic force microscopy

During recent years, atomic force microscopy has become a powerful technique for studying the mechanical properties (such as stiffness, viscoelasticity, hardness and adhesion) of various biological materials. The unique combination of high-resolution imaging and operation in physiological environment made it useful in investigations of cell properties. In this work, the microscope was applied to measure the stiffness of human red blood cells (erythrocytes). Erythrocytes were attached to the poly-L-lysine-coated glass surface by fixation using 0.5% glutaraldehyde for 1 min. Different erythrocyte samples were studied: erythrocytes from patients with hemolytic anemias such as hereditary spherocytosis and glucose-6-phosphate-dehydrogenase deficiency patients with thalassemia, and patients with anisocytosis of various causes. The determined Young's modulus was compared with that obtained from measurements of erythrocytes from healthy subjects. The results showed that the Young's modulus of pathological erythrocytes was higher than in normal cells. Observed differences indicate possible changes in the organization of cell cytoskeleton associated with various diseases.     

Transverse elasticity of myofibrils of rabbit skeletal muscle studied by atomic force microscopy

Surface structure of myofibrils of rabbit skeletal muscle and their transverse elasticity were studied by atomic force microscopy. Images of myofibrils had a periodic structure characteristic of sarcomeres of skeletal muscle fibers. The transverse elasticity distribution in the sarcomere was determined based on force-distance curves measured at various loci of single myofibrils. The Z-line in rigor myofibrils was the most rigid in all the loci of myofibrils studied under various physiological conditions. The overall transverse elasticity of myofibrils decreased in the order in rigor solution > +AMPPNP solution > relaxing solution. The "apparent" transverse Young's modulus of myofibrils estimated at the overlap region between thin and thick filaments was 84.0 +/- 18.1, 37.5 +/- 14.0, and 11.5 +/- 3.5 kPa in rigor, +AMPPNP, and relaxing solution respectively.     

Metastable and stable states of xanthan polyelectrolyte complexes studied by atomic force microscopy

The compaction of the semiflexible polysaccharide xanthan with selected multi- and polyvalent cations was studied. Polyelectrolyte complexes prepared at concentrations of 1-2 microg/ml were observed by tapping mode atomic force microscopy. High-molecular-weight xanthan compacted with chitosan yields a blend of mainly toroidal and metastable structures and a small fraction of rod-like species. Polyelectrolyte complexes of xanthan with polyethylenimine and trivalent chromium yielded similar structures or alternatively less well packed species. Racquet-type morphologies were identified as kinetically trapped states occurring on the folding path toward the energetically stable state of the toroids. Thermal annealing yielded a shift of the distribution of xanthan-chitosan morphologies toward this stable state. Ensembles of toroidal and rod-like morphologies of the xanthan-chitosan structures, collected using an asphericity index, were analyzed. The mean height of the toroids increased upon heating, with a selective increase in the height range above 2 nm. It is suggested that the observed metastable structures are formed from the high-molecular-weight fraction of xanthan and that these are driven toward the toroidal state, being a low-energy state, following annealing. Considered a model system for condensation of semiflexible polymers, the compaction of xanthan by chitosan captures the system at various stages in the folding toward a low-energy state and thus allows experimental analyses of these intermediates and their evolution.     

Shear-induced self-assembly of native silk proteins into fibrils studied by atomic force microscopy

Noncontact mode atomic force microscopy was used to investigate native silk proteins prepared in different ways. Low protein concentrations revealed that single protein molecules exhibit a simple, round shape with apparent diameters of 20-25 nm. Shearing the native protein solutions after extraction from the gland and prior to drying led to a beads-on-a-string assembly at the nanometer scale. Protein concentration had a significant effect on the morphology of the protein assemblies. At higher protein concentrations, shear-induced alignment into nanofibrils was observed, while lower concentrations lead to the formation of much thinner fibrils with a width of about 8 nm.     

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.     

Cellular secretion studied by force microscopy

Using the optical microscope, real adventures in cellular research began in earnest in the latter half of the nineteenth century. With the development of the electron microscope, ultramicroscopy, and improved cell staining techniques, significant advances were made in defining intracellular structures at the nanometer level. The invention of force microscopy, the atomic force microscope (AFM) in the mid 1980s, and the photonic force microscope (PFM) in the mid 1990s, finally provided the opportunity to study live cellular structure-function at the nanometer level. Working with the AFM, dynamic cellular and subcellular events at the molecular level were captured in the mid 1990s, and a new cellular structure 'the porosome' in the plasma membrane of all secretory cells has been defined, where specific docking and fusion of secretory vesicles occur. The molecular mechanism of fusion of the secretory vesicle membrane at the base of the porosome membrane in cells, and the regulated release of intravesicular contents through the porosome opening to the extracellular space, has been determined. These seminal discoveries provide for the first time a molecular mechanism of cell secretion, and the possibility to ameliorate secretory defects in disease states.     

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。