Local rheology of human neutrophils investigated using atomic force microscopy

During the immune response, neutrophils display localized mechanical events by interacting with their environment through the micro-vascular transit, trans-endothelial, and trans-epithelial migration. Nano-mechanical studies of human neutrophils on localized nano-domains could provide the essential information for understanding their immune responsive functions. Using the Atomic Force Microscopy (AFM)-based micro-rheology, we have investigated rheological properties of the adherent human neutrophils on local nano-domains. We have applied the modified Hertz model to obtain the viscoelastic moduli from the relatively thick body regions of the neutrophils. In addition, by using more advanced models to account for the substrate effects, we have successfully characterized the rheological properties of the thin leading and tail regions as well. We found a regional difference in the mechanical compliances of the adherent neutrophils. The central regions of neutrophils were significantly stiffer (1,548 ± 871 Pa) than the regions closer to the leading edge (686 ± 801 Pa), while the leading edge and the tail (494 ± 537 Pa) regions were mechanically indistinguishable. The frequency-dependent elastic and viscous moduli also display a similar regional difference. Over the studied frequency range (100 to 300 Hz), the complex viscoelastic moduli display the partial rubber plateau behavior where the elastic moduli are greater than the viscous moduli for a given frequency. The non-disparaging viscous modulus indicates that the neutrophils display a viscoelastic dynamic behavior rather than a perfect elastic behavior like polymer gels. In addition, we found no regional difference in the structural damping coefficient between the leading edge and the cell body. Thus, we conclude that despite the lower loss and storage moduli, the leading edges of the human neutrophils display partially elastic properties similar to the cell body. These results suggest that the lower elastic moduli in the leading edges are more favorable for the elastic fluctuation of actin filaments, which supports the polymerization of the actin filaments leading to the active protrusion during the immune response.     

Selection of DNA aptamers using atomic force microscopy

Atomic force microscopy (AFM) can detect the adhesion or affinity force between a sample surface and cantilever, dynamically. This feature is useful as a method for the selection of aptamers that bind to their targets with very high affinity. Therefore, we propose the Systematic Evolution of Ligands by an EXponential enrichment (SELEX) method using AFM to obtain aptamers that have a strong affinity for target molecules. In this study, thrombin was chosen as the target molecule, and an ‘AFM-SELEX’ cycle was performed. As a result, selected cycles were completed with only three rounds, and many of the obtained aptamers had a higher affinity to thrombin than the conventional thrombin aptamer. Moreover, one type of obtained aptamer had a high affinity to thrombin as well as the anti-thrombin antibody. AFM-SELEX is, therefore, considered to be an available method for the selection of DNA aptamers that have a high affinity for their target molecules.     

High-resolution atomic force microscopy of DNA

A method using high resolution atomic force microscopy for imaging DNA has been elaborated. Using super-sharp probes and modified graphite as support for molecule adsorption, DNA molecule images were obtained whose resolution made possible the observation of their fine structure with repeated helical motifs. The method can be used to visualize individual spread molecules of single-stranded DNA.     

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     

Photodynamic effect on melanoma cells investigated by atomic force microscopy

Atomic force microscopy (AFM) is a modern experimental method for imaging of conducting or non-conducting samples. New trends in the application of scanning probe microscopy (SPM) give us the ability to scan live cells directly in their ingenuous surroundings or in air. Our apparatus was replenished with an inverse optical microscope, so we could observe the position of the scanning tip in every individual cell. The aim of the presented study is to picture the cell surface in air. A dry scanner in non-contact or tapping mode was used in the biological application of AFM. In our work the cell line G361 was used as a biological sample. We imaged the cell line before and after induction of a photodynamic effect (PDE) by irradiation of ZnTPPS4-loaded cells with a light dose of 15 J/cm(2). Individual cells before PDE induction had a smooth surface without protrusion on the entire surface. Cells after PDE induction did not have a smooth surface but their surface was rough with protrusion and in some places cleaved.     

Sequence-specific binding of luzopeptin to DNA.

We have examined the binding of luzopeptin, an antitumor antibiotic, to five DNA fragments of varying base composition. The drug forms a tight, possibly covalent, complex with the DNA causing a reduction in mobility on nondenaturing polyacrylamide gels and some smearing of the bands consistent with intramolecular cross-linking of DNA duplexes. DNAase I and micrococcal nuclease footprinting experiments suggest that the drug binds best to regions containing alternating A and T residues, although no consensus di- or trinucleotide sequence emerges. Binding to other sites is not excluded and at moderate ligand concentrations the DNA is almost totally protected from enzyme attack. Ligand-induced enhancement of DNAase I cleavage is observed at both AT and GC-rich regions. The sequence selectivity and characteristics of luzopeptin binding are quite different from those of echinomycin, a bifunctional intercalator of related structure.     

Quantum dot binding to DNA: Single-molecule imaging with atomic force microscopy

The interaction between nanoparticles (NPs) and DNA is of significance for both application and implication research of NPs. In this study, a single-molecule imaging technique based on atomic force microscopy (AFM) was employed to probe the NP-DNA interactions with quantum dots (QDs) as model NPs. Reproducible high-quality images of single DNA molecules in air and in liquids were acquired on mica by optimizing sample preparation conditions. Furthermore, the binding of QDs to DNA was explored using AFM. The DNA concentration was found to be a key factor influencing AFM imaging quality. In air and liquids, the optimal DNA concentration for imaging DNA molecules was approximately 2.5 and 0.25 μg/mL, and that for imaging DNA binding with QDs was 0.5 and 0.25 μg/mL, respectively. In the presence of QDs, the DNA conformation was altered with the formation of DNA condensates. Finally, the fine conformation of QD-DNA binding sites was examined to analyze the binding mechanisms. This work will benefit investigations of NP-DNA interactions and the understanding of the structure of NP-DNA bioconjugates. See accompanying article by Wang DOI: 10.1002/biot.201200309     

Modeling of DNA binding to the condensin hinge domain using molecular dynamics simulations guided by atomic force microscopy

The condensin protein complex compacts chromatin during mitosis using its DNA-loop extrusion activity. Previous studies proposed scrunching and loop-capture models as molecular mechanisms for the loop extrusion process, both of which assume the binding of double-strand (ds) DNA to the hinge domain formed at the interface of the condensin subunits Smc2 and Smc4. However, how the hinge domain contacts dsDNA has remained unknown. Here, we conducted atomic force microscopy imaging of the budding yeast condensin holo-complex and used this data as basis for coarse-grained molecular dynamics simulations to model the hinge structure in a transient open conformation. We then simulated the dsDNA binding to open and closed hinge conformations, predicting that dsDNA binds to the outside surface when closed and to the outside and inside surfaces when open. Our simulations also suggested that the hinge can close around dsDNA bound to the inside surface. Based on these simulation results, we speculate that the conformational change of the hinge domain might be essential for the dsDNA binding regulation and play roles in condensin-mediated DNA-loop extrusion.     

Towards nanomicrobiology using atomic force microscopy

At the cross-roads of nanoscience and microbiology, the nanoscale analysis of microbial cells using atomic force microscopy (AFM) is an exciting, rapidly evolving research field. Over the past decade, there has been tremendous progress in our use of AFM to observe membrane proteins and live cells at high resolution. Remarkable advances have also been made in applying force spectroscopy to manipulate single membrane proteins, to map surface properties and receptor sites on cells and to measure cellular interactions at the single-cell and single-molecule levels. In addition, recent developments in cantilever nanosensors have opened up new avenues for the label-free detection of microorganisms and bioanalytes.     

Biomolecular imaging using atomic force microscopy

Atomic force microscopy (AFM) has become a well-established technique for imaging single biomacromolecules under physiological conditions. The exceptionally high spatial resolution and signal-to-noise ratio of the AFM enables the substructure of individual molecules to be observed. In contrast to other methods, specimens prepared for AFM remain in a plastic state, which enables direct observation of the dynamic molecular response, creating unique opportunities for studying the structure–function relationships of proteins and their functionally relevant assemblies. This review presents recent advances in methods and applications of AFM to imaging biological samples. It is clear that AFM will become an increasingly important tool for probing both the structural and kinetic properties of biological macromolecules.     

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.     

Applications for atomic force microscopy of DNA.

Tapping mode atomic force microscopy (AFM) of DNA in propanol, dry helium, and aqueous buffer each have specific applications. Resolution is best in propanol, which precipitates and immobilizes the DNA and provides a fluid imaging environment where adhesive forces are minimized. Resolution on exceptional images of DNA appears to be approximately 2 nm, sufficient to see helix turns in detail, but the smallest substructures typically seen on DNA in propanol are approximately 6-10 nm in size. Tapping AFM in dry helium provides a convenient way of imaging such things as conformations of DNA molecules and positions of proteins on DNA. Images of single-stranded DNA and RecA-DNA complexes are presented. In aqueous buffer DNA molecules as small as 300 bp have been imaged even when in motion. Images are presented of the changes in shape and position of circular plasmid DNA molecules.     

Revealing the mode of action of DNA topoisomerase I and its inhibitors by atomic force microscopy

In this study, we used, for the first time, atomic force microscope (AFM) images to investigate the mode of action of DNA topoisomerase I (topo I) in the presence and absence of its inhibitors: camptothecin (CPT) and tyrphostin AG-1387. The results revealed that in the absence of the inhibitors, the enzyme relaxed supercoiled DNA starting from a certain point in the DNA molecules and proceeded in one direction towards one of the edges of the DNA molecule. In addition, the relaxation of the supercoiled DNA is subsequently followed by a knotting event. In the presence of CPT, enzyme-supercoiled DNA complexes in which the enzyme is locked inside a relaxed region of the supercoiled DNA molecule were observed. Tyrphostin AG-1387 altered the DNA relaxation process of topo I producing unique shapes of DNA molecules. AFM images of the topo I protein provided a picture of the enzyme, which resembles its known crystallographic structure. Thus, AFM images provide new information on the mode of action of topo I in the absence and presence of its inhibitors.     

DNA fragmentation by gamma radiation and electron beams using atomic force microscopy

Double-stranded pBS plasmid DNA was irradiated with gamma rays at doses ranging from 1 to 12 kGy and electron beams from 1 to 10 kGy. Fragment-size distributions were determined by direct visualization, using atomic force microscopy with nanometer-resolution operating in non-tapping mode, combined with an improved methodology. The fragment distributions from irradiation with gamma rays revealed discrete-like patterns at all doses, suggesting that these patterns are modulated by the base pair composition of the plasmid. Irradiation with electron beams, at very high dose rates, generated continuous distributions of highly shattered DNA fragments, similar to results at much lower dose rates found in the literature. Altogether, these results indicate that AFM could supplement traditional methods for high-resolution measurements of radiation damage to DNA, while providing new and relevant information.     

Probing membrane proteins using atomic force microscopy

To gain insights into how biological molecules function, advanced technologies enabling imaging, sensing, and actuating single molecules are required. The atomic force microscope (AFM) would be one of novel potential tools for these tasks. In this study, techniques and efforts using AFM to probe biomolecules are introduced and reviewed. The state-of-art techniques for characterizing specific single receptor using the functionalized AFM tip are discussed. An example of studying the angiotensin II type 1 (AT1) receptors expressed in sensory neuronal cells by AFM with a functionalized tip is given. Perspectives for identifying and characterizing specific individual membrane proteins using AFM in living cells are provided. Given that many diseases have their roots at the molecular scale and are best understood as a malfunctioning biological nanomachines, the prospects of these unique techniques in basic biomedical research or in clinical practice are beyond our imagination.     

Mechanical force analysis of peptide interactions using atomic force microscopy

Some peptides have previously been reported to bind low molecular weight chemicals. One such peptide with the amino acid sequence His-Ala-Ser-Tyr-Ser was selectively screened from a phage library and bound to a cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphine (TMpyP), with a binding constant of 10(5) M(-1) (J. Kawakami, T. Kitano, and N. Sugimoto, Chemical Communications, 1999, pp. 1765-1766). The proposed binding was due to pi-electron stacking from two aromatic amino acids of histidine and tyrosine. In this study, the weak interactions between TMpyP and the peptide were further investigated by force curve analysis using atomic force microscopy (AFM). The mechanical force required to unbind the peptide-porphyrin complex was measured by vertical movement of the AFM tip. Peptide self-assembled monolayers were formed on both a gold-coated mica substrate and a gold-coated AFM tip. The TMpyPs could bind between the two peptide layers when the peptide-immobilized AFM tip contacted the peptide-immobilized substrate in solution containing TMpyP. In the retracting process a force that ruptured the interaction between TMpyPs and peptides was observed. The unbinding force values correlated to the concentration of TMpyP. A detection limit of 100 ng/mL porphyrin was obtained for the force measurement, and was similar to surface plasmon resonance sensor detection limits. Furthermore, we calculated the product of the observed force and the length of the molecular elongation to determine the work required to unbind the complexes. The obtained values of unbinding work were in a reasonable range compared to the binding energy of porphyrin-peptide.     

The structure of intramolecular triplex DNA: atomic force microscopy study

We applied atomic force microscopy (AFM) for direct imaging of intramolecular triplexes (H-DNA) formed by mirror-repeated purine-pyrimidine repeats and stabilized by negative DNA supercoiling. H-DNA appears in atomic force microscopy images as a clear protrusion with a different thickness than DNA duplex. Consistent with the existing models, H-DNA formation results in a kink in the double helix path. The kink forms an acute angle so that the flanking DNA regions are brought in close proximity. The mobility of flanking DNA arms is limited compared with that for cruciforms and three-way junctions. Structural properties of H-DNA may be important for promoter-enhancer interactions and other DNA transactions.     

Imaging microtubules and kinesin decorated microtubules using tapping mode atomic force microscopy in fluids

The atomic force microscope has been used to investigate microtubules and kinesin decorated microtubules in aqueous solution adsorbed onto a solid substrate. The netto negatively charged microtubules did not adsorb to negatively charged solid surfaces but to glass covalently coated with the highly positively charged silane trimethoxysilylpropyldiethylenetriamine (DETA) or a lipid bilayer of 1,2-dipalmitoyl-3-dimethylammoniumpropane. Using electron beam deposited tips for microtubules adsorbed on DETA, single protofilaments could be observed showing that the resolution is up to 5 nm. Under conditions where the silane coated surfaces are hydrophobic, microtubules opened, presumably at the seam, whose stability is lower than that of the bonds between the other protofilaments. This led to a “sheet” with a width of about 100 nm firmly attached to the surface. Microtubules decorated with a stoichiometric low amount of kinesin molecules in the presence of the non-hydrolyzable ATP-analog 5′-adenylylimidodiphosphate could also be adsorbed onto silane-coated glass. Imaging was very stable and the molecules did not show any scan-induced deformation even after hundreds of scans with a scan frequency of 100 Hz. Received: 23 February 1999 / Revised version: 19 July 1999 / Accepted: 17 August 1999