Atsushi Ikai

Quantification of cell adhesion force with AFM: distribution of vitronectin receptors on a living MC3T3-E1 cell.

Distribution of vitronectin (VN) receptors on a living murine osteoblastic cell was successfully measured by atomic force microscopy (AFM). First, the distribution of the integrin beta(5) subunit which constitutes a part of the VN receptor on the cell was confirmed by conventional immunohistochemistry after fixing the cell. To visualize the distribution of the receptor on a living cell by an independent and potentially a more quantitative method, the AFM was used with a microbead attached to the cantilever tip to increase the area of contact and VN was immobilized on the microbead. Force measurements were then performed over a large area of a living murine osteoblastic cell using the microbead covered with VN.

mRNA analysis of single living cells.

Analysis of specific gene expression in single living cells may become an important technique for cell biology. So far, no method has been available to detect mRNA in living cells without killing or destroying them. We have developed here a novel method to examine gene expression of living cells using an atomic force microscope (AFM). AFM tip was inserted into living cells to extract mRNAs. The obtained mRNAs were analyzed with RT-PCR, nested PCR, and quantitative PCR. This method enabled us to examine time-dependent gene expression of single living cells without serious damage to the cells.

Quantification of fibronectin and cell surface interactions by AFM

Abstract The cell adhesion strength between FN and the living cell was successfully examined using an AFM cantilever having a FN-coated spherical microbead as a probe. The microbead was used to increase (by some 55-fold) the area of contact with the cell surface. Fibronectin (FN) was chosen as the ligand and immobilized on the bead. The area under the force-extension curve obtained during retraction of the FN-coated tip from the cell surface after contact was defined as ‘separation work’ and used as a parameter to evaluate the strength of the interaction. In previous studies, final rupture forces were taken to represent the strength of interactions, but in the present study using a large contact area, separation work was expected to reflect the overall adhesion strength in the contact area. The mean separation work between a FN-coated bead and mouse fibroblast cell was 0.69±0.45×10 −17 J/μm 2 . In the presence of 0.1 mg/ml FN in the scanning solution, this value decreased to 0.10±0.08×10 −17 J/μm 2 . The separation work between an unmodified microbead and cell was calculated to be 0.15±0.13×10 −17 J/μm 2 . Mapping of the separation work over a limited area of the cell surface was found to be reproducible for six repeated scans and the distribution of FN-binding sites on the cell surface was investigated by mapping over a large area. The results showed that separation work is a good measure of specific interactions between FN and the cell surface, and the system developed here is a promising quantitative method to evaluate cell adhesion force.

Self-oscillation technique for AFM in liquids

Abstract A simple self-oscillation circuit was built into a commercially available atomic force microscope (AFM) apparatus and used for imaging sample surfaces with a high rigidity in liquid environments. Imaging experiments were conducted with an AFM tip recommended for the normal tapping (intermittent contact) mode in liquids. In spite of the existence of many artificial resonance peaks, the self-oscillation could be successfully achieved with our simple electronic circuits. Force curves obtained in liquids showed that, as the tip contacted the surface, the resonance frequency began to increase steeply without a clear reduction of the vibration amplitude. Image of the sample surface was stably obtained under the condition of the positive frequency shift within a few Hertzs, which corresponded to a contact force comparable with Q -control operation.

A Review on: Atomic Force Microscopy Applied to Nano-mechanics of the Cell

Since its introduction in 1986, AFM has been applied to biological studies along with its widespread use in physics, chemistry and engineering fields. Due to its dual capabilities of imaging nano-materials with an atomic level resolution and of directly manipulating samples with high precision, AFM is now considered an indispensable instrument for nano-technological researchers especially in physically oriented fields. In biology in general, however, and in biotechnology in particular, its usefulness must be critically examined and, if necessary as it certainly is, further explored from a practical point of view. In this review, a new trend of applying AFM based technology to elucidate the mechanical basis of the cellular structure and its interaction with the extracellular matrix including cell to cell interaction is reviewed. Some of the recent studies done by using other force measuring or force exerting methods are also covered in the hope that all the nano-mechanical work on the cellular level will eventually contribute to the emergence of the mechano-chemical view of the cell in a unified manner.

Atomic force microscopy for cellular level manipulation: imaging intracellular structures and DNA delivery through a membrane hole

The atomic force microscope (AFM) is a versatile tool for imaging, force measurement and manipulation of proteins, DNA, and living cells basically at the single molecular level. In the cellular level manipulation, extraction, and identification of mRNAs from defined loci of a cell, insertion of plasmid DNA and pulling of membrane proteins, for example, have been reported. In this study, AFM was used to create holes at defined loci on the cell membrane for the investigation of viability of the cells after hole creation, visualization of intracellular structure through the hole and for targeted gene delivery into living cells. To create large holes with an approximate diameter of 5–10u2009µm, a phospholipase A2 coated bead was added to the AFM cantilever and the bead was allowed to touch the cell surface for approximately 5–10u2009min. The evidence of hole creation was obtained mainly from fluorescent image of Vybrant DiO labeled cell before and after the contact with the bead and the AFM imaging of the contact area. In parallel, cells with a hole were imaged by AFM to reveal intracellular structures such as filamentous structures presumably actin fibers and mitochondria which were identified with fluorescent labeling with rhodamine 123. Targeted gene delivery was also attempted by inserting an AFM probe that was coated with the Monster Green Fluorescent Protein phMGFP Vector for transfection of the cell. Following targeted transfection, the gene expression of green fluorescent protein (GFP) was observed and confirmed by the fluorescence microscope. Copyright

Mechanical perturbation-induced fluorescence change of green fluorescent protein

The force curve measurement mode of the atomic force microscope (AFM) is a powerful experimental technique in biotechnology. However, it is more effective if the spectroscopic properties of the biomolecule in the contact area can be simultaneously measured. Thus, we developed a confocal laser scanning microscope/AFM system. In this study, we simultaneously measured the fluorescence spectra of green fluorescent protein with the application of an external force in order to investigate the stability and dynamics of the β-barrel structure. Consequently, the fluorescence was quenched by applying both compression and extension forces and the quenching efficiencies differed in each case.

Direct Detection of Cellular Adaptation to Local Cyclic Stretching at the Single Cell Level by Atomic Force Microscopy

The cellular response to external mechanical forces has important effects on numerous biological phenomena. The sequences of molecular events that underlie the observed changes in cellular properties have yet to be elucidated in detail. Here we have detected the responses of a cultured cell against locally applied cyclic stretching and compressive forces, after creating an artificial focal adhesion under a glass bead attached to the cantilever of an atomic force microscope. The cell tension initially increased in response to the tensile stress and then decreased within ∼1xa0min as a result of viscoelastic properties of the cell. This relaxation was followed by a gradual increase in tension extending over several minutes. The slow recovery of tension ceased after several cycles of force application. This tension-recovering activity was inhibited when cells were treated with cytochalasin D, an inhibitor of actin polymerization, or with (-)-blebbistatin, an inhibitor of myosin II ATPase activity, suggesting that the activity was driven by actin-myosin interaction. To our knowledge, this is the first quantitative analysis of cellular mechanical properties during the process of adaptation to locally applied cyclic external force.

Single molecular dynamic interactions between glycophorin A and lectin as probed by atomic force microscopy.

Glycophorin A (GpA) is one of the most abundant transmembrane proteins in human erythrocytes and its interaction with lectins has been studied as model systems for erythrocyte related biological processes. We performed a force measurement study using the force mode of atomic force microscopy (AFM) to investigate the single molecular level biophysical mechanisms involved in GpA-lectin interactions. GpA was mounted on a mica surface or natively presented on the erythrocyte membrane and probed with an AFM tip coated with the monomeric but multivalent Psathyrella velutina lectin (PVL) through covalent crosslinkers. A dynamic force spectroscopy study revealed similar interaction properties in both cases, with the unbinding force centering around 60 pN with a weak loading rate dependence. Hence we identified the presence of one energy barrier in the unbinding process. Force profile analysis showed that more than 70% of GpAs are free of cytoskeletal associations in agreement with previous reports.

Molecular shape and binding force of Mycoplasma mobile's leg protein Gli349 revealed by an AFM study.

Recent studies of the gliding bacteria Mycoplasma mobile have identified a family of proteins called the Gli family which was considered to be involved in this novel and yet fairly unknown motility system. The 349kDa protein called Gli349 was successfully isolated and purified from the bacteria, and electron microscopy imaging and antibody experiments led to the hypothesis that it acts as the leg of M. mobile, responsible for attachment to the substrate as well as for gliding motility. However, more precise evidence of the molecular shape and function of this protein was required to asses this theory any further. In this study, an atomic force microscope (AFM) was used both as an imaging and a force measurement device to provide new information about Gli349 and its role in gliding motility. AFM images of the protein were obtained revealing a complex structure with both rigid and flexible parts, consistent with previous electron micrographs of the protein. Single-molecular force spectroscopy experiments were also performed, revealing that Gli349 is able to specifically bind to sialyllactose molecules and withstand unbinding forces around 70pN. These findings strongly support the idea that Gli349 is the leg protein of M. mobile, responsible for binding and also most probably force generation during gliding motility.