Takanori Kihara

Measurement of biomolecular diffusion in extracellular matrix condensed by fibroblasts using fluorescence correlation spectroscopy.

The extracellular matrix (ECM) comprises the heterogeneous environment outside of cells in a biological system. The ECM is dynamically organized and regulated, and many biomolecules secreted from cells diffuse throughout the ECM, regulating a variety of cellular processes. Therefore, investigation of the diffusive behaviors of biomolecules in the extracellular environment is critical. In this study, we investigated the diffusion coefficients of biomolecules of various sizes, measuring from 1 to 10 nm in radius, by fluorescence correlation spectroscopy in contracted collagen gel caused by fibroblasts, a traditional culture model of dynamic rearrangement of collagen fibers. The diffusion coefficients of the biomolecules in control collagen gel without cells decreased slightly as compared to those in solution, while the diffusion coefficients of biomolecules in the contracted gel at the cell vicinity decreased dramatically. Additionally, the diffusion coefficients of biomolecules were inversely correlated with molecular radius. In collagen gels populated with fibroblasts, the diffusion coefficient at the cell vicinity clearly decreased in the first 24 h of culture. Furthermore, molecular diffusion was greatly restricted, with a central focus on the populated cells. By using the obtained diffusion coefficients of biomolecules, we calculated the collagen fiber condensation ratio by fibroblasts in the cell vicinity at 3 days of culture to represent a 52-fold concentration. Thus, biomolecular diffusion is restricted in the vicinity of the cells where collagen fibers are highly condensed.

Effect of composition, morphology and size of nanozeolite on its in vitro cytotoxicity.

The extensive applications of nanoparticle materials in biomedical and biotechnological fields trigger the rapid development of nanotoxicology, because nanoparticles are reported to cause more damage than larger ones when human exposure to them. In the present manuscript, we prepared a series of zeolite nanocrystals with different frameworks, sizes, compositions and shapes, and provided the first report on their toxic difference. As our results, the toxicities of zeolite nanoparticles depend on their size, composition and shape when they are exposed to HeLa cells. The pure-silica nanozeolite silicalite-1 displays nontoxicity, but aluminum-containing nanozeolites, such as ZSM-5, LTL, and LTA, show a dose-dependent toxic manner. The different shapes of nanozeolites can lead to different cytotoxicities, while the influences of the surface charge differences of various nanozeolites on their toxicities are unconspicuous. More importantly, caspase-3 activity and LDH released assays showed that the toxic nanozeolites seem to induce cell necrosis rather than cell apoptosis by the damnification for the cell membranes. These results are expected to direct the applications of nanozeolites with different structures and shapes in biomedicine and clinic science.

The mechanical properties of a cell, as determined by its actin cytoskeleton, are important for nanoneedle insertion into a living cell.

Previously, we reported that a nanoneedle of 200 nm diameter manipulated by an atomic force microscope apparatus could be inserted into a living cell. The insertion probabilities varied according to cell type. However, the nanoneedle was never successfully inserted into artificial liposomes. In the current study, we found that the stress fibers and actin filaments comprising the plasmalemmal undercoat are important, determining factors as to whether a nanoneedle can be successfully inserted into a cell. Depolymerization of microtubules increased both the number of stress fibers and insertion efficiency in NRK cells. These results indicate that the insertion efficiency of a nanoneedle (200 nm in diameter) into a cell with a smaller actin meshwork in its plasmalemmal undercoat is enhanced and the formation of stress fibers obviously contributes to this incremental enhancement. These facts are not only important as technical information to improve the efficiency of cell manipulation but also as observations of the mechanical properties of the native cell cortex.

Development of a novel method to detect intrinsic mRNA in a living cell by using a molecular beacon-immobilized nanoneedle.

We developed a novel nanobiosensor for monitoring mRNA expression in a single living cell by using an atomic force microscope (AFM) equipped with a nanoprobe. The nanoprobe was constructed by immobilizing a biotin-modified molecular beacon onto an ultrathin needle (nanoneedle) via neutravidin. In order to evaluate the effectiveness of the nanoprobe, we selected glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as the target. A single HeLa cell contained approximately 1000 copies of GAPDH mRNA. The nanoprobe was directly inserted into living HeLa cells, and it reacted with the intrinsic target mRNA within the cells. The nanoprobe could be renatured by pulling it out of the cells. Further, we successfully used the nanoprobe for continuous detection of GAPDH mRNA in multiple cells, i.e., the nanoprobe was highly specific and sensitive for the detection of intrinsic mRNA in single living cells. mRNAs are thought to be highly condensed because of the large number of organelles and complexes present in cells and the limited space available for distribution. Therefore, direct analysis of intrinsic mRNAs in living cells would be advantageous, and our novel nanoprobe is highly suitable for monitoring the RNAs in living cells.

Development of a method to evaluate caspase-3 activity in a single cell using a nanoneedle and a fluorescent probe.

A method to detect an enzymatic reaction in a single living cell using an atomic force microscope equipped with an ultra-thin needle (a nanoneedle) and a fluorescent probe molecule was developed. The nanoneedle enables the low-invasive delivery of molecules attached onto its surface directly into a single cell. We hypothesized that an enzymatic reaction in a cell could be profiled by monitoring a probe immobilized on a nanoneedle introduced into the cell. In this study, a new probe substrate (NHGcas546) for caspase-3 activity based on fluorescent resonance energy transfer (FRET) was constructed and fixed on a nanoneedle. The NHGcas546-modified nanoneedle was inserted into apoptotic cells, in which caspase-3 is activated after apoptosis induction, and a change in the emission spectrum of the immobilized probe could be observed on the surface of the nanoneedle. Thus, we have developed a successful practical method for detecting a biological phenomenon in a single cell. We call the method MOlecular MEter with Nanoneedle Technology (MOMENT).

Fabrication of in vitro three-dimensional multilayered blood vessel model using human endothelial and smooth muscle cells and high-strength PEG hydrogel.

We fabricated a three-dimensional multilayered blood vessel model using human cells and high-strength PEG hydrogel. The hydrogel tube was physically suitable for perfusion culture, and cells were cultured on the hydrogel surface by binding with fibronectin. Using the layer-by-layer cell multilayered technique, we successfully constructed an artificial blood vessel.

Physical properties of mesenchymal stem cells are coordinated by the perinuclear actin cap.

Mesenchymal stem cells (MSCs) have been extensively investigated for their applications in regenerative medicine. Successful use of MSCs in cell-based therapies will rely on the ability to effectively identify their properties and functions with a relatively non-destructive methodology. In this study, we measured the surface stiffness and thickness of rat MSCs with atomic force microscopy and clarified their relation at a single-cell level. The role of the perinuclear actin cap in regulating the thickness, stiffness, and proliferative activity of these cells was also determined by using several actin cytoskeleton-modifying reagents. This study has helped elucidate a possible link between the physical properties and the physiological function of the MSCs, and the corresponding regulatory role of the actin cytoskeleton.

Mechanical force-based probing of intracellular proteins from living cells using antibody-immobilized nanoneedles

We developed a method combining atomic force microscopy (AFM) and antibody-immobilized nanoneedles to discriminate living cells by probing intracellular cytoskeletal proteins without the need for cell labeling. The nanoneedles are ultra-thin AFM probes sharpened to 200 nm in diameter. While retracting a nanoneedle inserted into a cell, we measured the mechanical force needed to unbind the antibody-target protein complex. Using this method, the intermediate filament protein, nestin and neurofilament were successfully detected in mouse embryonic carcinoma P19 cells and rat primary hippocampal cells within a minute for a single cell and cell differentiation states could be determined. Additionally, the measured magnitude of the force detecting nestin was indicative of the malignancy of breast cancer cells. This method was shown to affect neither the doubling time of cells nor does it leave extrinsic antibodies within the examined cells, allowing to be used in subsequent analyses in their native state.

Actin-based biomechanical features of suspended normal and cancer cells

The mechanical features of individual cells have been regarded as unique indicators of their states, which could constantly change in accordance with cellular events and diseases. Particularly, cancer progression was characterized by the disruption and/or reorganization of actin filaments causing mechanical changes. Thus, mechanical characterization of cells could become an effective cytotechnological approach for early detection of cancer. To develop mechanical cytotechnology, it would be necessary to clarify the mechanical properties in various cell adhesion states. In this study, we investigated the surface mechanical behavior of cancer and normal cells in the adherent and suspended states using atomic force microscopy. Adherent normal stromal cells showed high surface stiffness due to developed actin cap structures on their apical surface, whereas cancer cells did not have developed filamentous actin structures, and their surface stiffness was low. Upon cell detachment from the substrate, filamentous actin structures of adherent normal stromal cells reorganized to the cortical region and their surface stiffness decreased consequently however, the stiffness of suspended normal cells remained higher than that of cancer cells. These suspended state actin structures were similar, regardless of the cell type. Furthermore, the mechanical responses of the cancer and normal stromal cells to perturbation of the actin cytoskeleton were different, suggesting distinct regulatory mechanisms for actin cytoskeleton in cancer and normal cells in both adherent and suspended states. Therefore, cancer cells possess specific mechanical and actin cytoskeleton features different from normal stromal cells.

Regulation of cysteine-rich protein 2 localization by the development of actin fibers during smooth muscle cell differentiation

Cysteine-rich protein 2 (CRP2) is a cofactor for smooth muscle cell (SMC) differentiation. Here, we examined the mechanism of CRP2 distribution dynamics during SMC differentiation. CRP2 protein directly associated with F-actin through its N-terminal LIM domain and Gly-rich region, as determined by ELISA. In undifferentiated cells that contain few actin stress fibers, CRP2 was broadly distributed throughout the whole cell, including the nucleus. After induction of SMC differentiation, CRP2 localized to actin stress fibers as they formed. The stress fiber-localized CRP2 entered the nucleus because of induced actin depolymerization. These CRP2 dynamics were reproduced by in silico simulation. CRP2 localization dynamics, which affect CRP2 function, are regulated by the formation of actin stress fibers in conjunction with SMC differentiation.