Hidetake Miyata

Tying a molecular knot with optical tweezers

Filamentous structures are abundant in cells. Relatively rigid filaments, such as microtubules and actin, serve as intracellular scaffolds that support movement and force, and their mechanical properties are crucial to their function in the cell. Some aspects of the behaviour of DNA, meanwhile, depend critically on its flexibility—for example, DNA-binding proteins can induce sharp bends in the helix. The mechanical characterization of such filaments has generally been conducted without controlling the filament shape, by the observation of thermal motions or of the response to external forces or flows. Controlled buckling of a microtubule has been reported, but the analysis of the buckled shape was complicated. Here we report the continuous control of the radius of curvature of a molecular strand by tying a knot in it, using optical tweezers to manipulate the strands ends. We find that actin filaments break at the knot when the knot diameter falls below 0.4 µm. The pulling force at breakage is around 1 pN, two orders of magnitude smaller than the tensile stress of a straight filament. The flexural rigidity of the filament remained unchanged down to this diameter. We have also knotted a single DNA molecule, opening up the possibility of studying curvature-dependent interactions with associated proteins. We find that the knotted DNA is stronger than actin.

The role of actomyosin contractility in the formation and dynamics of actin bundles during fibroblast spreading.

We studied the process of formation of stress fibres and involvement of phosphorylation of myosin-II during spreading of Swiss 3T3 fibroblasts. In cells that were allowed to spread for 1 h on a glass surface, circular bundles of actin and myosin-II filament were present. At 2-3 h after the plating, cells showed a polygonal and polarized shape. The proportion of the cells having circular bundles was decreased, whereas that of the cells with straight bundles of actin filaments was increased. At 4 h after the plating, cells were completely polarized and stress fibres were present at the periphery and the dorsal and ventral surfaces of the cells. Thus, spreading cells possessed different forms of actomyosin bundles corresponding to the cell shape. In circular bundles and stress fibres, myosin regulatory light chains were diphosphorylated. Formation of circular bundles and stress fibres was suppressed after the treatment of the cells with Y-27632, a Rho-kinase inhibitor, or blebbistatin, a myosin-II inhibitor. In digitonin-extracted cells, circular bundles as well as stress fibres contracted following the addition of Mg-ATP. These results suggest that circular bundles are contractile structures containing actin and phosphorylated myosin-II filaments, and the formation of circular bundles is regulated by Rho-kinase.

Probing the Cell Peripheral Movements by Optical Trapping Technique

Swiss 3T3 fibroblasts cultured on a poly-L-lysine-coated coverslip was stimulated with 0.5 micro M phorbol myristate acetate, and the movements of the peripheral membranes were probed with a 1- micro m polystyrene bead held in an optical trap. The bead brought into contact with the cell edge occasionally moved away from and returned to the original position. The movement ranged over 100 nm and occurred mainly in one direction, suggesting that the protruding cell membrane pushed the bead. The maximum velocities derived from individual pairs of protrusive and withdrawal movements exhibited a correlation, which is consistent with the previous reports. Acceleration and deceleration occurred both in the protrusive and withdrawal phases, indicating that the movements were regulated. Movement of the membrane occurred frequently with an ensemble-averaged maximum speed of 23 nm/s at the trap stiffness of 0.024 pN/nm, but it was strongly suppressed when the trap stiffness was increased to 0.090 pN/nm. Correlation of the protrusive and withdrawal velocities and the acceleration and deceleration both in the protrusive and withdrawal phases can be explained by the involvement of myosin motor at least in the withdrawal process. However, the fact that the movements were suppressed at higher trap stiffness implies a stochastic nature in the creation of the gap between the peripheral cell membrane and the actin network underlying it.

The effect of a 50-Hz sinusoidal magnetic field on nitric oxide (NO) production by human umbilical vein endothelial cells (HUVECs)

To investigate the effect of magnetic fields on living systems, we analyzed the effect of a 50-Hz, 1-mT sinusoidal magnetic field on nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs). Statistically significant differences in NO production were identified between exposed and unexposed (sham) cells. This finding confirms previous studies showing that magnetic fields can influence NO production; however, unlike previous studies, our results demonstrate two-way deviation of NO production under the influence of a magnetic field. Thus, in some cases, NO production was higher for the exposed group than the sham group, while in other cases the reverse was true. We suggest that the magnetic field affected the homeostasis of NO levels in a complex manner. Thus, the resulting NO level was dependent on the condition of the cell in each experiment or cells in each experiment were at a different stage in the cell cycle and therefore exhibited a different response when exposed to the magnetic field.

Evaluation of cell viability, DNA single-strand breaks, and nitric oxide production in LPS-stimulated macrophage RAW264 exposed to a 50-Hz magnetic field

Abstract Purpose: Synergistic effects between cellular oxidative stress and magnetic fields may explain the adverse biological effects of 50/60 Hz magnetic fields. To determine whether this hypothesis holds in macrophage RAW264 cells, we measured DNA single-strand breaks (SSB), cell viability, and nitric oxide (NO) production in cells with or without exposure to 0.5-mT, 50-Hz magnetic fields for 24 h and with or without simultaneous stimulation via the bacterial endotoxin, lipopolysaccharide (LPS). Materials and methods: Macrophages stimulated with 10 ng/ml LPS for 1 h were exposed to or not exposed to a magnetic field and were then subjected to (1) the alkaline comet assay to measure SSBs, (2) trypan-blue exclusion assay for cell viability, and (3) measurements of NO for evaluation of oxidative stress. Results: The 50-Hz magnetic field enhanced DNA SSB and decreased cell viability only in the LPS-stimulated macrophages in which NO production was greatly enhanced. The magnetic field alone did not alter NO production. Conclusion: Co-stimulation of the cell with LPS and a 50-Hz magnetic field promoted SSB and lowered cell viability, but these were not mediated by LPS-induced NO production.

3D Coupling of Fibronectin Fibril Arrangement with Topology of Ventral Plasma Membrane

Abstract Interaction of integrins with extracellular matrices is essential for cell adhesion to substrata. Ventral surfaces of fibroblasts adhering to flat substrata are not flat but have uneven 3D topology. However, spatial relationship between the topology of the ventral cell surface and arrangement of extracellular matrix fibrils remains unclear. Here, we report a novel and simple method based on total internal reflection fluorescence microscopy to quantify the distance between the ventral plasma membrane and the glass substratum. We observe that the distance varies from < 25 nm at focal adhesions to 40–50 nm at close contacts and > 80 nm in other regions. Furthermore, by applying this novel method, we show that fibronectin fibrils are also separated from the substratum in regions where the ventral cell surface-substratum distance is > 80 nm. Our results reveal that fibronectin fibrils are not simply adsorbed to the glass substratum but follow the ventral cell surface topology.

DNA Strand Breaks in Fibroblasts Exposed to a 50-Hz Magnetic Field

Bodies are exposed to environmental magnetic fields of low frequency produced by objects such as power lines. A large number of studies have investigated the biological effects of environmental magnetic fields [1]. However, the mechanism of magnetic field action on biological systems is not well understood, partly because of a lack of reproducibility. For example, studies based on the comet assay have reported DNA strand breaks caused by low-frequency magnetic fields at 1 mT [2], whereas some of later studies failed to confirm these results. Because DNA integrity is essential for the activities of life, the present study focused on the effect of low-frequency magnetic fields on DNA integrity.

Transcriptional effects of 50 Hz magnetic fields at 1.2 μT and 100 μT on human breast cancer MCF-7 cells

The International Agency for Research on Cancer (IARC) classified power frequency magnetic fields as a possible human carcinogen. Alteration in transcription programs is a fundamental feature of cancer. Here, using DNA array technology, we examined the transcriptional effects of 50 Hz magnetic fields on human breast cancer MCF-7 cells. It was found that expression of several oncogenes was significantly altered by magnetic-field exposure and that gene expression profilings were similar in MCF-7 cells exposed to magnetic fields at 1.2 μT and 100 μT for 1 week.

Lipid Bilayer Vesicles with Numbers of Membrane-Linking Pores

We report that phospholipid membranes spontaneously formed in aqueous medium giant unilamellar vesicles (GUVs) possessing many membranous wormhole-like structures (membrane-linking pores, MLPs). By phase contract microscopy and confocal fluorescence microscopy, the structures of the MLPs, consisting of lipid bilayer, were resolvable, and a variety of vesicular shapes having many MLPs (a high genus topology) were found. These vesicles were stable but easily deformed by micromanipulation with a microneedle. We also observed the size reduction of the MLPs with the increase in membrane tension, which was qualitatively consistent with a prediction from a simple dynamical model.

Methods for Physical Properties of Biomembranes and Cells

Various methods underlying progress of life science which include biophysics of cell and biomembrane are introduced in this chapter. Measurements of bulk system such as X-ray diffraction, differential scanning calorimetry and optical spectroscopy are changed to observation of specific region by use of imaging technique. Bio-imaging system such as X-ray CT and MRI is developed by need of medical diagnosis.