Hideyuki Yoshimura

Electroporation of cell membrane visualized under a pulsed-laser fluorescence microscope

Controlled permeability can be conferred to cell membranes by exposing cells to a microsecond electric pulse of sufficient intensity (electroporation). By constructing a fluorescence microimaging system with a submicrosecond time resolution we have been able to resolve temporally and spatially the events in a single cell under a microsecond electric pulse. An enormous membrane conductance, corresponding to a loss of 0.01-0.1% of the membrane area, was observed in those membrane regions where the transmembrane potential induced by the electric pulse exceeded a critical value. The conductance decreased to a low level in a submillisecond after the pulse, leaving a moderately electroporated cell.

Nucleation and growth of two-dimensional colloidal crystals

Abstract We first grow two-dimensional crystals of nanometer latex particles from a thin film of water suspension placed on a flat solid substrate. The crystallization is performed by controlling the evaporation rate of water and the meniscus profile. The kinetic observations of the crystallization demonstrate two distinct processes: nucleation, preceded by thinning of the thin liquid film, and crystal growth. The crystal grows linearly in size, and its area increases as a quadratic function of time, due to the convective influx of particles from the suspension. A simple model of crystal growth interprets the kinetic data.

Two-dimensional crystallization of proteins on mercury

Abstract A new technique to obtain two-dimensional (2D) crystals of proteins is reported. A clean surface of mercury was utilized for monolayer formation and crystallization of the protein. Two different proteins, ferritin from a horse spleen and F1-ATPase from a thermophilic bacterium (TF1), were crystallized on the clean surface of mercury in an oxygen atmosphere. Two dimensional crystals were analyzed by electron crystallography. Ferritin was found to form a hexagonal lattice with unit cell parameters a = b = 12 nm and γ = 60°. The 2D crystal of ferritin at a 2.0 nm resolution in negative stain revealed a subunit formation with P3m1 symmetry. TF1 also formed a hexagonal lattice with unit cell parameters a = b = 10 nm and γ = 60°. The reconstructed image of TF1 at a 1.8 nm resolution showed a ring structure with six peripheral constituents and minor components located in the middle hole.

Torsional motion of eosin-labeled F-actin as detected in the time-resolved anisotropy decay of the probe in the sub-millisecond time range

The internal motion of F-actin in the time range from 10(-6) to 10(-3) second has been explored by measuring the transient absorption anisotropy of eosin-labeled F-actin using laser flash photolysis. The transient absorption anisotropy of eosin-F-actin at 20 degrees C has a component that decays in the submicrosecond time scale to an anisotropy of about 0.3. This anisotropy then decays with a relaxation time of about 450 microseconds to a residual anisotropy of about 0.1 after 2 ms. When the concentration of eosin-F-actin was varied in the range from 7 to 28 microM, the transient absorption anisotropy curves obtained were almost indistinguishable from each other. These results show that the anisotropy decay arises from internal motion of eosin-F-actin. Analysis of the transient absorption anisotropy curves indicates that the internal motion detected by the decay in anisotropy is primarily a twisting of actin protomers in the F-actin helix; bending of the actin filament makes a minor contribution only to the measured decay. The torsional rigidity calculated from the transient absorption anisotropy is 0.2 X 10(-17) dyn cm2 at 20 degrees C, which is about an order of magnitude smaller than the flexural rigidity determined from previous studies. Thus, we conclude that F-actin is more flexible in twisting than in bending. The calculated root-mean-square fluctuation of the torsional angle between adjacent actin protomers in the actin helix is about 4 degrees at 20 degrees C. We also found that the torsional rigidity is approximately constant in the temperature range from 5 to approximately 35 degrees C, and that the binding of phalloidin does not appreciably affect the torsional motion of F-actin.

Formation of two-dimensional colloid crystals in liquid films under the action of capillary forces

When two similar small particles are attached to a liquid interface they attract each other due to a lateral capillary force. This force appears because the gravitational potential energy of the floating particles decreases when they are approaching each other. This force is proportional to R6 (R is the particle radius), so it decreases very fast with particle size and becomes negligible for R<10 mu m. We found that the situation is quite different when the particles (instead of being freely floating) are partially immersed in a liquid layer on a substrate. In this case the energy of capillary attraction is proportional to R2 and turns out to be much larger than kT even with particles of diameter about 10 nm. The effect is related to the particle three-phase contact angle, i.e. to the intermolecular forces, rather than to gravity. The experiments show that the lateral capillary forces can bring about the formation of a two-dimensional array (2D-crystal) from both micrometre-size and submicrometre particles: latex spheres, protein globules, etc.

Development of a,b-plane-oriented hydroxyapatite ceramics as models for living bones and their cell adhesion behavior

In vertebrate bones and tooth enamel surfaces, the respective a,b-planes and c-planes of hydroxyapatite (HAp) crystals are preferentially exposed. However, the reason why the HAp crystals show different orientations depending on the type of hard tissues is not yet understood. To clarify this question, appropriate ceramic models with highly preferred orientation are necessary. In the present study, dense HAp ceramic models which have the same orientation as living bones were fabricated using composite powders of c-axis-oriented single-crystal apatite fibers (AF) and wet-synthesized apatite gels (AG). The results of crystalline identification and ultrastructural observation showed that the resulting HAp ceramics maintained the c-axis orientation of the AF particles, and their high a,b-plane orientation degrees could be maintained with small additive amounts of AG; however, when the AG content was over 30 mass%, this value decreased. The influence of orientation degree on the surface characteristics was investigated by evaluating the surface zeta-potential and wettability. These results show that increasing the a,b-plane orientation degree shifted the surface charge from negative to positive, and decreased the surface wettability. Initial cell-attachment assays were performed on these resulting ceramics using MC3T3-E1 cells as models of osteoblasts. The results show that the cell-attachment efficiency decreased with increasing a,b-plane orientation degree.

MONOLAYER CRYSTALLIZATION OF FLAGELLAR L-P RINGS BY SEQUENTIAL ADDITION AND DEPLETION OF LIPID

The L-P ring complex is thought to be a molecular bushing that supports flagellar motor rotation at about 10,000 revolutions per minute with presumably very little friction. Structural studies of this complex have been limited because only very small amount of samples are available. Therefore devising an efficient method of crystallization was essential. The addition of a phospholipid and its subsequent slow depletion by phospholipase A2 have been used to successfully grow well-ordered monolayer crystals that extend up to about 10 micrometers. The interaction of the L-P ring complex with lipid membranes was also visualized during this process.

Size control of catalytic nanoparticles by thermal treatment and its application to diameter control of single-walled carbon nanotubes

The authors report size control of catalytic nanoparticles by thermal annealing for diameter-controlled growth of single-walled carbon nanotubes (SWNTs). They found that Co nanoparticle-size gradually decreased through repetitive annealing at 1000°C in Ar ambient. Results of x-ray photoelectron spectroscopy and secondary ion mass spectroscopy show that thermal evaporation is responsible for the decrease. After SWNT growth using this phenomenon, the authors found that thinner SWNTs with a narrower diameter distribution grew as the nanoparticles became smaller. Their results provide a rational and straightforward technique to prepare catalysts having a desirable size and uniformity toward diameter-controlled SWNT growth.

Effect of nanoparticle density on narrow diameter distribution of carbon nanotubes and particle evolution during chemical vapor deposition growth

Single-walled carbon nanotubes (SWNTs) were synthesized by chemical vapor deposition (CVD) using catalytic nanoparticles both on the substrates and above the substrates in order to investigate the effect of nanoparticle density on diameter-controlled SWNT growth. As the density of the catalytic nanoparticles increased, tube-diameter distribution broadened and the diameter itself also increased. SWNTs observed in this study were grown by the base-growth mechanism and their diameters were much smaller than those of the nanoparticles. Based on elaborate diameter measurements, we reasonably conjecture that the time evolution of catalytic nanoparticles during CVD growth can explain these large size differences.

Electron Cryomicroscopy of Bacteriorhodopsin Vesicles: Mechanism of Vesicle Formation

We obtained vesicles from purple membrane of Halobacterium halobium at different suspension compositions (pH, electrolytes, buffers), following the procedure of Kouyama et al. (1994) (J. Mol. Biol. 236:990-994). The vesicles contained bacteriorhodopsin (bR) and halolipid, and spontaneously formed during incubation of purple membrane suspension in the presence of detergent octylthioglucoside (OTG) if the protein:OTG ratio was 2:1 by weight. The size distribution of the vesicles was precisely determined by electron cryomicroscopy and was found to be almost independent on the incubation conditions (mean radius 17.9-19 nm). The size distribution in a given sample was close to the normal one, with a standard deviation of approximately +/- 1 nm. During dialysis for removal of the detergent, the vesicles diminished their radius by 2-2.5 nm. The results allow us to conclude that the driving force for the formation of bR vesicles is the preferential incorporation of OTG molecules in the cytoplasmic side of the membrane (with possible preferential delipidation of the extracellular side), which creates spontaneous curvature of the purple membrane. From the size distribution of the vesicles, we calculated the elasticity bending constant, K(B) approximately 9 x 10(-20) J, of the vesicle wall. The results provide some insight into the possible formation mechanisms of spherical assembles in living organisms. The conditions for vesicle formation and the mechanical properties of the vesicles could also be of interest with respect to the potential technological application of the bR vesicles as light energy converters.