Hiromi Miyoshi

Control of highly migratory cells by microstructured surface based on transient change in cell behavior

Cell migration control techniques have been proposed for cells with relatively low migratory activity, based on static analyses performed with cells that attain a temporally homogenous state after being exposed to a cell guiding stimulus. To elucidate new functions of substrate topography, we investigated the transient change in the behavior of highly migratory cells coming from a flat surface to a grooved surface on a silicon substrate covered with SiO(2). A single line groove (1.5 μm in width, 20 μm in depth) and intersecting grooves (1.5 μm in width, 5 μm in spacing, 20 μm in depth) functioned as an effective cell repellent. In the case of wider grooves, a single line groove (4 μm in width; 20 μm in width) had no specified function. In contrast, intersecting grooves (4 μm in width, 5 μm in spacing) functioned as a trap for the cells. Our findings yield a new design concept of cell repelling and trapping surfaces which are applicable to cell guiding methods and single or multiple cell confinement on cell culture substrates, and thus may contribute to development of more advanced biomaterials.

Characteristics of motility-based filtering of adherent cells on microgrooved surfaces.

Topographical features are known to physically affect cell behavior and are expected to have great potential for non-invasive control of such behavior. To provide a design concept of a microstructured surface for elaborate non-invasive control of cell migration, we systematically analyzed the effect of microgrooves on cell migration. We fabricated silicon microstructured surfaces covered with SiO(2) with microgrooves of various sizes, and characterized the behavior of cells moving from the flat surface to the grooved surface. The intersecting microgrooves with well-defined groove width absorbed or repelled cells precisely according to the angle of approach of the cell to the boundary with the grooved surface. This filtering process was explained by the difference in the magnitude of the lamellar dragging effect resulting from the number of the grooves interacting with the lamella of the cell. This study provides a framework to tailor the microgrooved surface for non-invasive control of cell migration with label-free detection of a specific property of the target cells. This should offer significant benefits to biomedical research and applications.

Characteristics of trajectory in the migration of Amoeba proteus

Summary. We investigated the behavior of migration of Amoeba proteus in an isotropic environment. We found that the trajectory in the migration of A. proteus is smooth in the observation time of 500-1000 s, but its migration every second (the cell velocity) on the trajectory randomly changes. Stochastic analysis of the cell velocity and the turn angle of the trajectory has shown that the histograms of the both variables well fit to Gaussian curves. Supposing a simple model equation for the cell motion, we have estimated the motive force of the migrating cell, which is of the order of piconewton. Furthermore, we have found that the cell velocity and the turn angle have a negative cross-correlation coefficient, which suggests that the amoeba explores better environment by changing frequently its migrating direction at a low speed and it moves rectilinearly to the best environment at a high speed. On the other hand, the model equation has simulated the negative correlation between the cell velocity and the turn angle. This indicates that the apparently rational behavior comes from intrinsic characteristics in the dynamical system where the motive force is not torquelike.

Spatiotemporal coordinated hierarchical properties of cellular protrusion revealed by multiscale analysis

We present a methodology for integrative multiscale analysis to highlight hierarchical properties of cellular protrusion and mechanochemical interactions in cellular protrusion based on live cell imaging data with high spatiotemporal resolution. As an appropriate experimental system, we selected non-polarized full-moon-shaped keratocytes that present balanced protrusion around the entire cell periphery at the cellular scale simultaneously with active protrusion and retraction at the subcellular scale. We achieved the observation of a whole cell with sub-micrometer spatial precision and sub-second time resolution for three minutes or more. The multiscale characteristics of cell peripheral activity and those of the cell peripheral shape were extracted from an identical image sequence by estimating the cell protrusion rates and the cell peripheral curvatures at various differential intervals. The spatiotemporal maps of the cell protrusion rates demonstrated a spatiotemporally nested structure of travelling waves of active protruding regions at the cellular and subcellular scales. Moreover, correlation analysis demonstrated the relationship between the cell protrusion rate and peripheral curvature at the subcellular scale. The novel integrative methodology presented here well highlighted the hierarchical properties of organized cellular protrusion, and further provided insight about the underlying mechanochemical interactions between the cell membrane and the actin filaments under the membrane.

Three-dimensional modulation of cortical plasticity during pseudopodial protrusion of mouse leukocytes.

Leukocytes can rapidly migrate virtually within any substrate found in the body at speeds up to 100 times faster than mesenchymal cells that remain firmly attached to a substrate even when migrating. To understand the flexible migration strategy utilized by leukocytes, we experimentally investigated the three-dimensional modulation of cortical plasticity during the formation of pseudopodial protrusions by mouse leukocytes isolated from blood. The surfaces of viable leukocytes were discretely labeled with fluorescent beads that were covalently conjugated with concanavalin A receptors. The movements of these fluorescent beads were different at the rear, central, and front surfaces. The beads initially present on the rear and central dorsal surfaces of the cell body flowed linearly toward the rear peripheral surface concomitant with a significant collapse of the cell body in the dorsal-ventral direction. In contrast, those beads initially on the front surface moved into a newly formed pseudopodium and exhibited rapid, random movements within this pseudopodium. Bead movements at the front surface were hypothesized to have resulted from rupture of the actin cytoskeleton and detachment of the plasma membrane from the actin cytoskeletal cortex, which allowed leukocytes to migrate while being minimally constrained by a substrate.

Chaotic behavior in the locomotion of Amoeba proteus

SummaryThe locomotion ofAmoeba proteus has been investigated by algorithms evaluating correlation dimension and Lyapunov spectrum developed in the field of nonlinear science. It is presumed by these parameters whether the random behavior of the system is stochastic or deterministic. For the analysis of the nonlinear parameters, n-dimensional time-delayed vectors have been reconstructed from a time series of periphery and area ofA. proteus images captured with a charge-coupled-device camera, which characterize its random motion. The correlation dimension analyzed has shown the random motion ofA. proteus is subjected only to 3–4 macrovariables, though the system is a complex system composed of many degrees of freedom. Furthermore, the analysis of the Lyapunov spectrum has shown its largest exponent takes positive values. These results indicate the random behavior ofA. proteus is chaotic and deterministic motion on an attractor with low dimension. It may be important for the elucidation of the cell locomotion to take account of nonlinear interactions among a small number of dynamics such as the sol-gel transformation, the cytoplasmic streaming, and the relating chemical reaction occurring in the cell.

Dynamics of Actin Stress Fibers and Focal Adhesions during Slow Migration in Swiss 3T3 Fibroblasts: Intracellular Mechanism of Cell Turning

To understand the mechanism regulating the spontaneous change in polarity that leads to cell turning, we quantitatively analyzed the dynamics of focal adhesions (FAs) coupling with the self-assembling actin cytoskeletal structure in Swiss 3T3 fibroblasts. Fluorescent images were acquired from cells expressing GFP-actin and RFP-zyxin by laser confocal microscopy. On the basis of the maximum area, duration, and relocation distance of FAs extracted from the RFP-zyxin images, the cells could be divided into 3 regions: the front region, intermediate lateral region, and rear region. In the intermediate lateral region, FAs appeared close to the leading edge and were stabilized gradually as its area increased. Simultaneously, bundled actin stress fibers (SFs) were observed vertically from the positions of these FAs, and they connected to the other SFs parallel to the leading edge. Finally, these connecting SFs fused to form a single SF with matured FAs at both ends. This change in SF organization with cell retraction in the first cycle of migration followed by a newly formed protrusion in the next cycle is assumed to lead to cell turning in migrating Swiss 3T3 fibroblasts.

Characteristics of motive force derived from trajectory analysis of Amoeba proteus.

Summary.We used a monochromatic charge-coupled-device camera to observe the migration behavior of Amoeba proteus every 5 s over a time course of 10000 s in order to investigate the characteristics of its centroid movement (cell velocity) over the long term. Fourier transformation of the time series of the cell velocity revealed that its power spectrum exhibits a Lorentz type profile with a relaxation time of a few hundred seconds. Moreover, some sharp peaks were found in the power spectrum, where the ratios of any two frequencies corresponding to the peaks were expressed as simple rational numbers. Analysis of the trajectory using a Langevin equation showed that the power spectrum reflects characteristics of the cell’s motive force. These results suggest that some phenomena relating to the cell’s motility, such as protoplasmic streaming and the sol–gel transformation of actin filaments, which seem to be independent phenomena and have different relaxation times, interact with each other and cooperatively participate in the generation process of the motive force.

Innovative approaches to cell biomechanics

Actin Cytoskeletal Structure in Migrating Cells.- Actin Cytoskeleton Generates Mechanical Forces for Cell Migration.- Multi-scale Mechanochemical Interactions between Cell Membrane and Actin Filaments.- Actin Network Flow and Turnover are coupled in Migrating Cells.- Mechanical Strain is involved in Actin Network Reorganization.- Actin Network Dynamics is Regulated by Actomyosin Interactions.- Biophysical Interactions between Cells and Extracellular Matrix.- Cell Migration in Engineered Micro-/Nano-environments with Controlled Physical Properties.- Engineered Biomaterial for Cell Manipulation.

The tension at the top of the animal pole decreases during meiotic cell division.

Meiotic maturation is essential for the reproduction procedure of many animals. During this process an oocyte produces a large egg cell and tiny polar bodies by highly asymmetric division. In this study, to fully understand the sophisticated spatiotemporal regulation of accurate oocyte meiotic division, we focused on the global and local changes in the tension at the surface of the starfish (Asterina pectinifera) oocyte in relation to the surface actin remodeling. Before the onset of the bulge formation, the tension at the animal pole globally decreased, and started to increase after the onset of the bulge formation. Locally, at the onset of the bulge formation, tension at the top of the animal pole began to decrease, whereas that at the base of the bulge remarkably increased. As the bulge grew, the tension at the base of the bulge additionally increased. Such a change in the tension at the surface was similar to the changing pattern of actin distribution. Therefore, meiotic cell division was initiated by the bulging of the cortex, which had been weakened by actin reduction, and was followed by contraction at the base of the bulge, which had been reinforced by actin accumulation. The force generation system is assumed to allow the meiotic apparatus to move just under the membrane in the small polar body. Furthermore, a detailed comparison of the tension at the surface and the cortical actin distribution indicated another sophisticated feature, namely that the contraction at the base of the bulge was more vigorous than was presumed based on the actin distribution. These features of the force generation system will ensure the precise chromosome segregation necessary to produce a normal ovum with high accuracy in the meiotic maturation.