菅原 路子

Epithelial-mesenchymal transition (EMT), a qualitative change in cell migration behavior during cancer invasion and metastasis, is becoming a new target for anticancer drugs. Therefore, it is crucial to develop in vitro assays for the evaluation of the abilities of drug candidates to control EMT progression. We herein report on a method for the quantification of the EMT based on particle image velocimetry and correlation functions. The exponential fitting of the correlation curve gives an index (lambda), which represents transforming growth factor (TGF)-beta 1-induced EMT progression and its suppression by inhibitors. Moreover, real-time monitoring of the lambda value illustrates a time-dependent EMT progressing process, which occurs earlier than the bio-chemical changes in an EMT marker protein expression. The results demonstrate the usefulness of the present method for kinetic studies of EMT progression as well as EMT inhibitor screening.

A particle-fluid flow under alternating current (ac) electrokinetics was numerically simulated to investigate the three-dimensional (3-D) particle motion in a complex electric field of a high conductivity medium generated by an electrode-multilayered microfluidic device. The simulation model coupling thermal-fluid-electrical and dispersed particle problems incorporates three ac electrokinetics (ACEK) phenomena, namely, the ac electrothermal effect (ACET), thermal buoyancy (TB), and dielectrophoresis (DEP). The electrode-multilayered microfluidic device was fabricated with 40 electrodes exposed at the flow channel sidewalls in five cross sections. The governing equations of the simulation model are solved by the Eulerian-Lagrangian method with finite volume discretization. Fluid flow simulations in three cases with or without consideration of ACET and TB are performed to clarify the contributions of these phenomena. The fluid flow is found to be composed of short-range vortices due to ACET and long-range circulation due to TB based on the features of the electrode-multilayered microfluidic device. The 3-D particle trajectory influenced by the fluid flow is compared with four values of the real part of the Clausius-Mossotti (CM) factor to evaluate the DEP phenomenon. The simulation model is validated by experiments using a cell suspension. The pattern of cell trajectories in the upper part of the flow channel measured by particle tracking velocimetry agrees with the simulated pattern. By comparison of the simulation and experiment, it is found that the cells moving straight away from the electrode on the focal plane are decelerated within the region of 60 mu m from the electrode by positive-DEP with Re[K(omega)] = 0.08-0.11. Furthermore, the 3-D DEP-effective region and the ACET and TB dominant regions for the cells are predicted by evaluating the particle-fluid relative velocity due to DEP force with Re[K(omega)] = 0.10. Consequently, the flow mechanism and dominant region of each ACEK phenomenon in the device are clarified from the 3-D simulation validated by the experiments.

The distinct motion of GFP-tagged histone expressing cells (Histone-GFP type cells) has been investigated under ac electrokinetics in an electrode-multilayered microfluidic device as compared with Wild type cells and GFP type cells in terms of different intracellular components. The Histone-GFP type cells were modified by the transfection of green fluorescent protein-fused histone from the human lung fibroblast cell line. The velocity of the Histone-GFP type cells obtained by particle tracking velocimetry technique is faster thanWild type cells by 24.9% andGFP type cells by 57.1%. This phenomenon is caused by the more amount of proteins in the intracellular of single Histone-GFP type cell than that of theWild type and GFP type cells. The more amount of proteins in the Histone-GFP type cells corresponds to a lower electric permittivity ec of the cells, which generates a lower dielectrophoretic force exerting on the cells. The velocity of Histone-GFP type cells is well agreed with Eulerian-Lagrangian two-phase flow simulation by 4.2% mean error, which proves that the fluid motion driven by thermal buoyancy and electrothermal force dominates the direction of cells motion, while the distinct motion of Histone-GFP type cells is caused by dielectrophoretic force. The fluidmotion does not generate a distinct dragmotion for Histone-GFP type cells because the Histone-GFP type cells have the same size to theWild type and GFP type cells. These results clarified the mechanism of cells motion in terms of intracellular components, which helps to improve the cell manipulation efficiency with electrokinetics.

Persistent directional cell migration is involved in animal development and diseases. The small GTPase Rac1 is involved in F-actin and focal adhesion dynamics. Local Rac1 activity is required for persistent directional migration, whereas global, hyperactivated Rac1 enhances random cell migration. Therefore, precise control of Rac1 activity is important for proper directional cell migration. However, the molecular mechanism underlying the regulation of Rac1 activity in persistent directional cell migration is not fully understood. Here, we show that the ubiquitin ligase mind bomb 1 (Mib1) is involved in persistent directional cell migration. We found that knockdown of MIB1 led to an increase in random cell migration in HeLa cells in a wound-closure assay. Furthermore, we explored novel Mib1 substrates for cell migration and found that Mib1 ubiquitinates Ctnnd1. Mib1-mediated ubiq-uitination of Ctnnd1 K547 attenuated Rac1 activation in cultured cells. In addition, we found that posterior lateral line primordium cells in the zebrafish mib1(ta52b) mutant showed increased random migration and loss of directional F-actin-based protrusion formation. Knock down of Ctnnd1 partially rescued posterior lateral line primordium cell migration defects in the mib1(ta52b) mutant. Taken together, our data suggest that Mib1 plays an important role in cell migration and that persistent directional cell migration is regulated, at least in part, by the Mib1-Ctnnd1-Rac1 pathway.

A method was developed for photocontrolling cell adhesion on a gel substrate with defined mechanical properties. Precise patterning of geometrically controlled cell clusters and their migration induction became possible by spatiotemporally controlled photo-irradiation of the substrate. The clusters exhibited unique collective motion that depended on substrate stiffness and cluster geometry.