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A03 IWADATE, Yoshiaki |Proposed Research Projects (2014-2015)

Paper | Original Paper

2016

Ayane Sonoda, Chika Okimura, and *Yoshiaki Iwadate,
Shape and area of keratocytes are related to the distribution and magnitude of their traction forces,
Cell Structure and Function 41(1), 33-43 (2016).

[Summary] Fish epidermal keratocytes maintain an overall fan shape during their crawling migration. The shape-determination mechanism has been described theoretically and experimentally on the basis of graded radial extension of the leading edge, but the relationship between shape and traction forces has not been clarified. Migrating keratocytes can be divided into fragments by treatment with the protein kinase inhibitor staurosporine. Fragments containing a nucleus and cytoplasm behave as mini-keratocytes and maintain the same fan shape as the original cells. We measured the shape of the leading edge, together with the areas of the ventral region and traction forces, of keratocytes and mini-keratocytes. The shapes of keratocytes and mini-keratocytes were similar. Mini-keratocytes exerted traction forces at the rear left and right ends, just like keratocytes. The magnitude of the traction forces was proportional to the area of the keratocytes and mini-keratocytes. The myosin II ATPase inhibitor blebbistatin decreased the forces at the rear left and right ends of the keratocytes and expanded their shape laterally. These results suggest that keratocyte shape depends on the distribution of the traction forces, and that the magnitude of the traction forces depends on the area of the cells.

Chika Okimura, Kazuki Ueda, Yuichi Sakumura, *Yoshiaki Iwadate,
Fast-crawling cell types migrate to avoid the direction of periodic substratum stretching,
Cell Adhesion & Migration 10, 331-341 (2016).

[Summary] To investigate the relationship between mechanical stimuli from substrata and related cell functions, one of the most useful techniques is the application of mechanical stimuli via periodic stretching of elastic substrata. In response to this stimulus, Dictyostelium discoideum cells migrate in a direction perpendicular to the stretching direction. The origins of directional migration, higher migration velocity in the direction perpendicular to the stretching direction or the higher probability of a switch of migration direction to perpendicular to the stretching direction, however, remain unknown. In this study, we applied periodic stretching stimuli to neutrophil-like differentiated HL-60 cells, which migrate perpendicular to the direction of stretch. Detailed analysis of the trajectories of HL-60 cells and Dictyostelium cells obtained in a previous study revealed that the higher probability of a switch of migration direction to that perpendicular to the direction of stretching was the main cause of such directional migration. This directional migration appears to be a strategy adopted by fast-crawling cells in which they do not migrate faster in the direction they want to go, but migrate to avoid a direction they do not want to go.

Takako Nakata, Chika Okimura, Takafumi Mizuno, and *Yoshiaki Iwadate,
The role of stress fibers in the shape determination mechanism of fish keratocytes,
Biophysical Journal 110, 481-492 (2016).

[Summary] Crawling cells have characteristic shapes that are a function of their cell types. How their different shapes are determined is an interesting question. Fish epithelial keratocytes are an ideal material for investigating cell shape determination, since they maintain a nearly constant fan-shape during their crawling locomotion. We compared the shape and related molecular mechanisms in keratocytes from different fish species to elucidate the key mechanisms that determine cell shape. Wide keratocytes from cichlids applied large traction forces at the rear due to large focal adhesions, and showed a spatially loose gradient associated with actin retrograde flow rate, whereas round keratocytes from black tetra applied low traction forces at the rear small focal adhesions and showed a spatially steep gradient of actin retrograde flow rate. Laser ablation of stress fibers (contractile fibers connected to rear focal adhesions) in wide keratocytes from cichlids increased actin retrograde flow rate and led to slowed leading edge extension near the ablated region. Stress fibers thus might play an important role in the mechanism of maintaining cell shape by regulating actin retrograde flow rate.

2015

Naoki Narematsu, Raymond Quek, *Keng‐Hwee Chiam, and *Yoshiaki Iwadate,
Ciliary metachronal wave propagation on the compliant surface of Paramecium cells,
Cytoskeleton 72, 633-646 (2015).

[Summary] Ciliary movements in protozoa exhibit metachronal wave-like coordination, in which a constant phase difference is maintained between adjacent cilia. It is at present generally thought that metachronal waves require hydrodynamic coupling between adjacent cilia and the extracellular fluid. To test this hypothesis, we aspirated a Paramecium cell using a micropipette which completely sealed the surface of the cell such that no fluid could pass through the micropipette. Thus, the anterior and the posterior regions of the cell were hydrodynamically decoupled. Nevertheless, we still observed that metachronal waves continued to propagate from the anterior to the posterior ends of the cell, suggesting that in addition to hydrodynamic coupling, there are other mechanisms that can also transmit the metachronal waves. Such transmission was also observed in computational modeling where the fluid was fully decoupled between two partitions of a beating ciliary array. We also imposed cyclic stretching on the surface of live Paramecium cells and found that metachronal waves persisted in the presence of cyclic stretching. This demonstrated that, in addition to hydrodynamic coupling, a compliant substrate can also play a critical role in mediating the propagation of metachronal waves.

Hitomi Nakashima, Chika Okimura, and *Yoshiaki Iwadate,
The molecular dynamics of crawling migration in microtubule-disrupted keratocytes,
Biophysics and Physicobiology 12, 21-29 (2015).

[Summary] Cell-crawling migration plays an essential role in complex biological phenomena. It is now generally believed that many processes essential to such migration are regulated by microtubules in many cells, including fibroblasts and neurons. However, keratocytes treated with nocodazole, which is an inhibitor of microtubule polymerization – and even keratocyte fragments that contain no microtubules – migrate at the same velocity and with the same directionality as normal keratocytes. In this study, we discovered that not only these migration properties, but also the molecular dynamics that regulate such properties, such as the retrograde flow rate of actin filaments, distributions of vinculin and myosin II, and traction forces, are also the same in nocodazole-treated keratocytes as those in untreated keratocytes. These results suggest that microtubules are not in fact required for crawling migration of keratocytes, either in terms of migrating properties or of intracellular molecular dynamics.