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Journal of Biochemistry Advance Access originally published online on November 13, 2008
Journal of Biochemistry 2009 145(2):137-150; doi:10.1093/jb/mvn151
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© The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

The Role of Actomyosin Contractility in the Formation and Dynamics of Actin Bundles During Fibroblast Spreading

Yosuke Senju* and Hidetake Miyata

Department of Physics, Graduate School of Science, Tohoku University, Sendai, Miyagi 980-8578, Japan

*To whom correspondence should be addressed. Tel: +81-22-795-6465, Fax: +81-22-795-6774, E-mail: yosuke{at}bio.phys.tohoku.ac.jp

Received October 13, 2008; Accepted October 22, 2008


  Abstract

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.

Key Words: actin, adhesion, dynamics, myosin, polarity

Abbreviations: GFP, green fluorescent protein; MLCK, myosin light chain kinase; MRLC, myosin regulatory light chains; 1P-MRLC, monophosphorylated MRLC; 2P-MRLC, diphosphorylated MRLC; TIRF, total internal reflection fluorescence


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