Stretching Actin Filaments within Cells Enhances their Affinity for the Myosin II Motor Domain Taro Q. P. Uyeda1,2*, Yoshiaki Iwadate3,4, Nobuhisa Umeki1, Akira Nagasaki1, Shigehiko Yumura3 1 Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan, 2 Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Koto, Tokyo, Japan, 3 Department of Functional Molecular Biology, Graduate School of Medicine, Yamaguchi University, Yamaguchi, Yamaguchi, Japan, 4 Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Kawaguchi, Saitama, Japan Abstract To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine- phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli. Citation: Uyeda TQP, Iwadate Y, Umeki N, Nagasaki A, Yumura S (2011) Stretching Actin Filaments within Cells Enhances their Affinity for the Myosin II Motor Domain. PLoS ONE 6(10): e26200. doi:10.1371/journal.pone.0026200 Editor: Maria Gasset, Consejo Superior de Investigaciones Cientificas, Spain Received June 28, 2011; Accepted September 22, 2011; Published October 13, 2011 Copyright: 2011 Uyeda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by Grants-in-Aid to Y.I. and T.U. from the Ministry of Education, Culture, Sports, Science and Technology, Japan (http:// www.mext.go.jp/english/). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: t-uyeda@aist.go.jp Introduction Actin filaments play a variety of important roles in eukaryotic cells, and each of their functions depends on a specific set of actin binding proteins. Indeed, it is generally believed that local regulation by actin binding proteins determines the function of the actin filaments in that area [1,2]. In polarized amoeboid cells, for instance, Arp2/3-dependent polymerization of actin filaments pushes the membrane of the leading edge forward. At the same time, cofilin is enriched in the area slightly behind the leading edge, where it promotes the disassembly and turnover of the actin filaments. In the posterior of those cells, active interaction between actin filaments and bipolar myosin II filaments contracts the cortex, assisting detachment of the cell rear from the substrate and propulsion of the cytoplasm in a forward direction. Similarly, active interaction between actin filaments and myosin II filaments constricts the contractile rings in dividing cells. Biochemical and biophysical studies of the interaction between actin filaments and various actin-binding proteins are providing insight into the mechanisms underlying the functional differenti- ation of actin filaments in vivo. Most importantly, it is now well established that actin filaments assume multiple conformations, depending upon the binding of nucleotides and/or actin binding proteins [3,4,5,6]. It has also been shown that in certain cases the conformational changes are highly cooperative in the sense that the binding of an actin binding protein to an actin subunit within a filament induces conformational changes in neighboring subunits. For instance, the binding of cofilin changes the conformation within individual actin subunits as well as the interaction between the subunits, leading to significant shortening of the helical pitch [7,8]. The binding of cofilin to actin filaments is highly cooperative, which leads to the formation of cofilin clusters along the filaments under certain conditions, and the changes in helical pitch induced by cofilin can extend well beyond the clusters into the bare zone of the filaments [8]. One way to interpret these observations is that cofilin binding induces cooperative conforma- tional changes in neighboring actin subunits, which in turn increases the affinity of the neighboring actin subunits for cofilin [9], leading to cluster formation. A slightly different way of interpreting these observations is that actin subunits within filaments thermally fluctuate among multiple semi-stable struc- tures, and cofilin binds to segments with a favorable structure, thereby stabilizing that structure [8]. This view is supported by the observations that pure actin filaments naturally have variable twist PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26200