[10]. Egelman and his colleagues [11] went on to demonstrate that subunits within native actin filaments take one of the six distinct conformations, and subunits within a segment of the filament take the same conformation, representing strong cooperativity. Al- though questioned by another recent, high resolution electron microscopic analysis [12], we feel cooperative polymorphism of pure actin filaments plausible because it is able to explain well- established cooperative conformational changes of unbound subunits induced by binding of actin binding proteins to neighbor subunits within the same filament. Studies of cooperative conformational changes to actin filaments induced by myosin have a longer history. For instance, skeletal heavy meromyosin (HMM)-induced increases in the signal from fluorescently labeled actin subunits saturate when the molar concentration of HMM is only 1/10 that of the actin subunits [13]. Similar saturating effects of HMM or its motor domain (subfragment 1 or S1) at significantly sub-stoichiometric concen- trations have been observed using several different techniques [14,15,16,17,18]. Furthermore, the binding of HMM to actin filaments is cooperative in vitro [19,20]. In the case of Ca2+-actin filaments in the absence of ATP, this cooperativity results in the clustering of HMM molecules in some parts of the filament, which leaves other parts of the filament bare [19]. In the case of physiological Mg2+-actin filaments in the presence of low concentrations of ATP, the cooperativity is weaker in that some of the actin filaments appear bare, while others are sparsely bound with HMM molecules [20]. This weaker cooperativity cannot be explained by direct interactions between HMM molecules because they are separated by unbound actin subunits; instead, it most likely involves cooperative conformational changes in the actin subunits that increase the affinity of neighboring actin subunits for HMM. If this weaker cooperative binding between HMM and actin filaments reflects the preferential binding of HMM to subunits with a favorable conformation among multiple semi-stable conformations, as was suggested for the cooperative binding of cofilin to actin filaments [8], it would lead to an interesting hypothesis that the myosin II motor domain selectively binds to specific subsets of actin filaments having a favorable conformation, which would contribute to the proper intracellular localization of myosin II filaments in vivo. This view is apparently inconsistent with the observation that filament formation is necessary for proper intracellular localization of myosin II in Dictyostelium [21,22] and Drosophila S2 cells [23], and that GFP-fused S1 of Dictyostelium myosin II is diffusely distributed in the cytoplasm (T. Uyeda, unpublished observation). We speculate that the myosin II motor domain has a stronger affinity for subsets of actin filaments with a favorable conformation, but detection of this preferential binding in vivo is difficult because the time-averaged affinity between the motor domain and the actin filaments in the presence of ATP is too weak in the absolute sense. In the present study, therefore, we expressed two GFP-fused S1 mutants with amino acid substitutions that enhanced its affinity for actin filaments in the presence of ATP. It was our expectation that these GFP-S1 mutants could serve as probes enabling detection of subsets of actin filaments having a higher affinity for the myosin II motor domain in vivo. The results demonstrate that these GFP-S1 mutants do indeed preferentially bind to subsets of actin filaments; more specifically, they bind to mechanically stretched subsets of the filaments in vivo. Here we present a novel scheme whereby stretch- induced changes in actin filament conformation and the resultant promotion of myosin II binding help amoeboid cells to stabilize front-to-rear polarity and to respond to external mechanical stimuli. Stretching Actin Filaments Attracts Myosin II Materials and Methods Cell culture and expression of fluorescently labeled proteins Wild-type Dictyostelium discoideum AX2 cells and mutant cells lacking mhcA (encoding myosin II heavy chain), ctxA (encoding cortexillin I) or pten (encoding PTEN) were grown in plastic Petri dishes containing HL-5 medium [24] supplemented with penicillin and streptomycin at 22uC. Cells were transfected by electropora- tion with the Dictyostelium expression vector pBIG [25], pTIKL [26], pDdNeo or pDdBsr (Fig. S1) harboring a gene encoding a GFP- or mCherry-fusion protein. Transfectants were selected and grown in HL-5 medium containing 12 mg/ml G418 and/or 10 mg/ml blasticidin S. The construction of the plasmids to express fluorescently labeled proteins is detailed in Text S1. Live cell observation using confocal fluorescence microscopy Live cell imaging was accomplished in the following two ways. Cells expressing GFP-mutant S1 or GFP-myosin II heavy chain were settled on plastic Petri dishes with thin glass bottoms (Iwaki Glass, Japan) and observed using an Olympus IX-70 microscope equipped with a PlanApo 1006 (NA = 1.35) oil-immersed objec- tive and a confocal laser scanning unit (CSU 10, Yokogawa, Japan). To obtain chemotactic cells, the cells were starved for 8– 10 h in 17 mM K+-Na+-phosphate buffer (pH 6.4) before imaging. To image cells undergoing cytokinesis C, the cells were incubated for 3 days in HL-5 medium containing 12 mg/mL G418 in a Teflon flask on a rotating shaker and then allowed to settle onto a glass-bottomed dish for 15 min. The medium was then replaced with K+-Na+-phosphate buffer, and the cells were imaged as above. To observe flattened cells live, the cells were overlaid with a thin agarose sheet, as described previously [27]. Observation of fixed cells using confocal fluorescence microscopy Cells on glass-bottomed dishes were simultaneously permeabi- lized and fixed by replacing the K+-Na+-phosphate buffer with a solution containing 10 mM Pipes (pH 6.8), 3 mM MgCl2, 1 mM EGTA, 1 mM DTT, 0.1% Triton X-100, and 1% glutaraldehyde. After fixing the cells for 10 min, they were stained for 1 h in PBS containing 3 nM rhodamine-phalloidin (Rh-Ph), rinsed in PBS containing 10 mM DTT, and observed using the IX-70 confocal microscope. Superimposition of two pseudocolored images (GFP and rhodamine) of the same cells was accomplished using ImageJ software (http://rsb.info.nih.gov/ij/). Alternatively, cells flattened with an agarose sheet were fixed in ethanol containing 1% formalin. They were then stained with Rh- Ph after washing with PBS, and observed using a confocal microscope (LSM510 Meta, Carl Zeiss) equipped with a 1006 Plan Neofluor objective (NA = 1.3). Argon (488 nm line) and HeNe (543 nm line) lasers were used for excitation of GFP and rhodamine, respectively. Ratiometric images were calculated from the GFP and rhodamine images of the same cells using Image Calculator in ImageJ. Aspiration assays Portions of cells co-expressing GFP-mutant S1 and mCherry- actin were aspirated into a pipette as described previously [28]. Briefly, a suction pipette with an inner diameter of 3 mm was made from a glass capillary (G-1, Narishige, Tokyo, Japan) using a PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26200