binding proteins and perform different functions. Two nonexclu- sive mechanisms have been proposed [75]. One is that the nucleators of actin polymerization ‘‘imprint’’ the structure of the resultant filament, which specifies the binding partner and, consequently, the function of the filament. The fact that the binding of one gelsolin molecule at the barbed end of a filament affects the structure of the filament over a long distance [86] implies that such an imprinting mechanism is highly plausible. The other mechanism depends on the mutually inhibitory binding of two actin binding proteins to actin filaments, coupled with long- range cooperative conformational changes to the filaments. More specifically, it was recently shown that actin filaments in fission yeast cells bind either fimbrin or tropomyosin [87]. This mutually exclusive binding of fimbrin or tropomyosin appears to depend on the ability of fimbrin to inhibit tropomyosin binding, and the long- range cooperativity of actin filaments ensures that neighboring subunits within a filament take the same conformation status. Here, we suggest that there is a third mechanism that is not exclusive with respect to the two mechanisms summarized above: mechanical stretch-induced long-range cooperative conformation- al changes to actin filaments. Finally, it is worth mentioning the extensibility of the thin filaments in skeletal muscle. Mechanical and X-ray diffraction measurements have established that active contraction stretches the thin filaments, which is accompanied by untwisting of the helix [88]. More recently, Tsaturyan et al. revealed that rigor binding of myosin heads, without significant tension, untwists the helix of thin filaments by ,0.2%, and applied tension further stretches the helix by a similar amount [71]. Although muscle is a complex and highly ordered system and interpretation of these results needs caution, S1-induced untwisting of actin filaments was observed in vitro as well (T. Yasunaga, personal communication). This implies that, at least in skeletal muscle, thin filaments are extensible springs, albeit rather stiff ones. Moreover, with the reasonable assumption that skeletal myosin heads possess a higher affinity for untwisted actin filaments, since the binding of skeletal myosin heads untwists the helix, it is further suggested that a positive feedback loop similar to what we proposed in Fig. 5B is formed in skeletal muscle. Conclusions Mechanical sensing and downstream signaling involving the cytoskeleton play important roles in cellular responses in both the short term and over long periods. A number of proteins involved in regulating the cytoskeleton [89,90,91,92], as well as the myosin motor [93,94], have been shown to possess mechanical sensitivity. In the present study, however, we suggest a new possibility, that actin filaments are themselves mechanical sensors, which further emphasizes the functional importance of the structural polymor- phism of actin filaments [11]. Supporting Information Figure S1 pDdNeo. The gene to be expressed in the form GFP- fusion protein is subcloned between the BamHI and SacI sites. Truncated DdpI is a 2,033 bp HindIII fragment of pBIG. pDdBsr carries a blasticidin S resistance cassette in place of G418 resis- tance cassette. (TIF) Figure S2 Comparison of the fluorescent probes for actin filaments. A: A wild-type Dictyostelium cell expressing GFP-Lifeact was permeabilized and fixed with 0.1% Triton X100 and 1% glutaraldehyde, stained with Rh-Ph, and observed using a confocal fluorescence microscope. B: A wild-type Dictyostelium cell express- Stretching Actin Filaments Attracts Myosin II ing GFP-actin was permeabilized/fixed, stained with Rh-Ph, and observed as above. The left, middle and right panel in each triplet show a GFP fluorescence image, rhodamine fluorescence image, and superimposition of the two pseudocolored images. Arrows show the direction of movement. Bars: 10 mm. (TIF) Figure S3 Comparison of GFP and rhodamine fluorescence intensities in wild-type cells expressing GFP-G680A S1 (A) and GFP-wild-type S1 (B) after permeabilization/fixation and staining with Rh-Ph. Live cells expressing GFP-wild-type S1 were brightly fluorescent (movie S1), but most of the fluorescence was lost during the permeabilization/fixation procedure, presumably because most of the GFP-wild-type S1 molecules were not bound to actin filaments in the cells. Therefore the original GFP fluorescence image in B was very dark and needed brightness enhancement for visualization. C: Starved and streaming wild-type cells expressing GFP-L596S S1DIQ observed as above. D: GFP-L596S S1DIQ - expressing myosin II-null cell grown in suspension for 3 days and then allowed to undergo cytokinesis C on a glass substrate was observed as above. The left, middle and right panel in each triplet shows a GFP fluorescence image, rhodamine fluorescence image, and superimposition of the two pseudocolored images. Bars: 10 mm. (TIF) Figure S4 Montage sequence of movie S7. Accumulations of GFP-L596S S1 along cytoplasmic strands during cytokinesis C and along the retracting cortices are marked by arrows and arrowheads, respectively. Numbers show elapsed time in min. Bar: 20 mm. (TIF) Figure S5 Localization of wild-type and mutant GFP-myoB- S1DIQ. Wild type cells expressing GFP- wild-type myoB-S1DIQ (A) or GFP-S332D/G607A myoB-S1DIQ (B) were observed by confocal microscopy. GFP- wild-type myoB-S1DIQ was mostly diffuse in the cytoplasm and only weakly concentrated in the ex- tending pseudopods (arrowheads). GFP-S332D/G607A myoB- S1DIQ was more prominently localized along the cell cortex (arrow) and in the extending pseudopods (arrowheads). Bar: 10 mm. (TIF) Movie S1 Starved wild-type cells expressing GFP-wild type S1. The width of this field is 85 mm, and the speed is 1056. (MOV) Movie S2 Starved wild -type cells expressing GFP-L596S S1. The width of this field is 85 mm, and the speed is 1056. (MOV) Movie S3 A wild-type cell expressing GFP-L596S S1 during cytokinesis under an agarose sheet. The width of this field is 33 mm, and the speed is 426. (MOV) Movie S4 Myosin-null cells expressing GFP-L596S S1 under- going cytokinesis C and retractions. Accumulations of GFP-L596S S1 along cytoplasmic strands during cytokinesis C and along the retracting cortices are marked in the montage sequence of this movie (Supplemental Fig. S4). The width of this field is 135 mm, and the speed is 4206. (MOV) Movie S5 Relocalization of GFP-L596S S1 and mCherry-actin in wild-type cells in response to local aspiration using a microcapillary. Speed: 506. This is the data set shown in Fig. 3A. (AVI) PLoS ONE | www.plosone.org 10 October 2011 | Volume 6 | Issue 10 | e26200