other model cells. These are, local assembly/disassembly of myosin II filaments [33,51,52], directional transport of myosin II filaments riding on the flow of cortical actin filaments [53,54,55], and association of the backbone of myosin II filaments with one or more components of the cell cortex [23,56,57,58,59]. Notably all three of these mechanisms require myosin II to be in the filament state, a notion that is supported by the observation that assembly- incompetent mutant myosin II is unable to localize in Dictyostelium [21,22] or Drosophila S2 cells [23]. Here, we demonstrated another mechanism, in which individual myosin II motor domains or S1 molecules preferentially bind to mechanically stretched subsets of actin filaments. Use of L596S or G680A S1 mutant was necessary because the time-averaged affinity of wild-type S1 for actin filaments was too weak in the presence of ATP to detect distinct intracellular localizations (movie S1 and Fig. S3). We believe that the localizations we observed with the GFP- S1 mutants reflect the intrinsic properties of the myosin II motor domain, as the two mutations appear to enhance the affinity for actin filaments through different molecular mechanisms. Consistent with this premise, GFP-fused S1 of non-muscle myosin IIB was shown to bind more strongly to stress fibers than to peripheral actin filaments in normal rat kidney cells [60]. Similarly, the myosin II motor domain of fission yeast (Myo2p) is reportedly enriched along contractile rings [58]. Intriguingly, within each of these cell types stress fibers and contractile rings are composed of mechanically stretched actin filaments, which suggests that preferential binding to mechanically stretched subsets of actin filaments is a common property of the myosin II motor domain, except that the actin affinity of the motor domain of rat and yeast myosin II in the presence of ATP is relatively stronger than that of Dictyostelium’s. Three different molecular mechanisms might contribute to the enhanced binding of the myosin II motor domain to stretched Stretching Actin Filaments Attracts Myosin II actin filaments (Fig. 5A). A conventional view would assume a mechanosensor that triggers a biochemical pathway that ultimate- ly leads to enhanced affinity of actin filaments for myosin II. For instance, it has been shown that tropomyosin isoforms differen- tially regulate the affinities of actin filaments for different classes of myosin motors [61,62,63]. According to Tang and Ostap [60], this differential regulation explains the enhanced binding of the rat non-muscle myosin IIB motor domain, but not that of the myosin I motor domain, to stress fibers along which tropomyosin is enriched. However, bona fide tropomyosin genes have not been identified in the completely sequenced Dictyostelium genome, making it difficult to speculate that biochemical signaling involving tropomyosin plays an important role in the stretch-induced recruitment of myosin II motors to actin filaments in Dictyostelium cells. In addition, although PTEN and cortexillin I have been implicated in the recruitment of myosin II filaments to stretched cortex in Dictyostelium [28,48], we found that neither of those molecules is required for the preferential binding of GFP-L596S S1 to stretched actin filaments. The second mechanism assumes stretch-induced higher order structural changes to the actin cytoskeleton. In the relaxed cell cortex, individual actin filaments are oriented more or less randomly [64], but mechanical stretching of the cortex would align the filaments in the direction of the stretch. The myosin II motor domain – e.g., the proteolytic muscle S1 [65] or recombinant His tagged Dictyostelium S1 (T. Uyeda, unpublished data) – tends to form bundles of actin filaments in the absence of ATP in vitro. It is thus possible that a GFP-S1 mutant carrying a mutation that increases its affinity for actin in the presence of ATP prefers to bind to the aligned actin filaments enriched in the stretched areas. On the other hand, the GFP-S1 mutants were not enriched in filopodia, which contain parallel bundles of actin filaments, as they were in the rear cortex of polarized cells. It is Figure 5. A: Three molecular mechanisms for recruiting myosin II S1 to stretched actin filaments in vivo. B: Possible physiological function of a three- component positive feedback loop consisting of stretch-induced conformational changes to actin filaments, preferential binding of the myosin II filaments to stretched actin filaments, and myosin II-dependent tension generation. doi:10.1371/journal.pone.0026200.g005 PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e26200