unlikely this is due to some unfavorable geometry of the filaments within the bundles, such as parallel vs. anti-parallel alignments, because exogenously-added GFP-S1 efficiently bound to filopodial actin filaments in Triton X-100-treated cells (S. Yumura, unpublished data). Moreover, GFP-S332D/G607A myoB- S1DIQ bound to similar degrees along filopodial actin bundles and cortical actin filaments (Fig. 2), ruling out the possibility that limited accessibility prevented the binding of the myosin II GFP- S1 mutants to the filopodial actin bundles. The third mechanism assumes that stretching induces structural changes in individual actin filaments at the atomic level. It is well established that actin filaments are able to assume multiple conformational states, in which individual actin subunits take on different structures (for review, see [5,11]). A number of actin binding proteins [6,66,67,68,69,70], including skeletal muscle myosin II [71] and brain myosin V [72], have been shown to change the structure of actin filaments. Those structural changes most likely increase the affinity of the filaments for that particular actin binding protein (cooperative binding), as has been demon- strated in the case of cofilin [8,73,74]. In addition, those conformational changes to the filament could modulate the affinity for other actin binding proteins, either positively or negatively [3] - e.g., so that the preferential binding of a specific group of actin binding proteins to a particular subset of actin conformers would lead to the exclusion of other actin binding proteins [75]. At present, there is no detailed information about stretch-induced conformational changes to actin subunits within filaments, but molecular dynamics simulations suggest that mechanical stretching of actin filaments with a physiologically relevant force (200 pN) is able to untwist the helix and change the mechanical properties of the filament [76]. Furthermore, Shimo- zawa and Ishiwata detected a fluorescence increase when they stretched tetramethylrhodamine-labeled actin filaments, signaling the occurrence of stretch-induced changes in the atomic structure of the actin subunits [77]. The functional relevance of these conformational changes were confirmed by Sokabe and his colleagues, who found that cofilin severs stretched actin filaments more inefficiently than relaxed ones in vitro [78], suggesting actin subunits within stretched filaments may assume a conformation having a lower affinity for cofilin. Intriguingly, cofilin binding not only changes the atomic structure of each subunit, but also reduces the helical pitch of the filaments (super twisting) [7,8]. Conversely, the binding of skeletal S1 slightly untwists the helix of actin filaments [71] (T. Yasunaga, personal communication), raising the possibility that the myosin II motor domain prefers to bind to untwisted actin filaments. As mechanical stretching is suggested to untwist the actin filaments [76], therefore, it is highly plausible that S1 prefers to bind to stretched actin filaments. On the other hand, a population of pure actin filaments exhibits a spectrum of helical pitches in the absence of external forces [8,10]. Thus, even in the absence of an applied force, a certain fraction of actin filaments will presumably possess a more untwisted conformation with a higher affinity for the myosin II motor domain; stretching induced by an applied force only increases the untwisted fraction. For these reasons, and because we previously demonstrated the cooperative binding of myosin II to Mg2+-actin filaments without additional proteins or alignment of the filaments in vitro [20], we favor the third mechanism, in which stretch-induced changes in the atomic structure of actin filaments and/or untwisting of the helix attract the myosin II motor domain. However, we do not exclude the possible contributions of either or both of the other two mechanisms. Needless to say, the aforementioned regulatory mechanisms involving the assembly Stretching Actin Filaments Attracts Myosin II of myosin II filaments also play important roles in the intracellular localization of myosin II. Interestingly, the myosin I motor domain did not preferentially bind to stretched actin filaments. This is again in line with the report from Tang and Ostap, who showed that GFP-fused myo1b, a rat myosin I, localized along the cell periphery but not along stress fibers [60]. Distinct intracellular localizations of members of the same family of actin binding proteins have been reported for calponin-homology proteins [79], coronin [80], tropomyosin [81] and talin [82]. Notably, distinct intracellular localizations along specific actin-containing structures were also observed with the GFP-fused, isolated ABDs of Dictyostelium a-actinin and filamin, both of which are calponin homology proteins [79]. This suggests that subtle differences in the actin binding face of homologous actin binding domains can result in preferential binding to different conformations of actin subunits. In this scenario, the filamin ABD, which binds to cortical actin cytoskeleton but not to those in protruding pseudopods [79], may share a similar preference for actin structures with the myosin II motor domain. Physiological relevance of the preferential binding of myosin II motor domain to stretched actin filaments In Dictyostelium, myosin II filaments interact with actin filaments located at the rear of polarized cells, at the tips of retracting pseudopods, and along the contractile rings in dividing cells, and drive local contraction [27,40]. Thus, if individual myosin II motor domains have a higher affinity for stretched actin filaments, that would lead to formation of a local positive feedback loop, consisting of accumulation of myosin II filaments, increased tension, and conformational changes within the actin filaments that attract additional myosin II filaments (Fig. 5B). The affinity between individual motor domains and actin filaments is too weak for stable association in the presence of ATP, which necessitated the use of S1 mutants in this study. However, myosin II filaments are able to stably associate with actin filaments in the presence of ATP because they contain large numbers of motor domains. It was suggested that stretching actin filaments in vitro reduces their affinity for cofilin [78]. Thus, stretching actin filaments would attract myosin II and repel cofilin. Conversely, along the leading edges of polarized cells, polymerization of actin filaments pushing against the cell membrane may axially compress the filaments, and prevent the binding of myosin II while attracting cofilin. This is consistent with the anterior localization of cofilin in polarized Dictyostelium cells [83] and fish keratocytes [84], which would further super-twist the actin filaments in the anterior region, forming another local positive feedback loop. Those two local positive feedback loops would contribute to the stabilization of cell polarity established by other biochemical stimuli. Additionally, the responsiveness of actin filaments to mechanical stretch would enable cells to respond to external mechanical stimuli or perturbations. Our aspiration experiments directly demonstrated such a possibility, in that the locally stretched portion of the cell cortex exhibited locally enhanced contractility, which enabled the cell to escape from the mechanical stimulus. When a portion of an unpolarized, round fragment of a fish keratocyte was pushed with the tip of a microneedle, the cell fragment gained front-rear polarity and started to move unidirectionally away from the microneedle [85]. Again, it may be that local deformation and stretching of the cell cortex enhanced the contractility at the site of deformation through recruitment of myosin II filaments, and made that portion of the cell the rear. One key unanswered question in modern cell biology is how different actin filaments within the same cell interact with different PLoS ONE | www.plosone.org 9 October 2011 | Volume 6 | Issue 10 | e26200