pipette puller (PG-1, Narishige) and a microforge (MF-830, Narishige). The pipette was then connected to a vertical open- ended glass tube and a 5 ml syringe via a silicone tube, and all three were filled with Bonner’s salt solution (10 mM NaCl, 10 mM KCl, 3 mM CaCl2). The syringe was then used to adjust the height of the water surface in the glass tube so that the hydrostatic pressure at the mouth of the suction pipette is 2.5 kPa. The cells were observed using the LSM510 confocal microscope. Results Probes used in this study To identify subsets of actin filaments with a higher affinity for the myosin II motor domain, we needed two types of probes: one that would accurately report local concentrations of total actin filaments and another that would preferentially bind to subsets of actin filaments having a higher affinity for the myosin II motor domain. To observe actin filaments within cells using fluorescence microscopy, three distinct classes of probes were available: GFP- actin [29,30,31], GFP-actin binding domain (ABD) of actin binding proteins (e.g., GFP-Lifeact) [32], and Rh-Ph. Staining patterns of GFP-actin, GFP-Lifeact and Rh-Ph were compared in fixed and permeabilized Dictyostelium cells, which demonstrated that those of GFP-actin and Rh-Ph were more similar to one another than those of GFP-Lifeact and Rh-Ph were (Text S2 and Fig. S2). Thus, we chose to stain cells with Rh-Ph after fixation and permeablization, in order to detect total actin filaments in a semiquantitative manner. Visualization of actin filaments having increased affinity for the myosin II motor domain required the use of a fluorescently labeled motor domain lacking the tail domain, since filament formation involving the tail domain is able to localize myosin II filaments in vivo (reviewed by [33]). However, GFP-fused myosin II S1 appeared to be always diffusely distributed in the cytoplasm (movie S1). This was presumably because in the presence of ATP, myosin II S1 spends most of its time in the ATPase cycle carrying ADP and phosphate, and associates only weakly with actin filaments. Slow, actin-stimulated release of phosphate from the S1- ADP-Pi complex establishes strong binding to the actin filament, followed by a rapid power stroke and ADP release. In the presence of physiological concentrations of ATP, rebinding of the nucleotide is rapid, and S1-ATP almost immediately dissociates from actin filaments, so that the time spent strongly bound to the actin is relatively short [34,35], which makes the time-averaged affinity of S1 for actin in the presence of ATP very low. Several S1 mutations that enhance its affinity for actin in the presence of ATP have been reported. In Dictyostelium, G680A myosin II S1 exhibits very slow actin-stimulated ADP release, which extends the strongly bound state and increases its time-averaged affinity for actin in the presence of ATP [36,37]. The corresponding G699A mutant skeletal myosin II also exhibits strong affinity for actin in the presence of ATP [38]. L596S S1 exhibits a very high affinity for actin in the weakly bound state and accelerated transition to the strongly bound state, again resulting in a higher time-averaged affinity for actin in the presence of ATP. The mechanism by which the L596S mutation increases the affinity of S1 for actin in the weakly bound state is unknown, but it is presumably allosteric, since Leu596 is not situated on myosin’s actin binding face [39]. We incorporated the G680A or L596S mutation into S1 fused N- terminally to GFP and C-terminally to a FLAG tag. When expressed in wild-type Dictyostelium cells, each of the two GFP-S1 mutants showed a distinct cortical localization, and was particu- larly abundant along the front and rear cortex in polarized cells Stretching Actin Filaments Attracts Myosin II (movie S2). This pattern of localization was similar to that reported for actin filaments, which was consistent with the enhanced affinity of the S1 mutants for actin filaments in the presence of ATP. We therefore decided to use these GFP-S1 mutants as probes for actin filaments having increased affinity for the myosin II motor domain. Comparison of the patterns of GFP-S1 mutant localization and Rh-Ph staining To semi-quantitatively compare the local abundances of the GFP-S1 mutants and actin filaments in vivo, cells expressing a GFP- S1 mutant were simultaneously permeabilized and fixed in the presence of Triton X-100 and glutaraldehyde, then stained with Rh-Ph and observed under a confocal fluorescence microscope. At first glance, the distributions of Rh-Ph and GFP-L596S S1 fluorescence appeared similar in all of the cells examined. However, more careful comparison clearly demonstrated that there were distinct and reproducible differences between the two distributions. Superimposition of pseudocolored GFP and rhoda- mine images of the same cells showed that Rh-Ph fluorescence was stronger along the front edge and along filopod-like thin projections, whereas the GFP fluorescence was stronger along the sides and the rear cortex in polarized cells (Fig. 1A, B). A similar pattern was prominent in chemotactically streaming cells during the development phase (Fig. 1C). Because the distributions of GFP-G680A S1 and GFP-L596S S1 were very similar (supplemental Fig. S3A), we mainly used GFP-L596S S1 as the probe for actin filaments with a higher affinity for the myosin II motor domain in subsequent experiments. The relative intensities of the Rh-Ph and GFP fluorescence signals were also visualized through ratiometric representation, which clearly showed the GFP signal divided by the rhodamine signal to be stronger along the sides of the cell and in the rear cortex (Fig. 1D). In a dividing cell, GFP fluorescence was enriched in both the equatorial and polar regions (movie S3), but ratiometric images showed that the GFP signal was relatively stronger in the equatorial region than the polar regions (Fig. 1E). In contrast, GFP fluorescence was distributed mainly in the cytoplasm when cells expressing GFP-wild-type S1 were processed in the same manner, and it was difficult to compare the relative intensities of the Rh-Ph and GFP fluorescences between different parts of the cortex because of the weakness of the GFP signals (supplemental Fig. S3B). These results suggested that actin filaments along the sides and rear cortices in polarized cells and the equatorial cortices in dividing cells had higher affinities for GFP-L596S S1 than actin in other areas. These higher affinity actin filaments are typically bound to endogenous myosin II filaments [27,40], and were thus presumed to be mechanically stretched. This led us to speculate that conformational changes in actin filaments induced by either mechanical stretching or biochemical changes related to the recruitment of myosin II enhanced the affinity of the filaments for GFP-L596S S1. One might speculate that GFP-S1 probes bound to myosin II or to some other actin binding protein on actin filaments, rather than directly to the actin filaments. This possibility was unlikely, however, because robust binding of the GFP-S1 probes to actin filaments in vivo required either of the two mutations that enhanced the affinity of the probe for purified actin filaments in the presence of ATP in vitro. Furthermore, to experimentally rule out the possibility that the GFP-S1 probes bound to specific actin filaments through direct interaction with myosin II, the localization of GFP-L596S S1 was characterized in myosin II-null cells. These cells are unable to divide in suspension culture and so become very large and highly multinucleate after 3 PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26200