Stretching Actin Filaments Attracts Myosin II microcapillary (movie S5 and Fig. 3A). Dual color live imaging of wild-type cells co-expressing mCherry-actin and GFP-L596S S1 showed that actin also accumulates along the aspirated cortex (movie S5 and Fig. 3A), but detailed comparison of the time- dependent changes in the fluorescence profile revealed that there was a poor correlation between the accumulation of GFP-L596S S1 and mCherry-actin, and that accumulation of GFP-L596S S1 usually preceded that of mCherry-actin (Fig. 3B). These results indicated that at least the initial increase in GFP-L596S S1 was not dependent on an increase in actin filaments, which was consistent with the idea that GFP-L596S S1 preferentially binds to stretched actin filaments, whether the stretch is due to the cell’s own force or to an externally applied force. In parallel experiments, GFP-S332D/G607A myoB-S1DIQ expressed in wild-type cells was mostly cytoplasmic, and we were unable to detect distinct localization along the cortex inside or outside the aspirated areas of live cells (movie S6). Effects of knocking out genes known to affect myosin II localization Knocking out pten [28] or ctxA [48] gene in Dictyostelium impairs stretch-induced local accumulation of myosin II in vivo. To explore the possible involvement of their products, PTEN and cortexillin I, respectively, in the preferential binding of the myosin II motor domain to stretched actin filaments in vivo, we investigated the behavior of GFP-L596S S1 in pten- and ctxA- cells. Like myosin II- (mhcA-) cells, pten- cells failed to divide efficiently and became multinucleate during 3 days in suspension culture [49], and then underwent typical cytokinesis C on glass substrates. In those cells, GFP-L596S S1 accumulated extensively along the cytoplasmic strands, as in myosin II-null cells (Fig. 4A). ctxA- cells also frequently failed to divide in suspension culture [50] and then underwent cytokinesis C on glass substrates; and again GFP- L596S S1 accumulated along the cytoplasmic strands (Fig. 4B). Starved and chemotactically streaming ctxA- cells appeared Figure 3. Relocalization of GFP-L596S S1 and mCherry-actin in wild-type cells in response to local aspiration using a microcapillary. A: Accumulation of GFP-L596S S1 and mCherry-actin at the cortex in the portions of the cell deformed by aspiration (arrowheads). The numbers indicate time after initiation of suction. Bar: 10 mm. This result is representative of 8 experiments. B: Time sequence showing the accumulation of GFP- L596S S1 and mCherry-actin at the cortex in the aspirated portion of the cell shown in panel A. Fluorescence intensities in the cytoplasmic area and hemispherical tip area of the aspirated portion in the rectangle were quantified, and the edge/cytoplasm ratios were calculated for each fluorescence image. Accumulation of GFP fluorescence (green line) is evident at 15 s, while increase in mCherry fluorescence (red line) is detectable only after 105 s. Overall, the edge/cytoplasm ratio was higher for GFP than mCherry (black line), and there was no strong correlation between the accumulations of GFP and mCherry fluorescence in this or any other sequences (not shown). doi:10.1371/journal.pone.0026200.g003 PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26200