cells [44]. This prompted us to ask whether the myosin I motor domain prefers to bind to the same subset of actin filaments as the myosin II motor domain. To address that question, we initially expressed a GFP-fused myoB motor domain lacking the light chain binding domain (myoB-S1DIQ). However, after we failed to detect significant intracellular localization of the GFP fluorescence in a preliminary experiment, two point mutations expected to increase the protein’s affinity for actin in the presence of ATP were introduced (GFP-S332D/G607A myoB-S1DIQ). S332D is an activating mutation at the so-called ‘‘TEDS rule’’ site [45]. Given that Gly607 of myoB corresponds to Gly680 of myosin II, and this Gly residue between the so-called SH1-SH2 helices is absolutely conserved among diverse myosins, we presumed that G607A likely increases the affinity of myosin I for ADP and, hence, its time- averaged affinity for actin in the presence of ATP. Although we have no biochemical data as to the consequences of those two mutations, the combination resulted in more pronounced localization of GFP-myoB-S1DIQ along the cortex and in the leading pseudopods (Fig. S5). In wild-type cells that were permeabilized, fixed and stained as above, GFP-S332D/G607A myoB-S1DIQ was found mainly in Stretching Actin Filaments Attracts Myosin II the cytoplasm, distributed in a punctate manner, but was also localized along the cortical actin filaments and in the filopodia (Fig. 2A, B). However, superimposition of pseudocolored GFP and rhodamine images of the same cell shows that, unlike GFP-L596S S1, GFP-S332D/G607A myoB-S1DIQ was not concentrated along specific subsets of actin filaments. Likewise, GFP-S332D/ G607A myoB-S1DIQ was not enriched along the cytoplasmic strands to a greater degree than Rh-Ph during cytokinesis C in myosin II-null cells (Fig. 2C). Response to aspiration-induced cortical stretching We next tested whether GFP-L596S S1 would also preferen- tially bind to cortical actin filaments when the cell cortex was stretched due to an external stimulus. For this experiment, we used a microcapillary to apply negative pressure to the cell cortex. We and others previously showed that myosin II transiently accumu- lates along the cortex when it is sucked into a capillary, and suggested that myosin II-dependent cortical contraction then contributes to the escape of the cell from the capillary [28,46,47]. Live fluorescence imaging showed that GFP-S1-L596S was also enriched along the cortex near the tip of the area drawn into the Figure 2. Relative signal intensities from localized Rh-Ph and GFP-myosin I motor domain. Cells expressing GFP-S332D/G607A myoB- S1DIQ were permeabilized/fixed and stained with Rh-Ph. A: Starved and streaming wild-type cells. The arrow shows the direction of movement. B: A myosin II-null cell with numerous filopodia. C: A large, multinucleate myosin II-null cell undergoing cytokinesis C. The left, middle and right image of each triplet is a GFP fluorescence image, rhodamine fluorescence image and superimposition of the two pseudocolored images. Bars: 10 mm. doi:10.1371/journal.pone.0026200.g002 PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e26200