Stretching Actin Filaments Attracts Myosin II Figure 4. Relative signal intensities of localized Rh-Ph and GFP-L596S S1 (A, B, D) or GFP-myosin II (C) in various knockout cells. Knockout cells lacking PTEN (pten-) or cortexillin I (ctxA-) and expressing GFP-L596S S1or GFP-myosin II heavy chain were permeabilized/fixed and stained with Rh-Ph. The left, middle and right panel of each triplet is a GFP fluorescence image, rhodamine fluorescence image, and superimposition of the two pseudocolored images. A: Large, multinucleate pten- cell undergoing cytokinesis C. B and C: Large, multinucleate ctxA- cells undergoing cytokinesis C. D: A starved and streaming ctxA- cell. The arrow shows the direction of movement. Bars: 10 mm. doi:10.1371/journal.pone.0026200.g004 different from wild-type cells in that the mutants had numerous protrusions along their sides. Nonetheless, the fluorescence from the GFP-L596S S1 was relatively stronger in the rear cortex in these cells than the Rh-Ph fluorescence (Fig. 4D). These results indicated that neither PTEN nor cortexillin I plays an essential role in the preferential binding of GFP-L596S S1 to stretched actin filaments in vivo. Finally, we expressed GFP-myosin II heavy chain in ctxA- and pten- cells induced to undergo cytokinesis C as above, and found that in both cases the GFP-myosin II accumulated along the cytoplasmic strands during cytokinesis C (Fig. 4C and movie S7). Discussion Mechanism of preferential binding of the myosin II motor domain to stretched actin filaments Non-muscle myosin II transiently forms bipolar filaments and associates with specific subsets of actin filaments to drive local contraction of the cell cortex. This leads to a number of important cellular activities, including contraction of contractile rings and retraction of the rear of polarized cells. To fulfill those functions, myosin II filaments must selectively bind to appropriate subsets of actin filaments within the cell, and three different mechanisms have been suggested to play roles in this process in Dictyostelium and PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e26200