Stretching Actin Filaments Attracts Myosin II Figure 1. Relative signal intensities from localized Rh-Ph and myosin II GFP-S1. Cells expressing GFP-L596S S1 were permeabilized/fixed and stained with Rh-Ph. A: A starved and polarized wild-type cell. The arrow shows the direction of movement, and the left, middle and right images of this and panels B, C and F are GFP fluorescence image, rhodamine fluorescence image, and superimposition of the two pseudocolored images. B: A starved wild-type cell moving in a keratocyte-manner [31]. C: Starved and streaming wild-type cells. The cell indicated by the arrowhead is bi-axial, with both ends of the cell advancing. D: Similar to A, except that this cell was flattened by a sheet of agarose and the right panel shows a GFP/Rh ratiometric image. E: A dividing wild-type cell under an agarose sheet, with a ratiometric image on the right. The arrowhead shows the cleavage furrow. F: A large, multinucleate myosin II-null cell undergoing cytokinesis C. The arrowhead shows the cytoplasmic strand. Bars: 10 mm. doi:10.1371/journal.pone.0026200.g001 days. When subsequently placed on a substrate, they quickly adhere and different parts of the large cells move in different directions. Eventually, a thin cytoplasmic strand is formed between each cell fragment and the rest of the cell, which is severed after further pulling by the movement of the cell fragment, effectively resulting in cell cycle-uncoupled cell division (cytokinesis C or traction-mediated cytofission) [41,42,43]. During this process, the cytoplasmic strands are greatly stretched in an apparently passive manner, without myosin II. Live confocal imaging revealed that GFP-L596S S1 fluorescence was significantly enriched along the cortex of the cytoplasmic strands during this stretching (movie S4 and arrows in Supplemental Fig. S4). It was also noted that GFP- L596S S1 was enriched along the retracting cortex in those myosin II-null cells (movie S4 and arrowheads in Supplemental Fig. S4). Double labeling of permeabilized, fixed cells and superimposition of pseudocolored GFP and rhodamine images of the same cell demonstrated that, although Rh-Ph fluorescence was enriched along the cortex of the stretched cytoplasmic strands, the enrichment of GFP-L596S S1 in that area was far more pronounced (Fig. 1F). Thus, the enhanced binding of the GFP- L596S S1 to a subset of actin filaments reflects conformational changes in the actin filaments, rather than direct interaction with endogenous myosin II. To test the possibility that the C-terminal FLAG tag or the two light chain binding domains with the light chains bound were involved in the localization of GFP-L596S S1, another mutant, GFP-L596S S1DIQ, which lacked both the light chain binding domains and the FLAG tag, was expressed in wild-type cells. GFP fluorescence from this chimeric protein was also enriched along the sides and the posterior of polarized cells, and along the cytoplasmic strands during cytokinesis C (Supplemental Fig. S3C, D). Based on these observations, we conclude that the GFP-S1 probes recognized the conformation of a subset of actin filaments with enhanced affinity for the myosin II motor domain. GFP-fused myosin I motor domain Although myosin II is normally localized along the sides and posterior of polarized cells, other classes of myosin show different intracellular distributions. Most notably, myosin I (myoB and myoD) localizes along the leading edges of polarized Dictyostelium PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26200