EpCAM regulates phospho-myosin levels along cortical F-actin and formation of phospho-myosin-rich leader cells.

<p><b>(A)</b> Low-density, two-day cell cultures on collagen stained for F-actin (green), phospho-myosin (pMLC, red) and DNA (blue), and single channel images for F-actin, and for phospho-myosin (pMLC). Phospho-myosin-rich areas of cortical F-actin at the edge of colonies are marked with arrows and phospho-myosin-rich multicellular junctions inside colonies are marked with arrowheads. Bars = 50 μm. <b>(B)</b> Magnification of images in (A) showing the phospho-myosin rich junctions marked with arrowheads. Bars = 25 μm. <b>(C)</b> Phospho-myosin (pMLC) / F-actin ratios in cortical F-actin areas were determined for each cell line shown in (A, B) with at least 30 images of 0.087mm<sup>2</sup>. Error bars = S.E.M.; ***<i>p</i> < 0.0001 for MDCK and MhE16; ***<i>p</i> = 0.000114 for ctrl sh and Esh2 in two-tailed Mann-Whitney test. <b>(D)</b> Merged images showing edges of epithelial sheets with leader cells (arrows) at 6 hours migration after well removal stained for F-actin (green), phospho-myosin (pMLC, red) and DNA (blue). Bars = 50 μm. <b>(E)</b> Average number of leader cells formed at 6 hours migration in a monolayer with a starting area of 0.35 cm<sup>2</sup>. Error bars = S.E.M. of four experiments; ***<i>p</i> < 0.0001 for MDCK and MhE16 and **<i>p</i> = 0.005 for ctrl sh and Esh2 in unpaired Student’s <i>t</i> test. <b>(F)</b> Percent cell sheet edge with lamellipodium. Error bars = S.E.M. of four experiments; ***<i>p</i> < 0.0213 for MDCK and MhE16; ***<i>p</i> = 0.0037 for ctrl sh and Esh2 in unpaired Student’s <i>t</i> test.</p>