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

(A) 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) Magnification of images in (A) showing the phospho-myosin rich junctions marked with arrowheads. Bars = 25 μm. (C) 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 0.087mm2. Error bars = S.E.M.; ***p p = 0.000114 for ctrl sh and Esh2 in two-tailed Mann-Whitney test. (D) 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. (E) Average number of leader cells formed at 6 hours migration in a monolayer with a starting area of 0.35 cm2. Error bars = S.E.M. of four experiments; ***p p = 0.005 for ctrl sh and Esh2 in unpaired Student’s t test. (F) Percent cell sheet edge with lamellipodium. Error bars = S.E.M. of four experiments; ***p p = 0.0037 for ctrl sh and Esh2 in unpaired Student’s t test.