Fig. 3

Podocalyxin stimulates collective breast tumor cell migration in 2-D monolayer culture. a Confocal XZ vertical images show that, as expected, podocalyxin alters the architecture of the apical membrane surface of MCF-7-podo cells maintained in 2-D culture. As a result it causes the cells to assume different shapes within the monolayers, which are much more uniform in the controls. The basolateral localization of E-cadherin is relatively unaffected in the podocalyxin-expressing cells and tight junctions are still present apically as indicated by discrete puncta of localized ZO-1, although the location of the latter varies within the vertical plane given the change in cell architecture. Podocalyxin does not cause a loss of epithelial keratin filaments but it does disrupt the uniformity of their localization at the apical surface. Scale = 10 μm. b Serum-starved MCF-7-control and MCF-7-podo cell monolayers attached to a rigid collagen I-coated substratum (0.25 μg/cm²) were subjected to a wound assay under growth factor-stimulated conditions (EGF 100 ng/ml). The ability of the cells to close the wound after 16 hours was monitored by phase microscopy, and photomicrographs of the same wound area after that period are shown (upper panel, 0 hours after wounding; bottom panels, 16 hours after wounding at low and high power). There was little difference in wound closure in non-EGF-stimulated conditions (data not shown), but the podocalyxin-expressing cells were able to close the wound more readily in response to EGF treatment compared with controls (graph, mean ± SD, unpaired Student’s t test, *p >0.05). Data shown are from one of three representative experiments. Scale = 50 μm. c MCF-7 cells were subjected to wounding as in b and after 16 hours they were fixed and immunostained for podocalyxin (red) and E-cadherin (green). Projections of confocal stacks at the leading edge of the wound show that podocalyxin-expressing cells continued to form adherens junctions. Scale = 10 μm. d MCF-7 cells were subjected to wounding as in b, and after 16 hours they were fixed and immunostained for podocalyxin (red) and f-actin (green). Projections of x/y-axis confocal stacks show that MCF-7-podo cells have enhanced, f-actin-rich lamellipodia at their leading migratory edges compared with MCF-7-control cells (upper three panels). X–Z-axis images (sliced along the white line shown in the x/y merged image) show that podocalyxin is polarized to the free, apical surface membrane (arrow), but does not extend completely into the enhanced f-actin rich lamellipodia (lower panels). Scale = 10 μm