TROP2 Defines Branch Formation in Fetal Kidney Figure 5. Attachment and migration of MDCK cells. (A) Cell spreading assay. Equal numbers of MDCK-control cells and MDCK-TROP2 cells, which expressed GFP and TROP2, respectively, were plated on collagen-coated glass coverslips. At 1 and 2.5 h after plating, spreading cells in several random fields were counted under a phase-contrast microscope. Arrowheads indicate spreading cells. Scale bars = 100 mm. (B) The percentage of spreading cells (n = 200). (C) MDCK-control cells and MDCK-TROP2 cells were plated on collagen-coated glass coverslips for 24 h and stained with Alexa555-Phalloidin. Arrowheads indicate the membrane ruffling at the edge of cells. Scale bars = 50 mm. (D) Wound healing assay. Cells grown as a monolayer on collagen-gel were scratched with a pipette tip. Photomicrographs were taken at 0, 6 and 16 h to record the healing process. Representative images are shown. An arrowhead indicates the unrecovered area. Scale bars = 100 mm. (E) The average migratory distance at 6 h after the scratch was measured manually (n = 3). Error bars, s.d. * p,0.05, ** p,0.01. doi:10.1371/journal.pone.0028607.g005 collagen-I-binding integrin subunit do not show severe defects in the branching of ureteric buds [35,36]. However, collagen-gel cultures of MDCK cells show that integrin a1 and a2 are essential for generating new branches and tubular structures [30,37]. We have demonstrated here that collagen-I is present around the ureteric bud and there is a gradient of its expression from the tip to trunk during branching (Figure 3 and Figure S3). These results together suggest that collagen-I plays an important role in branching morphogenesis in fetal kidney. An important finding of this study is the restricted expression and co-localization of TROP2 and collagen-I at the trunk of the ureteric duct. In contrast to collagen-I, where expression is restricted to that region, laminin is expressed almost uniformly around the ureteric bud (Figure S5). These expression patterns suggest a functional link between TROP2 and collagen-I. In fact, TROP2high trunk cells showed reduced adhesion and spreading on collagen-I-coated plates compared to TROP2low tip cells, though levels of collagen-binding integrin receptors a1, a2 and b1 were comparable between the trunks and tips (Figure 3B). To reveal the role in TROP2 for the binding and spreading of ureteric bud cells on collagen-I, we overexpressed TROP2 in TROP2low tip cells and found the binding and spreading to be reduced. We also demonstrated that TROP2 expression inhibits the spreading and migration of MDCK cells on collagen-I. PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e28607