Figure 7.
SCRIB and NOS1AP regulate cell polarity during wound-healing migration. (a, b) Representative images of MDA-MB-231 monolayers, which were fixed 4 h after wounding to induce cell migration. Fixed cells were incubated with antibodies detecting (a) SCRIB and NOS1AP, and (b) SCRIB and VANGL. Images are oriented to show cell lamellipodia extending toward the wound edge, which runs parallel to the top of each image. Arrows indicate areas where SCRIB and NOS1AP, or SCRIB and VANGL, colocalize to the leading edge. (c) MDA-MB-231 cells were fixed immediately or 4 h after wounding and then incubated with a GM130 antibody to visualize the Golgi apparatus, and phalloidin to visualize the actin cytoskeleton. Cells with correctly oriented Golgi relative to the nucleus and the wound edge are marked by a white asterisk. (d) Percent of MDA-MB-231 shRNA-expressing cells with proper Golgi orientation determined immediately and 4 h after wounding. The horizontal line indicates the level of Golgi polarization at the beginning of wound-healing migration. The data represent the mean of four independent experiments ±s.e.m. *P<0.05 and **P<0.01 (Student's t-test). See Materials and methods for more information on Golgi polarity scoring. (e) Confocal images of wounded MDA-MB-231 cell monolayers expressing either PLKO.1 (top panel) or SCRIB shRNAs stained with an NOS1AP antibody. (f) Confocal images of wounded MDA-MB-231 cell monolayers expressing either PLKO.1 (top panel) or NOS1AP shRNAs stained with a SCRIB antibody. (g) Confocal images of wounded MDA-MB-231 cell monolayers expressing PLKO.1, SCRIB or NOS1AP shRNAs stained with a VANGL antibody. In panels e–g arrows indicate areas where SCRIB, NOS1AP or VANGL localize to the leading edge. In all images scale bars are 30 μm.