Fig. 1. Rac is required for apical localization of E-Cadherin and negatively regulates protein levels in the cadherin cell adhesion system. (A-B) Optical sections of the ventral epidermis of stage 15 embryos labeled for E-Cadherin (green) and Fasciclin 3 (purple). Grayscale images of each channel are shown below. In wild-type embryos, the apical concentration of E-Cadherin marks a distinct Fasciclin 3-free domain (A). In embryos zygotically null for Rac1 and Rac2 (Rac1, 2) this distinction is lost and many cells become flattened, with E-Cadherin localized over the entire cell surface (B). (C-D) Head epidermis of stage 6 embryos stained for E-Cadherin (green) and F-actin (purple). Cells in D have formed multiple cell layers and E-Cadherin is mislocalized. Grayscale images of each channel are shown below. (E) Western blot analysis of cadherins and their associated molecules in embryos with different levels of Rac activity. Arrows and arrowheads indicate mature and degradation products of each molecule, respectively. Arrowheads with an asterisk indicate degradation products of catenins, which were decreased in amount when Rac activity was reduced. Protein extracts of 15 embryos of each genotype were analyzed, and protein loading was monitored by Coomassie Brilliant Blue staining of the gel, which varied within a range of 10%. (F) Protein and RNA quantification. Protein was quantified by densitometric scanning of the films exposed to chemiluminescence. mRNA amounts were measured by quantitative RT-PCR. The amounts of cadherins and catenins were expressed as fold increase from the values for the control y w strain and were standardized by assuming that the amount of PP2A_A remained constant. Asterisks on the bars for the amounts of ß-Catenin protein indicate that the measurement was underestimated because of overexposure of the chemiluminescence. Scale bars: in A, 10 µm for A-B; in C, 10 µm for C-D.