FIGURE 5:

Context-dependent effects of phosphatidic acid–binding residues on Bazooka activity and localization in vivo. (A) DGK overexpression has no effect on the localization of BazΔOD,PDZ1^5A,ΔPDZ2-3-GFP or BazΔOD,ΔPDZ1-2,PDZ3^5A-GFP. The cortical localization of these proteins relies on sequences in PDZ1 and PDZ3, respectively, outside of their peptide-binding pockets (McKinley et al., 2012). For each Baz construct, each image shown was collected and adjusted with the same settings. Signal intensities were quantified by determining average grayscale values across single xy-planes (200 by 200 pixels). Means and standard deviations are shown for these quantifications from the numbers of embryos indicated. (B) Mutation of the three positive amino acid residues responsible for PA binding to alanine in PDZ3 of BazΔOD,ΔPDZ1-2,PDZ3^5A-GFP has no effect on the localization of the protein. Each image shown was collected and adjusted with the same settings. Signal intensities were quantified as in A. (C) BazΔPDZ1-2 partially rescues the baz mutant cuticle phenotype, but mutation of the three positive amino acid residues responsible for PA binding in PDZ3 to alanine abrogates this activity. Each data set is an average of two separate experiments (N = 122–393 embryonic cuticles per experiment). (D) Baz∆PDZ1-2-GFP and Baz∆PDZ1-2,PDZ3^3A-GFP both localize around the apical cortex of baz mutant epithelial cells, although Baz∆PDZ1-2,PDZ3^3A-GFP does so at lower levels. Each image shown was collected and adjusted with the same settings. Signal intensities were quantified as in A. In C and D, baz, gal4 is short form for baz, maternal-α4-tubulin-GAL4-VP16. (E) Expression of Baz∆PDZ1-2-GFP and Baz∆PDZ1-2,PDZ3^3A-GFP with a stronger GAL4 driver in a wild-type background led to a greater difference in their cortical levels. Each image shown was collected and adjusted with the same settings. Signal intensities were quantified as in A.