FIGURE 2:

Rap1 is regulated by PDZ-GEF and is required for border cell migration. (A, B) GFP-Rap1 is expressed in border cells. Representative examples of stage 9 egg chambers expressing GFP-Rap1 (green) and costained for DAPI (blue in A) to label nuclear DNA, phospho-Jun (red in A) to label nuclei, or E-cadherin (E-cad; magenta in B) to label cell membranes. (A) Arrow points to border cells. GFP-Rap1 is also expressed in follicle cells and nurse cells. (B) Close-up view of a border cell cluster showing that GFP-Rap1 is membrane-enriched and partly colocalizes with E-cadherin in border cells and polar cells (left panel: asterisks; colocalization in white). (C) Activity pull-down assay demonstrates that PDZ-GEF regulates Rap1 activity in Drosophila S2 cells. GST-RalGDS-RBD beads were used to pull down GTP-bound active Rap1 from S2 cells in the presence of wild-type levels of PDZ-GEF (control gal80 RNAi) or when PDZ-GEF was knocked down (v27107 and TRiP.HM05139 RNAi; see Materials and Methods). The relative amount of active Rap1 pulled down was identified by Western blot using a Rap1 antibody. Relative band intensities were measured as a percentage of the control, which represents the amount of maximally active Rap1 in this assay. (D, E) Inhibition of Rap1 activity by dominant-negative Rap1 (Rap1^N17) or knockdown by RNAi disrupts border cell migration. (D) Expression of Rap1^N17 disrupts border cell migration. Example of a stage 10 c306>Rap1^N17 egg chamber (c306-GAL4 tsGAL80/+; UAS-Rap1^N17/+) stained for Fascin (red) to label border cells (arrow), phalloidin to label F-actin (green) and DAPI (blue) to label nuclear DNA. (E) Quantification of complete (green), incomplete (pink), and no (blue) migration in stage 10 control (slbo-Gal4/+), Rap1^WT (slbo-Gal4/+; +/UAS-Rap1^WT), Rap1^N17 (slbo-Gal4/UAS-Rap1^N17), and Rap1 RNAi (c306-Gal4 tsGAL80/+; +/UAS-Rap1 RNAi v33437) egg chambers. Migration distance as in Figure 1B. Values consist of five trials, with each trial assaying n ≥ 50 egg chambers (total n ≥ 250 egg chambers per genotype); ns, not significant, p ≥ 0.05; ****p < 0.0001; unpaired two-tailed t test comparing “complete” migration. (F, F′) Rap1 mosaic mutant border cells do not complete their migration to the oocyte. Stage 10 Rap1^CD3 mosaic mutant egg chamber stained for Fascin (green) to label the border cells (arrow) and DAPI (blue) to visualize nuclear DNA. His2Av.mRFP (red fluorescent protein, RFP; red) marks wild-type cells; colocalization with DAPI appears as magenta. Loss of RFP marks the homozygous mutant cells, including border cells (arrowheads in F′). (F′) Magnified view of the Rap1^CD3 mosaic mutant border cell cluster from (F). Three cells, likely the pair of polar cells (yellow asterisk) and one border cell, are wild-type (red), while the remaining border cells are mutant (loss of red fluorescence). (G) Extent of migration when border cells are mosaic mutant for a loss-of-function allele of Rap1. Quantification of complete (green), incomplete (pink), and no (blue) migration in stage 10 control (FRT 2A) and Rap1^CD3 FRT 2A mosaic mutant egg chambers. Migration distance as in Figure 1B. Values consist of four trials, with each trial assaying n ≥ 75 egg chambers (total n ≥ 300 egg chambers per genotype); ***p = 0.0001; unpaired two-tailed t test comparing “complete” migration. (H) Expression of Rap1^N17 partially suppresses the migration defects caused by PDZ-GEF overexpression. Quantification of complete (green), incomplete (pink), and no (blue) migration in stage 10 egg chambers expressing PDZ-GEF and GFP (c306-GAL4/+; UAS-PLC∆PH-GFP/UAS-PDZ-GEF), Rap1^N17 and GFP (c306-GAL4/+; UAS-Rap1^N17/+; +/UAS-PLC∆PH-GFP), or Rap1^N17 and PDZ-GEF (c306-GAL4/+; UAS-Rap1^N17/+; +/UAS-PDZ-GEF). Values consist of three trials, with each trial assaying n ≥ 42 egg chambers per genotype (total n ≥ 176 egg chambers per genotype); ns, p ≥ 0.05; **p < 0.01; one-way analysis of variation (ANOVA) comparing “incomplete” migration. Error bars in E, G, and H: ± SEM.