Figure 3. Nrg and Ankyrin Cooperate to Drive Cell Reintegration.
(A and B) Genetic disruption of ankyrin results in failed cell reintegration. Expression of ankyrin shRNA, driven by Traffic Jam-GAL4, results in ~2 popped-out cells per egg chamber. Ankyrin shRNA potentiates the reintegration failure phenotype seen in Fas2-null tissue, but not Nrg-null tissue. Representative images are shown in (A) and quantification in (B). Quantification and statistical tests were performed as in Figure 1. Scale bars in (A) correspond to 20 µm. Average and error bars correspond to mean and SD. p values from left to right: p = 0.0086, p = 0.4663, p < 0.0001.
(C) Popped-out cells, indicating reintegration failure, are apparent in FE tissue mutant for β-spectrinFY18, but not βHEAVY-spectrin. Scale bars correspond to 20 µm.
(D) Fluorescence recovery curves of Nrg::YFP after photobleaching. Lateral FE cell junctions were bleached in egg chambers expressing ankyrin-shRNA driven by Traffic Jam-GAL4 or egg chambers with the driver alone (control). Fluorescence intensity was normalized to account for inherent photobleaching due to imaging. Experiments were performed independently with number of experimental repeats indicated. Curves were calculated by fitting a one-phase association curve. Error bars represent SEM. The 95% confidence interval (CI) of the control plateau is 41–44. This does not overlap with the 95% CI of the ankyrin-shRNA plateau, which is 48–51.
(E) Frames from an example FRAP experiment, showing the fluorescence of Nrg::YFP at a cell junction prior to and post bleaching in control and ankyrin-shRNA-expressing egg chambers. Red boxes indicate photobleached region over which fluorescence intensity was quantified. Scale bars correspond to 1 µm.
(F) We propose a refined model for IgCAM-mediated cell reintegration in epithelia. Both extracellular adhesion and intracellular connection to the cytoskeleton is necessary for the reintegration of cells into epithelial layers. Specifically, we reveal that the spectrin-based cytoskeleton stabilizes Nrg cell-cell adhesion through ankyrin to facilitate reintegration. We propose that the SBMS mechanically stabilizes the trans-interactions of Nrg at the “leading edge” of integrating cells to facilitate the progression of cell reintegration. Nrg-ankyrin interactions thereby provide a traction force (grip).
See also Figure S3.