not is required for normal actin polarity in migratory BCs. (A) Confocal micrographs of egg chambers harboring WT, not¹, or rescued not¹ GFP-labeled clones (not¹; tub>not^+r) labeled with phalloidin to visualize F-actin (red) and TO-PRO-3 to visualize nuclei (blue). Egg chambers are stage 10 except a WT control, which is shown at mid-migration at stage 9 (dotted line indicates expected position of the cluster at this stage of migration). BC clusters are indicated with arrows. In WT, F-actin is normally polarized, with high levels around the cortex, at BC–nurse cell junctions. In contrast, in not¹ clusters, F-actin predominantly accumulates at internal BC–BC junctions; this is rescued by transgenic overexpression of not^+r. Scale bars are 25 µm (red, green, and blue images) and 10 µm for magnified grayscale images of F-actin. (B) Representative line scans (indicated with yellow line in A) of the same genotypes showing signal intensities of F-actin from anterior (left) to posterior (right) and the change in F-actin profile in not¹ clusters. (C) Dot plots of area under curve for back, middle, and front (BMF) of the BC cluster derived from line scans through the cluster, with mean ± SEM derived from means of n > 5 egg chambers/replicate, showing a consistent defect in F-actin polarization in not¹ clusters. F-actin polarization was restored by not^+r overexpression (tub>not^+r; not¹). ****, P < 0.0001 by one-way ANOVA. st., stage.