Figure 7.
Bbg controls p-Sqh levels and Jub cortical recruitment. (A and B) bbg (A) or kst (B) mutant clones (outlined in yellow) marked by the absence of the β-Gal marker (red) and the apical accumulation of the MLC regulatory subunit Sqh (Sqh-GFP; green). (C) Western blot of cleared lysates from third instar larval homozygous mutant wing discs reveals a decrease in p-Sqh (anti-pMLC) levels in bbg mutants (0.76 ± 0.20 times), whereas the levels were up-regulated in kst (1.44 ± 0.24) and kst bbg double mutants (1.51 ± 0.23). Tubulin (anti-Tub) was used as a loading control and for normalization. Molecular masses are given in kilodaltons. (D) Quantification of the mean apical cell size in overexpression clones for bbgRNAi (left) and for bbgRNAi with a constitutively active form of Sqh (SqhEE; right) and shown as the mean ratio between GFP-positive tissues (overexpressing) and GFP-negative tissues (nonexpressing controls) in individual experimental wing discs (see the Cell numbers and apical cell size section of Materials and methods). SEM is shown; unpaired two-tailed Student’s t test; n = 12 area pairs from independent discs. (E) Quantification of the wing area after SqhEE overexpression. Each genotype is expressed as a ratio compared with the mean wing area of the nubG4>UAS GFP controls (Ctrl). SD is shown; unpaired two-tailed Student’s t test based on raw data; ***, P < 0.001; n = 17–20 independent female wings. (F and G) Effect of bbg depletion marked by the absence of Bbg (red) on the apical recruitment of the Hippo-negative regulator Jub (Jub-GFP; green). Cells depleted for bbg (F) had slightly lower levels of Jub-GFP. This effect was suppressed when an activated form of Sqh (SqhEE) was coexpressed (G). Bars, 10 µm. (H) Model for the action of Bbg on the regulation of actin and growth in the Drosophila wing imaginal discs. Bbg bound to the spectrin cytoskeleton (Kst) accumulates at the cortex of epithelial cells, where it opposes the effect of Kst on MLC phosphorylation. Bbg, through its role on the actin cytoskeleton, promotes cortical tension and Jub recruitment, negatively impinging on the Hippo signaling pathway to promote proper growth of the tissue.