Abstract
The Drosophila compound eye is formed from a sheet of epithelial tissue that undergoes extensive morphogenesis to form a pattern of regularly spaced ommatidia. These dramatic cell shape changes are regulated by reorganization of cytoskeleton via various of actin-binding proteins (ABPs). CAP/Capulet (Capt), Slingshot (Ssh) and Cofilin/Twinstar (Tsr) are ABPs that restrict actin polymerization. Previously, it was shown that low resolution analyses of loss-of-function mutations in capt, ssh and tsr all show ectopic F-actin accumulation in various Drosophila tissues. Here, we compared their loss-of-function phenotype at single-cell resolution, using a sheet of epithelial cells in the Drosophila eye imaginal disc as a model system. Surprisingly, we found that capt and ssh, but not tsr, mutant cells within and posterior to the morphogenetic furrow (MF) shared similar phenotypes. The capt/ssh mutant cells possessed: (1) hexagonal cell packing with discontinuous adherens junctions(AJs); and (2) largely complementary accumulation of excessive phosphorylated myosin light chain (p-MLC) and F-actin rings at the apical cortex. We further showed that the capt/ssh mutant phenotypes depended on the inactivation of protein kinase A (PKA) and activation of Rho. We also found that the accumulation of F-actin is dependent on the removal of Ci75. Conversely, Capt/Ssh negatively regulated Ci155 levels within the MF at a step upstream of protein kinase A-mediated Ci155 proteolysis. Significantly, unlike ssh mutant cells, a marked reduction of phosphorylated cofilin (p-cofilin) was detected in capt mutant cells. Although most studies have focused on the role of the C-terminal actin-binding domain of Capt, we found that overexpressing the N-terminal region of Capt that recycles cofilin and a constitutively active form of cofilin rescued the capt and ssh mutant phenotypes. We conclude that Capt and Ssh act at distinct steps to recycle and dephosphorylate cofilin, respectively, and then alter the cell shape changes and progression of the MF that in turn precisely organize the pattern of ommatidia during Drosophila eye morphogenesis.