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<p>Abstract</p> <p>Background</p> <p>CAP/Capulet (Capt), Slingshot (Ssh) and Cofilin/Twinstar (Tsr) are actin-binding proteins that restrict actin polymerization. Previously, it was shown that low resolution analyses of loss-of-function mutations in <it>capt, ssh</it> and <it>tsr</it> all show ectopic F-actin accumulation in various <it>Drosophila</it> tissues. In contrast, <it>RNAi</it> depletion of <it>capt, tsr</it> and <it>ssh</it> in <it>Drosophila</it> S2 cells all affect actin-based lamella formation differently. Whether loss of these three related genes might cause the same effect in the same tissue remains unclear.</p> <p><b>Methods</b></p> <p>Loss-of-function mutant clones were generated using the MARCM or EGUF system whereas overexpression clones were generated using the Flip-out system. Immunostaining were then performed in eye imaginal discs with clones. FRAP was performed in cultured eye discs.</p> <p>Results</p> <p>Here, we compared their loss-of-function phenotype at single-cell resolution, using a sheet of epithelial cells in the <it>Drosophila</it> eye imaginal disc as a model system. Surprisingly, we found that <it>capt</it> and <it>ssh</it>, but not <it>tsr</it>, mutant cells within and posterior to the morphogenetic furrow (MF) shared similar phenotypes. The <it>capt</it>/<it>ssh</it> mutant cells possessed: (1) hexagonal cell packing with discontinuous adherens junctions; 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 <it>capt/ssh</it> mutant phenotypes depended on the inactivation of protein kinase A (PKA) and activation of Rho.</p> <p>Conclusions</p> <p>Although Capt, Ssh and Tsr were reported to negatively regulate actin polymerization, we found that Capt and Ssh, but not Tsr, share overlapping functions during eye morphogenesis.</p>