aPKC Phosphorylates Miranda to Polarize Fate Determinants during Neuroblast Asymmetric Cell Division

Abstract

Summary Background Asymmetric cell divisions generate daughter cells with distinct fates by polarizing fate determinants into separate cortical domains. Atypical protein kinase C (aPKC) is an evolutionarily conserved regulator of cell polarity. In Drosophila neuroblasts, apically restricted aPKC is required for segregation of neuronal differentiation factors such as Numb and Miranda to the basal cortical domain. While Numb is polarized by direct aPKC phosphorylation, Miranda asymmetry is thought to occur via a complicated cascade of repressive interactions (aPKC –| Lgl –| Myosin II –| Miranda). Results Here we provide biochemical, cellular, and genetic data to show that aPKC directly phosphorylates Miranda to exclude it from the cortex and Lgl antagonizes this activity. Miranda is phosphorylated by aPKC at several sites in its cortical localization domain and phosphorylation is necessary and sufficient for cortical displacement suggesting that the repressive cascade model is incorrect. In investigating key results that led to this model, we found that Y-27632, a Rho Kinase inhibitor used to implicate Myosin II, efficiently inhibits aPKC. Lgl3A, a non-phosphorylatable Lgl variant used to implicate Lgl in this process, inhibits the formation of apical aPKC crescents in neuroblasts. Furthermore, Lgl directly inhibits aPKC kinase activity. Conclusions Miranda polarization during neuroblast asymmetric cell division occurs by displacement from the apical cortex by direct aPKC phosphorylation. Lgl does not mediate Miranda cortical displacement but instead promotes Par-6/aPKC asymmetry by directly inhibiting aPKC. The role of Myosin II in this process, if any, is unknown.