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

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. Whereas Numb is polarized by direct aPKC phosphorylation, Miranda asymmetry is thought to occur via a complicated cascade of repressive interactions (aPKC -vertical bar LgI -vertical bar myosin II -vertical bar Miranda). Results: Here we provide biochemical, cellular, and genetic data showing that aPKC directly phosphorylates Miranda to exclude it from the cortex and that LgI 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. LgI3A, a nonphosphorylatable LgI variant used to implicate LgI in this process, inhibits the formation of apical aPKC crescents in neuroblasts. Furthermore, LgI directly inhibits aPKC kinase activity. Conclusions: Miranda polarization during neuroblast asymmetric cell division occurs by displacement from the apical cortex by direct aPKC phosphorylation. Rather than mediating Miranda cortical displacement, LgI instead promotes aPKC asymmetry by regulating its activity. The role of myosin II in neuroblast polarization, if any, is unknown.