660B Poster - 10. Cell biology: Cytoskeleton, organelles and trafficking
Friday April 08, 2:00 PM - 4:00 PM

Cullin 3 promotes polarization of aPKC phosphorylated differentiation determinants during asymmetric neuroblast division


Authors:
Cheng-yu Lee; Hideyuki Komori; Noemi Rives-Quinto; John Bugay

Affiliation: Univ Michigan

Keywords:
b. cell polarity; c. neural stem cells

The aPKC/Par-6 complex is widely used in polarizing cortical localization of protein determinants that promote proper specification of cell identity and cell functionality. The prevailing model suggests that aPKC phosphorylation displaces its target proteins from the aPKC/Par-6 cortical domain into cytoplasm by perturbing their interactions with phospholipids or adaptors, leaving unphosphorylated proteins enriched in the opposite cell cortex. It remains unclear if phosphorylation-induced exclusion from the cortex is a generalizable mechanism for aPKC-mediated polarization of numerous downstream proteins during development and homeostasis. During asymmetric neuroblast division, aPKC kinase activity in the apical cortex regulates basal localization of Notch antagonists including Numb and their segregation into neuroblast progeny where they promote differentiation by downregulating Notch signaling. In contradiction of the prevailing model, we found that aPKC kinase activity levels positively correlate with polarized Numb accumulation in the basal cortex of mitotic neuroblasts. Analyses of novel fly or human numb alleles that encode missense mutations at two conserved aPKC phosphosides indicate that the phosphomimetic but not the non-phosphorylatable form of Numb protein asymmetrically localizes to the basal cortex of mitotic neuroblasts. These results indicate that aPKC phosphorylation positively induces polarization of Numb. We screened for genes that are required for Numb segregation during asymmetric neuroblast division in numb-hypomorphic brains, which show a mild supernumerary neuroblast phenotype due to reduced Numb activity in neuroblast progeny destined to differentiate. We identified the cullin 3 (cul3) gene as a novel regulator of Numb polarization in mitotic neuroblasts. Mitotic cul3-null neuroblasts show aPKC and Numb localized uniformly throughout the cortex, reducing Numb levels in neuroblast progeny and leading to their reversion into supernumerary neuroblasts due to defects in downregulation of Notch signaling. Consistent with defects in aPKC-induced Numb polarization, increased aPKC kinase activity levels restore Numb polarization in the basal cortex of mitotic cul3-null neuroblasts and restore differentiation in their progeny. We propose that the non-proteolytic function of Cul3 promotes aPKC phosphorylation-induced Numb polarization in mitotic neuroblasts by facilitating efficient Numb phosphorylation by aPKC.