Numb proteins specify asymmetric cell fates via an endocytosis- and proteasome-independent pathway.

Numb proteins are evolutionarily conserved signaling molecules that make the daughter cells different after asymmetric divisions by segregating to only one daughter. They contain distinct binding motifs for alpha-adaptin (alpha-Ada) and proteins with Eps15 homology (EH) domains, which regulate endocytosis, and for E3 ubiquitin ligases, which target proteins for proteasome-mediated degradation. In Drosophila melanogaster, Numb acts by inhibiting Notch activity to cause a bias in Notch-mediated cell-cell communication. These findings have led to the hypothesis that Numb modulates Notch signaling by using endocytosis and proteasomes to directly reduce Notch protein levels at the cell surface. Here we show that two Drosophila EH proteins, Eps15 homologue 1 (EH1) and the dynamin-associated 160-kDa protein (Dap160), negatively regulate Notch signaling. However, neither elimination of the binding motifs for endocytic proteins nor simultaneous reduction of proteasome activity affects the activity of Numb proteins. Our findings indicate that an endocytosis- and proteasome-independent pathway may mediate Numb signaling in asymmetric cell fate specification.