Endocytic internalization routes required for delta/notch signaling.

The internalization of transmembrane receptors from the cell surface plays a central role in signal regulation. Receptor internalization can occur through different routes; however, because of the difficulty in selectively blocking these routes in vivo, their roles in signaling are poorly understood. Here we use null mutations in Drosophila dynamin, clathrin, and AP-2 adaptor subunits to analyze internalization requirements for the Delta ligand and its receptor, Notch. Bulk Notch is internalized via AP-2-dependent endocytosis, but signaling by Notch requires AP-2-independent clathrin-dependent endocytosis, highlighting a distinction between Notch endocytic routes required for degradation versus signaling activation. Signaling by Delta requires dynamin, but whether this generates a pulling force of Delta on Notch or allows for Delta entry into a recycling pathway to gain signaling competence is widely debated. Surprisingly, we show that signaling by Delta in germline cells can occur by clathrin-independent endocytosis, when endosomal entry is blocked, and when activity of Rab11 or its effectors is reduced, suggesting that Delta need not pass through a recognized recycling pathway to achieve signaling competence. The absolute requirement for dynamin-dependent endocytosis but not endosomal entry or Rab11 activity supports "pulling force" rather than "recycling" models for Delta activation.