Scribble (Scrib) module proteins are major regulators of cell polarity, but how they influence membrane traffic is not known. Endocytosis is also a key regulator of polarity through roles that remain unclear. Here we link Scrib to a specific arm of the endocytic trafficking system. Drosophila mutants that block AP-2-dependent endocytosis share many phenotypes with Scrib module mutants, but Scrib module mutants show intact internalization and endolysosomal transport. However, defective traffic of retromer pathway cargo is seen, and retromer components show strong genetic interactions with the Scrib module. The Scrib module is required for proper retromer localization to endosomes and promotes appropriate cargo sorting into the retromer pathway via both aPKC-dependent and -independent mechanisms. We propose that the Scrib module regulates epithelial polarity by influencing endocytic itineraries of Crumbs and other retromer-dependent cargo.

Signaling through the transmembrane receptor Notch is widely used throughout animal development and is a major regulator of cell proliferation and differentiation. During canonical Notch signaling, internalization and recycling of Notch ligands controls signaling activity, but the involvement of endocytosis in activation of Notch itself is not well understood. To address this question, we systematically assessed Notch localization, processing, and signaling in a comprehensive set of Drosophila melanogaster mutants that block access of cargo to different endocytic compartments. We find that gamma-secretase cleavage and signaling of endogenous Notch is reduced in mutants that impair entry into the early endosome but is enhanced in mutants that increase endosomal retention. In mutants that block endosomal entry, we also uncover an alternative, low-efficiency Notch trafficking route that can contribute to signaling. Our data show that endosomal access of the Notch receptor is critical to achieve physiological levels of signaling and further suggest that altered residence in distinct endocytic compartments could underlie pathologies involving aberrant Notch pathway activation.