Lgl Regulates Notch Signaling via Endocytosis, Independently of the Apical aPKC-Par6-Baz Polarity Complex

The Drosophila melanogaster junctional neoplastic tumor suppressor, Lethal-2-giant larvae (Lgl), is a regulator of apicobasal cell polarity and tissue growth. We have previously shown in the developing Drosophila eye epithelium that, without affecting cell polarity, depletion of Lgl results in ectopic cell proliferation and blockage of developmental cell death due to deregulation of the Hippo signaling pathway. Here, we show that Notch signaling is increased in lgl-depleted eye tissue, independently of Lgl’s function in apicobasal cell polarity. The upregulation of Notch signaling is ligand dependent and correlates with accumulation of cleaved Notch. Concomitant with higher cleaved Notch levels in lgl− tissue, early endosomes (Avalanche [Avl+]), recycling endosomes (Rab11+), early multivesicular bodies (Hrs+), and acidified vesicles, but not late endosomal markers (Car+ and Rab7+), accumulate. Colocalization studies revealed that Lgl associates with early to late endosomes and lysosomes. Upregulation of Notch signaling in lgl− tissue requires dynamin- and Rab5-mediated endocytosis and vesicle acidification but is independent of Hrs/Stam or Rab11 activity. Furthermore, Lgl regulates Notch signaling independently of the aPKC-Par6-Baz apical polarity complex. Altogether, our data show that Lgl regulates endocytosis to restrict vesicle acidification and prevent ectopic ligand-dependent Notch signaling. This Lgl function is independent of the aPKC-Par6-Baz polarity complex and uncovers a novel attenuation mechanism of ligand-activated Notch signaling during Drosophila eye development. [Display omitted] •Lgl restricts ligand-dependent Notch signaling in the developing eye epithelium•Lgl colocalizes with Notch and endocytic markers and has a role in endosomal maturation•Lgl attenuates Notch signaling via restriction of vesicle acidification•Lgl regulates Notch signaling independently of aPKC-Par6-Baz polarity complex Parsons et al. reveal a role for the Lgl tumor suppressor in modulating Notch signaling in the Drosophila developing eye, independent of Par complex regulation and cell polarity. They show that Lgl associates with cytoplasmic Notch and endosomes and regulates endosomal maturation and vesicle acidification, thereby affecting Notch signaling.