Notch signaling depends on regulated intracellular trafficking of the receptor and its ligands (Kopan and Ilagan, Cell 137:216–233, 2009; Le Borgne et al., Development 132:1751–1762, 2005). Here we describe two methods to study the intracellular trafficking of Notch and Delta in Drosophila. First, an ex vivo antibody uptake assay is used to monitor endocytosis of Notch and Delta by living cells in dissected explants (Le Borgne and Schweisguth, Dev Cell 5:139–148, 2003). Second, real-time imaging of fluorescent proteins that are expressed at physiological levels is used to study trafficking of Notch in living flies (Venken et al., Science 314:1747–1751, 2006; Couturier et al., Nat Cell Biol 14, 131–139, 2012).
Live imaging Antibody uptake Endocytosis assay Notum dissection Drosophila
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
This work was supported by the Fondation pour la Recherche Médicale (DEQ20100318284). The antibody uptake assay was initially developed together with R. Le Borgne.
Le Borgne R, Bardin A, Schweisguth F (2005) The roles of receptor and ligand endocytosis in regulating Notch signaling. Development 132:1751–1762PubMedCrossRefGoogle Scholar
Gho M, Bellaiche Y, Schweisguth F (1999) Revisiting the Drosophila microchaete lineage: a novel intrinsically asymmetric cell division generates a glial cell. Development 126:3573–3584PubMedGoogle Scholar
Le Borgne R, Schweisguth F (2003) Unequal segregation of Neuralized biases Notch activation during asymmetric cell division. Dev Cell 5:139–148PubMedCrossRefGoogle Scholar
Rhyu MS, Jan LY, Jan YN (1994) Asymmetric distribution of Numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. Cell 76:477–491PubMedCrossRefGoogle Scholar
Couturier L, Vodovar N, Schweisguth F (2012) Endocytosis by Numb breaks Notch symmetry at cytokinesis. Nat Cell Biol 14: 131–139PubMedCrossRefGoogle Scholar
Couturier L, Mazouni K, Schweisguth F (2013) Numb localizes at endosomes and controls the endosomal sorting of notch after asymmetric division in Drosophila. Curr Biol 23:588–593PubMedCrossRefGoogle Scholar
Cotton M, Benhra N, Le Borgne R (2013) Numb inhibits the recycling of Sanpodo in Drosophila sensory organ precursor. Curr Biol 23:581–587PubMedCrossRefGoogle Scholar
Yamamoto S, Charng WL, Bellen HJ (2010) Endocytosis and intracellular trafficking of Notch and its ligands. Curr Top Dev Biol 92:165–200PubMedCrossRefGoogle Scholar
Daskalaki A, Shalaby NA, Kux K et al (2011) Distinct intracellular motifs of Delta mediate its ubiquitylation and activation by Mindbomb1 and Neuralized. J Cell Biol 195:1017–1031PubMedCentralPubMedCrossRefGoogle Scholar
Le Borgne R, Remaud S, Hamel S et al (2005) Two distinct E3 ubiquitin ligases have complementary functions in the regulation of Delta and Serrate signaling in Drosophila. PLoS Biol 3:e96PubMedCentralPubMedCrossRefGoogle Scholar
Venken KJ, He Y, Hoskins RA et al (2006) P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science 314:1747–1751PubMedCrossRefGoogle Scholar
Qi H, Rand MD, Wu X et al (1999) Processing of the Notch ligand Delta by the metalloprotease Kuzbanian. Science 283:91–94PubMedCrossRefGoogle Scholar
Diederich RJ, Matsuno K, Hing H et al (1994) Cytosolic interaction between deltex and Notch ankyrin repeats implicates deltex in the Notch signaling pathway. Development 120: 473–481PubMedGoogle Scholar
Jauffred B, Bellaiche Y (2012) Analyzing frizzled signaling using fixed and live imaging of the asymmetric cell division of the Drosophila sensory organ precursor cell. Methods Mol Biol 839:19–25PubMedCrossRefGoogle Scholar
Klueg KM, Parody TR, Muskavitch MA (1998) Complex proteolytic processing acts on Delta, a transmembrane ligand for Notch, during Drosophila development. Mol Biol Cell 9:1709–1723PubMedCentralPubMedCrossRefGoogle Scholar
Coumailleau F, Furthauer M, Knoblich JA et al (2009) Directional Delta and Notch trafficking in Sara endosomes during asymmetric cell division. Nature 458:1051–1055PubMedCrossRefGoogle Scholar