The role of Dropsophila auxilin in Notch signaling



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The goal of my graduate study is to understand the role of endocytosis for signaling receptor activation during development, especially ligand endocytosis for Notch activation. Notch is a transmembrane receptor which is conserved in metazoans. I am using the Drosophila model system. Notch is required in almost every developmental context and abnormality in Notch signaling components is related to many human diseases. Delta, one of the Notch ligands, is also a transmembrane protein. To activate Notch, endocytosis of Delta in the signaling cells is essential. However, the exact mechanism of how Delta endocytosis regulates Notch activation is not known. Liquid facets (Lqf) is an endocytic protein, called epsin in vertebrates, which is required only in the signaling cells for Delta endocytosis and Notch activation. Overexpression of Lqf in the eyes results in malformed eyes. Using this phenotype as a background, an EMS-mutagenesis screen was performed and auxilin mutants were isolated as enhancers of the eye phenotype. Auxilin is a J-domain protein involved in fission and uncoating of clathrin-coated vesicles. Mosaic clonal analysis showed that auxilin functions in Notch activation and that auxilin is required only in the signaling cells. The auxilin mutant phenotype was suppressed by addition of a clathrin heavy chain transgene. This result suggests that the auxilin phenotype is at least partly caused by clathrin depletion and that auxilin generates a pool of free clathrin which is required for Delta endocytosis. Auxilin is a multi-domain protein. Two C-terminal domains, the clathrin-binding and the J domains, are sufficient to function as auxilin in Drosophila. One of the popular models to explain why Delta endocytosis is required in the signaling cells is the 'recycling model' in which inactive Delta is endocytosed and recycled to the plasma membrane in active form. Rab11 is a small GTPase that regulates recycling. If the recycling model is correct, rab11 mutants may show a phenotype similar to auxilin, lqf and Delta mutants. The rab11 hypomorphs or expression of rab11 dominant negative result in fewer photoreceptor cells and less Delta protein in the eye. These phenotypes are the opposite of typical mutant phenotypes of Notch components. The rab11 mutant phenotype argues against the recycling model.