Genetic dissection of Drosophila auxilin function in Notch signaling
Abstract
Notch signaling is a highly conserved pathway that plays numerous roles during cell fate decisions and patterning events. Disruption of Notch signaling by genetic mutations can lead to diseases in humans affecting multiple organs and cancer. It is therefore important to understand the mechanism and regulation of the Notch pathway. The major components of the Notch pathway are the Notch receptor and its ligand, both of which are transmembrane proteins that interact from adjacent cells. The activation of Notch pathway requires endocytosis in both the signal-sending and the signal-receiving cells. To understand the role of endocytosis in Notch signaling, we have investigated the function of auxilin, which is a J-domain containing protein implicated in uncoating of clathrin-coated vesicles during clathrin-mediated endocytosis. In addition to the J-domain, auxilin has an N-terminal kinase domain, a PTEN-homology domain, and a clathrin-binding domain. By mutational analysis and domain deletion studies, the clathrin-binding domain and the J-domain appear to be the most critical for Drosophila auxilin (dAux) function in Notch signaling. By site-directed mutagenesis approach we have identified sequence motifs within the clathrin-binding domain that are essential for dAux function. Genetic data show that the function of dAux is required in multiple Notch-mediated processes suggesting that dAux is a general component of the Notch signaling pathway. Clonal analysis data show that dAux function is specifically required in the signal-sending cells, suggesting that Delta is the relevant cargo. Delta was indeed found to be accumulated at the cell surface in the dAux mutants while clathrin was found in large aggregates. The accumulation of Delta at the cell surface suggests a role for dAux at an earlier step than uncoating during clathrin-mediated endocytosis.
Degree
Ph.D.
Advisors
Chang, Purdue University.
Subject Area
Cellular biology
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