Browsing by Subject "Epsin"
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Item Function and regulation of Drosophila Epsin in notch signaling(2011-12) Xie, Xuanhua; Fischer, Janice Ann; Macdonald, Paul M.; O'Halloran, Theresa J.; Morgan, Jennifer R.; Thompson, Wesley J.Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. How Epsin promotes ligand endocytosis and thus Notch signaling is unclear. Here, by generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, I tested each of the five protein or lipid interaction modules of Epsin for a role in Notch activation by each of the two Drosophila ligands, Serrate and Delta. here are five main results of this work that impact present thinking about endocytic machinery/Epsin, Epsin/ligand, or ligand/receptor interactions at the plasma membrane. First, I discovered that deletion or mutation of both UIMs destroys Epsin’s function in Notch signaling and has a greater negative effect on Epsin’s ability to function than removal of any other module type. Second, only one of the two UIMs of Epsin is essential. Third, the lipid-binding function of the ENTH domain is required for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins are necessary collectively for Epsin activity, their functions are highly redundant. Finally, I detected no ligand-specific requirements for Epsin modules. Most unexpected was the finding that Epsin’s Clathrin binding motifs were dispensable. All of these observations are consistent with a model where Epsin’s essential function in ligand cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins.Item Identification of components involved in Epsin ubiquitination(2012-08) Bal, Sheila G.; Fischer, Janice Ann; Macdonald, PaulNotch signaling is a major signaling pathway that occurs in many tissues at in nearly all stages of development. In Drosophila, Notch and its ligands, Delta and Serrate, physically interact as a part of activation of the signal. Notch activation requires the endocytic adaptor protein Epsin to facilitate the endocytosis of the ligand Delta. Our laboratory has discovered that Epsin activity is regulated by ubiquitination. Liquid facets, the gene coding for the Drosophila protein related to Epsin, was discovered to be an enhancer of the fat facets (faf) mutant eye phenotype. faf codes for a deubiquitinating enzyme. Epsin has been determined to be a key substrate of the activity of Faf in the eye. An F1 screen for dominant suppressors of the faf phenotype was performed to identify the E3 ubiquitin ligase whose substrate is Epsin. The E2 ubiquitin-conjugating enzyme UbcD1 was isolated as a strong suppressor of the faf mutant eye defect. UbcD1 has previously been identified as a strong suppressor of faf. An RNAi approach was used to study UbcD1’s role in Epsin ubiquitination further. The data that I obtained do not elucidate the UbcD1’s role in Epsin ubiquitination, but instead suggest an alternative role that should be considered.Item Identification of genes that interact with liquid facets(2012-08) Van Der Ende, Gerrit Alexander; Fischer, Janice AnnThe protein Liquid facets (Lqf) promotes endocytosis at the plasma membrane1. Lqf activity is required for proper Notch signaling, likely through facilitating the endocytosis of Notch ligand by indirectly linking ligand to clathrin. A genetic modifier screen to identify genes that interact with lqf was performed by a previous graduate student. Genes identified in the screen might provide new insights into how Lqf promotes endocytosis or how Notch signaling is regulated. In this work, I performed genetic mapping techniques to identify the genes mutated in each complementation group of the screen. I identified the gene mutated in complementation group 6 as mitochondrial alanyl tRNA synthetase (Aats-ala-m). tRNA synthetases link a tRNA to its cognate amino acid during translation. Mitochondrial tRNA synthetases function in the mitochondria in translation. Aats-ala-m genetically interacts with lqf, suggesting the two genes function in the same pathway. In this work, I also identified chromosomal regions where the genes mutated in complementation groups 1,2, and 9 are located.Item The role of auxilin and endocytosis in delta signaling(2012-05) Banks, Susan Marie-Louise; Fischer, Janice Ann; Huibregtse, Jon; Macdonald, Paul; Morgan, Jennifer; O'Halloran, TheresaNotch signaling is important for cell-cell signaling during development. Notch signaling is highly conserved across all metazoans and failure in Notch signaling is causative in many human diseases. In the Drosophila eye, activation of the Notch pathway requires Lqf (Drosophila Epsin)-dependent and Clathrin-dependent internalization of the Notch receptor ligands, Delta or Serrate, by the signal-sending cells. However, it is unclear why ligand must be internalized into the signal-sending cells to activate Notch signaling in the signal-receiving cells. Evidence suggests that in addition to Clathrin and Epsin, Auxilin is essential for signaling and is indirectly required for internalization of the Notch receptor ligand Delta. Auxilin functions in uncoating Clathrin-coated vesicles to maintain a pool of free Clathrin and Epsin in the cell. auxilin mutants were used as an entryway to identify previously unknown components of the Notch signaling pathway. An F1, FLP/FRT, EMS screen was performed and enhancers of an auxilin mutant rough eye defect were isolated. The enhancers ultimately formed one complementation group on the 2nd chromosome and fourteen complementation groups on the 3rd chromosome. Three of the 3rd chromosome complementation groups were each identified as Delta, lqf, or hsc70. A single allele was identified as faf. Delta and Epsin have known roles in signaling cells to activate Notch as described above. Hsc70 is an ATPase that functions with Auxilin to uncoat Clathrin-coated vesicles and Faf is a deubiquitinating enzyme that maintains levels of active Epsin in the cell. These results suggest I have isolated mutations in genes closely tied to Notch signaling or functioning directly with Auxilin. Mutations in two genes previously undescribed in Notch signaling in the developing Drosophila eye were also isolated from the screen and identified. The second chromosome complementation group was identified as α-adaptin. α-Adaptin is a subunit of the heterotetrameric Clathrin adaptor protein AP-2. One of the third chromosome complementation groups was identified as crumbs. Crumbs is an integral membrane protein that functions at adherens junctions and in establishing apical/basal polarity in cells. Characterizing roles for α-Adaptin and Crumbs during Notch signaling may elucidate the purpose for Delta internalization to activate Notch signaling.