Notch signaling is required universally for development in every tissue in all metazoans, and defects in the mechanism are associated with many human developmental diseases and cancer. A peculiar feature of the Notch pathway is the extent to which its activation and regulation depend on endocytosis. A mysterious feature of Notch signaling is that ligand must be internalized by the signaling cells in order for them to activate the Notch receptor on adjacent cells. The research we propose seeks to determine why ligand endocytosis is necessary for signaling. In addition, we explore how regulation of endocytic factors contributes to regulation of signaling. Two endocytic proteins, Epsin and Auxilin, are absolutely necessary for ligand internalization and signaling. We propose to investigate how Epsin and Auxilin function in the signaling cells, and how they are regulated. In particular, regulation of Epsin activity by ubiquitination will be explored. Moreover, we propose to identify other endocytic proteins and regulators required in the signaling cells. The methodology we use includes Drosophila genetics, immunohistochemistry of developing eyes, and biochemistry. First, we will determine which protein interaction modules of Epsin are required for Delta signaling. We will generate transgenic flies that express a variety of mutant Epsin proteins, and determine which mutants support signaling, Delta endocytosis, Epsin ubiquitination, plasma membrane localization, and normal levels of Epsin accumulation. These experiments will resolve controversial issues regarding the function and regulation of Epsin. Second, we test two hypotheses as to why Auxilin is required for signaling. Auxilin has diverse roles in endocytosis, and if we can determine which role is important for signaling, we may be able to understand why Delta endocytosis is necessary. The approach is to test if expression of different transgenes in flies will obviate the requirement for Auxilin in signaling. In addition, we perform a screen for genes that interact with auxilin. Third, we propose to investigate the mechanism by which Ubiquitin regulates Epsin. Epsin is inactivated by ubiquitination, and deubiquitination by Fat facets activates Epsin.
We aim to understand the relevance to Delta signaling of this ubiquitination cycle. We propose to identify the Ubiquitin-ligase that ubiquitinates Epsin, to analyze the phenotypes of flies that lack the ligase, and to use the ligase in biochemical experiments to determine the mode of Epsin ubiquitination. In addition, we propose to use mass spectrometry to map the sites of ubiquitination on Epsin purified from flies, and to determine whether Ubiquitin chains are present, and if so, how they are linked. Fourth, we propose genetic and biochemical experiments to test the hypothesis that the Ral GTPase negatively regulates Delta signaling by depressing the levels of Epsin. Fifth, we plan to characterize nine genes that we identified in a screen for genes that interact with the Epsin gene, liquid facets. We think that some of these genes are likely to encode regulators of Delta signaling that function through Epsin.

Public Health Relevance

The Notch signaling pathway is used universally in metazoans to control cell proliferation, specification, differentiation, and growth of every cell type. Defects in Notch signaling are thus associated with a wide variety of human developmental diseases and cancer. For this reason, an understanding of Notch pathway regulation is critically important, and the combination of Drosophila genetics and biochemistry is a power manner in which to study these processes.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Project (R01)
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Development - 2 Study Section (DEV2)
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Henken, Deborah B
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University of Texas Austin
Schools of Arts and Sciences
United States
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Xie, Xuanhua; Cho, Bomsoo; Fischer, Janice A (2012) Drosophila Epsin's role in Notch ligand cells requires three Epsin protein functions: the lipid binding function of the ENTH domain, a single Ubiquitin interaction motif, and a subset of the C-terminal protein binding modules. Dev Biol 363:399-412
Cho, Bomsoo; Fischer, Janice A (2012) Ral inhibits ligand-independent Notch signaling in Drosophila. Small GTPases 3:186-91
Lee, Ji-Hoon; Fischer, Janice A (2012) Drosophila Tel2 is expressed as a translational fusion with EpsinR and is a regulator of wingless signaling. PLoS One 7:e46357
Banks, Susan M L; Cho, Bomsoo; Eun, Suk Ho et al. (2011) The functions of auxilin and Rab11 in Drosophila suggest that the fundamental role of ligand endocytosis in notch signaling cells is not recycling. PLoS One 6:e18259
Weinmaster, Gerry; Fischer, Janice A (2011) Notch ligand ubiquitylation: what is it good for? Dev Cell 21:134-44
Cho, Bomsoo; Fischer, Janice A (2011) Ral GTPase promotes asymmetric Notch activation in the Drosophila eye in response to Frizzled/PCP signaling by repressing ligand-independent receptor activation. Development 138:1349-59
Lee, Ji-Hoon; Overstreet, Erin; Fitch, Erin et al. (2009) Drosophila liquid facets-Related encodes Golgi epsin and is an essential gene required for cell proliferation, growth, and patterning. Dev Biol 331:1-13
Wu, Yaning; Bolduc, Francois V; Bell, Kimberly et al. (2008) A Drosophila model for Angelman syndrome. Proc Natl Acad Sci U S A 105:12399-404
Eun, Suk Ho; Banks, Susan M L; Fischer, Janice A (2008) Auxilin is essential for Delta signaling. Development 135:1089-95
Overstreet, Erin; Fitch, Erin; Fischer, Janice A (2004) Fat facets and Liquid facets promote Delta endocytosis and Delta signaling in the signaling cells. Development 131:5355-66

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