Janice A. Fischer - US grants

The long-term goal of the proposed research is to understand the novel pathway of cell communication in the developing Drosophila eye revealed by the fat facets (faf) gene. faf plays a key role in cell interactions that prevent particular cells present in the earliest stages of eye development from inappropriately becoming photoreceptors, and thus disturbing the symmetry that proper retinal function requires. A combination of molecular and genetic approaches will be used to answer four specific questions. 1. Where is FAF protein expressed and subcellularly localized? Anti-FAF antibodies and epitope-tagged FAF proteins will be generated and used to examine FAF protein expression in developing eyes with both light and electron microscopy. 2. When during eye development is faf function required and in which cells? Heterologous promoters will be used to express faf in portions of the developing eye at different times during development, and the effects on retinal morphogenesis assessed. 3. What are the functional domains of FAF protein? Evolutionarily conserved domains of FAF protein will be identified by isolating the Drosophila virilis faf gene and determining the DNA sequence of its coding region. Also, DNA sequence changes in many mutant faf alleles will be identified. FAF protein domains thus identified will be tested for function in transgenic flies. 4. What are the components of the cell communication pathway in which faf functions? A genetic screen in Drosophila will be conducted to find novel mutations that enhance or suppress the eye defects in faf mutant flies. A screen in yeast for direct protein-protein interactions between FAF protein domains and other eye proteins will be performed. In vertebrates and Drosophila, the use of homologous proteins for cell- cell interactions is commonplace. Thus, although the anatomy of the Drosophila and vertebrate eyes are quite different, the use of Drosophila as a model system will allow the discovery of cell communication mechanisms likely to be common to both.

The broad goal of the proposed research is to understand how organellar trafficking is regulated temporally and spatially during development. The developing Drosophila eye will be used as a model system and a gene called marbles (marb) will be used as a starting point. The marb gene product is required for particular nuclear migrations specific to differentiating cells in the developing eye; in eyes of flies with mutations in the marb gene, differentiating cells fail to undergo a stereotypical pattern of nuclear migrations, resulting in inappropriate cell shapes in the adult eye. There is evidence that MARB may be a cell type-specific component of a motor protein complex that facilitates nuclear migration. Many independent marb mutations have been generated and the marb gene has been molecularly cloned. These materials will be used to achieve three specific goals, using a combination of molecular biology histology, genetics, and biochemistry. First, using an antibody to the MARB protein, the expression pattern and subcellular localization of MARB protein in the developing eye will be determined. Second, genetic screens will be employed to identify Drosophila genes that interact with marb genetically. Finally, biochemical assays will be performed in order to test MARB protein for physical interactions with particular cytoskeletal proteins and the products of appropriate genes identified in the genetic screens. The identification of the marb gene provides the opportunity to study two critical cell biological problems from a unique starting point and in an ideal genetic system. First, active regulation of nuclear migration is a key feature of many important biological processes in vertebrates. For example, nuclear migration is required for cell migration in early human brain development; some brain disorders, like lissencephaly, result from defects in nuclear migration. Second, how molecular motor proteins attach to specific cargo is largely unknown. MARB protein may function as an adaptor protein that links the nucleus, either directly or indirectly, to a molecular motor. Almost without exception, every gene and biochemical pathway with an important role in Drosophila development exists and plays a similar role in human development.

The broad goal of the proposed research is to understand a novel cell communication pathway used in animal development. This cell communication pathway is regulated in a unique manner by ubiquitin- mediated proteolysis. Thus, the proposed research also provide important new information about ubiquitin pathway function and about how ubiquitin regulates development. The specific research proposed aims to identify and understand the interactions of the proteins that constitute the novel cell communication pathway used during Drosophila eye development defined by the fat facets (faf) gene. The faf gene encodes a deubiquitinating enzyme, or UBP, that regulates the ubiquitin-mediated proteolysis of specific proteins. In the absence of FAF protein, an unknown neural inhibition pathway fails to function, resulting in disorganized retinas containing many ectopic photoreceptors. Genetic screens will be used to identify the genes encoding the components of the cell communication pathway. Molecular cloning and transgenic fly technology will be used to isolate the genes and study the functions of the proteins they encode. These techniques will also be used to perform a structure/function analysis of the FAF protein. As mouse and human faf homologs have been identified, the cell communication pathway regulated by faf is likely to be used universally in animal development. Ubiquitin-mediated proteolysis regulates cell growth and some UBPs are oncogenes. Although ubiquitination may be used as widely as phosphorylation in regulating protein function, very little is known about the role of the ubiquitin pathway or UBPs in multicellular organisms. Drosophila faf mutants provide us with a unique opportunity to investigate a relatively unknown biochemical pathway in a powerful genetic system.

DESCRIPTION (provided by applicant): The broad goal of the research proposed is to understand how ubiquitination and endocytosis regulate particular cell communication pathways during animal development. Using a combination of genetic, molecular and biochemical techniques, a unique mechanism for the regulation of cell communication during animal development will be investigated. A cell signaling pathway essential for patterning the Drosophila compound eye has been identified; the signaling cells require the activity of a deubiquitinating enzyme called Fat facets (Faf). The function of Faf in these cells is to cleave a ubiquitin (Ub) chain from a protein called Liquid facets (Lqf). As the Ub chain targets Lqf for degradation, Faf activity increases the level of Lqf in the signaling cells. Faf is the only example of a deubiquitinating enzyme that removes a Ub chain from a specific protein, a function with respect to ubiquitination similar to that of a phosphatase with respect to phosphorylation. Moreover, Lqf is a Drosophila homolog of epsin, a vertebrate endocytic protein whose function is poorly understood. Thus, an endocytic protein is the target for regulating, via Ub, a cell communication pathway critical to cell determination. First, Drosophila molecular genetics will be used to identify additional components of the essential Faf/Lqf pathway in the Drosophila eye. Second, the substrates of Faf in its other roles in Drosophila development will be identified. Third, portions of Lqf protein required for its interaction with Faf, and for its other functions, will be localized. Fourth, a genetic screen is proposed to investigate the function of Lqf in endocytosis and other potential processes. Finally, a second Drosophila epsin gene, D-epsin2, will be analyzed; the structure and function of D-epsin2 will be investigated. As Faf and Lqf have human homologs, the pathways elucidated here will be universal for animal cells. Moreover, as Ub-mediated protein degradation and endocytosis are important for many cellular functions, several human disease genes, including oncogenes and also genes implicated in Parkinson?s and Alzheimer?s, encode proteins in these two pathways.

It is proposed to characterize the genes and gene products required for developmentally regulated nuclear migrations in photoreceptors of the Drosophila compound eye. Nuclear migration is of universal importance in animal development. For example, the human brain disorder, Lissencephaly, is the result of neural nuclei failing to migrate appropriately during brain development. Moreover, the Drosophila homolog of the human Lissencephaly gene, Lis1, is essential for photoreceptor nuclear migrations in Drosophila. Thus, the genes and proteins that will be identified and studied are directly relevant to human development and disease. Nuclear migration is an important phenomenon to understand also because of its relationship to other critical processes in eukaryotic development. Many of the proteins important for nuclear migration are also required for the transport of other organelles, establishment of cell polarity, and morphogen localization within the cell. There are four specific goals of the research proposed. The first is a structure/function analysis of Klarsicht, a protein required for photoreceptor nuclear migration. Transgenic flies expressing partial Klarsicht proteins will be used to correlate subcellular localization, protein binding, and organelle migration functions with protein structure. The second aim is to clone and characterize the egk1 gene, which like klarsicht, is essential for photoreceptor nuclear migration. Thirdly, antibodies to a variety of proteins will be used to order four nuclear migration genes, klarsicht, egk1, BicD and DLis-1, into a pathway. In addition, the possibility of physical interactions between Klarsicht and Egk1 proteins will be explored using in vivo and in vitro assays. Finally, a variety of different genetic screening approaches will be used to identify additional genes in the klarsicht/egk1 pathway.

DESCRIPTION (provided by applicant): Angelman syndrome is a neurological disorder affecting ~1/15,000 people that results in severe mental retardation. The disease is caused by loss-of-function mutations in Ube3a, which encodes a HECT-domain ubiquitin ligase or E3 protein. E3 proteins are specificity factors for ubiquitination; they bind specific substrates and bring the ubiquitination machinery to them. A simple model for the biochemistry of Angelman syndrome is that loss of UbeSa activity results in overexpression of the E3's normal substrates. The Drosophila genome contains a Ube3a homolog called CG6190. The goal of the research proposed is to generate a Drosophila model for Angelman syndrome that will allow a test of this hypothesis and the identification of the substrates relevant to the disease. First, it will be determined if Drosophila CG6190 knock-out mutants have an Angelman syndrome-like phenotype. Mutant flies will be analyzed for morphological and functional defects in their central nervous system. Second, the expression pattern of CG6190 mRNA and protein during Drosophila development will be described. In situ hybridization will be used to detect mRNA in whole organisms and anti-CG6190 antibodies will be generated and used to detect the protein. Third, it will be determined if Drosophila CG6190 and human UbeSa are functional homologs. By generating transgenes, the abilities of wild-type CG6190 and human UbeSa to complement the CG6190 mutant phenotype and to generate mutant phenotypes when overexpressed will be compared. In addition, site-directed mutagenesis will be used to generate alleles of the Drosophila and human genes that contain mutations identified in Angelman syndrome patients. The effects of expressing the resulting mutant alleles will be tested similarly. Finally, a genetic screen will be performed in Drosophila to identify candidate CG6190/Ube3a substrates. Overexpression of wild-type CG6190 in the Drosophila eye results in a severe mutant phenotype. Modifiers of the eye phenotype will be isolated and the mutant genes identified.

Project Summary 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.