The goal of my doctoral work is to understand how proteins involved in vesicle trafficking contribute to proper animal development. To understand aspects of this process, I studied how two vesicle trafficking proteins, Liquid facets(Lqf)/epsin1 and D-Epsin-Related, affect Drosophila eye development. I determined that Lqf, an endocytosis protein, together with Fat facets (Faf), a deubiquitinating enzyme, regulate the Notch and Delta signaling in the developing Drosophila eye. Notch signaling pathway is used in most developmental processes and is dependent on its ligand Delta. Faf deubiquitinates Lqf in the signaling cells, thereby increasing Lqf protein levels and also levels of Delta endocytosis. This event is necessary for Notch activation in neighboring cells. Lqf probably works in concert with the E3 ubiquitin ligase Neuralized (Neur), which ubiquitinates Delta. These conclusions are consistent with a relatively new model describing an obligate role for endocytosis in the signaling cells to effect activation in neighboring cells. To understand how Lqf functions mechanistically in this process, I performed a structure/function analysis of the Lqf protein. Lqf proteins with strategic deletions of certain functional domains were tested for their ability to function in vivo. The major result of these experiments is that the N-terminal ENTH domain of Lqf and a protein without the ENTH domain each retain significant activity. This suggests that Lqf has two functions: the ENTH domain function and the ENTH-less function. These data are in contrast with the most popular model suggesting that ENTH-less epsins are non-functional proteins. I present possible models for how ENTH-less epsins may retain function. The final part of my thesis focuses on D-Epsin-Related (D-Epsin-R) protein. I showed that D-Epsin-R is a Golgi protein, like its homologs. Surprisingly, D-Epsin-R ENTH domain is not required for function because an ENTH-less D-Epsin-R can substitute for endogenous D-Epsin-R. Also, D-Epsin-R has essential and probably specific developmental roles in the eye as D-Epsin-R mutants exhibit impaired cell growth. This work suggests that epsins are specific components of certain developmental pathways. / text

In eukaryotes, the process of nuclear migration is critical in fusion of haploid pronuclei after fertilization, in separation of daughter nuclei during mitosis, and in nuclear positioning in interphase cells. Experiments in several organisms have identified the basic protein requirements for nuclear migration and positioning: molecular motors that provide motive force; the cytoskeleton along which motors move nuclei, or to which the nuclei are anchored; and proteins of the outer and inner nuclear envelopes. These nuclear membrane proteins interact with the motors, the nuclear lamina and each other to effect nuclear migration and positioning. Proteins containing a SUN domain, which were first characterized in S. pombe Sad1 and C. elegans UNC-84, are inner nuclear envelope linkers of the nucleus to the cytoskeleton. In fungi, C. elegans, D. discoideum and vertebrates, these proteins are required not only for nuclear positioning, but also for maintaining the connection of the nucleus to the MTOC, for centrosomal duplication, for homologous pairing of chromosomes in meiosis, for distribution of nuclear pore complexes and for connecting the centrosome to chromatin to ensure genomic stability. The D. melanogaster genome has two genes, CG18584 and CG6589, which encode SUN domain proteins. The specific aims of my dissertation research were to generate null mutants in these genes, to characterize their null phenotypes, and to analyze where the genes are expressed. CG18584 = klaroid mutants are grossly normal, but adult eyes are mildly rough due to a defect in nuclear positioning that occurs during larval eye development. Klaroid protein is perinuclear in every cell of the eye, and functions by localizing the MTOC connector Klarsicht to the outer nuclear envelope. CG6589 = dspag4 null mutants are male sterile. In mature sperm, Dspag4 protein localizes rostrally to the sperm centriole. In the absence of Dspag4, most steps of gametogenesis occur normally, however, prior to the final steps of sperm maturation, the sperm nucleus dissociates from its centriole. Klaroid and Dspag4 thus have cellular roles typical for SUN domain proteins, and Dspag4 is unique in that its function is to attach nuclei to centrioles exclusively in maturing spermatids in the male germline. / text