Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 80-91).

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 126-138).

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from first page of PDF file (viewed on June 10, 2009). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [145]-174).

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 12, 2009). Includes bibliographical references (p. 136-146).

by Pik-ying Ng. / Bibliography: leaves 106-117 / Thesis (M.Phil.)--Chinese University of Hong Kong, 1983

Photoreceptors are highly compartmentalized neurons in the retina, and they function by detecting light and initiating signaling through the visual network. The photoreceptor contains several compartments including the outer segment (OS) which is a sensory cilium for detecting photons and the inner segment (IS) that carries out important modulatory functions via its resident channels and transporters. Those proteins are membrane proteins that function together to shape electrical properties of the cell membrane during both rest and active states. Therefore it is essential to maintain proper function of the membrane proteins in the IS. One important way to regulate the function of a membrane protein is via controlling its trafficking to ensure a proper amount of the protein in the proper cellular compartment. To date, little is known about how IS membrane protein trafficking is controlled in photoreceptors. In this study, our goal is to understand those mechanisms using cell biology and biochemistry approaches. To achieve the goal, we investigated trafficking of two unrelated IS resident proteins: the hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) that mediates a feedback current in photoreceptors, and the sodium potassium ATPase (NKA) which maintains the basic electrochemical property of the cell. In order to study trafficking of HCN1, we first investigated the dependence of HCN1 trafficking in photoreceptors on TRIP8b, an accessory subunit that influences trafficking of HCN1 in hippocampal neurons. By studying TRIP8b knockout mice we found that TRIP8b is dispensable for HCN1 trafficking in photoreceptors but required for maintaining the maximal expression level of HCN1. Since we revealed that HCN1 trafficking can be regulated in a cell-type specific manner, we subsequently focused on the amino acid sequence of HCN1 to identify novel trafficking signals that function in photoreceptors. By examining localization of a series of HCN1 mutants in transgenic Xenopus photoreceptors, we discovered a di-arginine ER retention motif and a leucine-based ER export motif. These two sequence motifs must function together to maintain equilibrium of HCN1 level between the endomembrane system and the cell surface. The study of HCN1 uncovered a mechanism for the photoreceptor to control membrane protein trafficking via the early secretory pathways. To reveal additional trafficking machineries in photoreceptors, we investigated trafficking of NKA. We first tested for an interaction with ankyrin, an adaptor protein that regulates NKA trafficking in epithelial cells, and found these proteins do not co-localize in photoreceptors. We then aimed to identify novel trafficking signals by studying the trafficking behavior of two NKA isozymes: NKA-α 3 and NKA-α 4. When expressed in transgenic Xenopus photoreceptors, these two proteins localize to the IS and the OS respectively. By studying localization of multiple chimeras and truncation mutants, we found that the distinct localization pattern is due to a VxP OS/ciliary targeting motif present in NKA-α 4. Since NKA-α 4 is naturally expressed in the ciliary compartment of the sperm, our finding in the photoreceptor suggests a mechanism for NKA-alpha 4 trafficking in its native environment. Overall, our studies of HCN1 and NKA together provide new insights into controlling membrane protein trafficking in photoreceptors and help establish the basics for future therapeutic intervention targeting trafficking pathways that are linked to about one third of proteins reported in retinal diseases.

Vertebrate photoreceptors are highly specialized sensory neurons that utilize a modified cilium known as the outer segment to detect light. Proper trafficking of proteins to the outer segment is essential for photoreceptor function and survival and defects in this process lead to retinal disease. In this dissertation I focus on two aspects of protein trafficking, intracellular vesicular trafficking in photoreceptors and retinal pigmented epithelial (RPE) cells and how it relates to the human disease choroideremia (CHM), and the trafficking of proteins through the photoreceptor cilium. The human retinal degenerative disease choroideremia (CHM) is caused by mutation of the Rab escort protein-1 (REP1) gene, which is required for proper intracellular vesicular trafficking. However, it was unclear whether photoreceptor degeneration in this disease is cell-autonomous, due to defective opsin transport within the photoreceptor, or is noncell-autonomous and a secondary consequence of defective RPE. Utilizing the technique of blastomere transplantation and a zebrafish line with a mutation in the rep1 gene, I show that photoreceptor degeneration in CHM is noncell-autonomous and is caused by defective RPE. The molecular machinery responsible for protein trafficking through the photoreceptor cilium remained unclear for a long time. Recent studies found Intraflagellar Transport (IFT) is the process that mediates cilia formation and transport of proteins through a cilium, and further analyses showed IFT is important for trafficking proteins to the outer segment. However, many details about how IFT works in photoreceptors remained unclear. By analyzing zebrafish harboring a null mutation in the ift57 gene, I show that Ift57 is only required for efficient IFT, and that the Ift57 protein plays a role in the ATP-dependent dissociation of kinesin II from the IFT particle. Lastly, I investigate the role of retrograde IFT in photoreceptors, a process that had yet to be investigated. By utilizing antisense morpholino oligonucleotides to inhibit expression of cytoplasmic dynein-2 (the molecular motor that mediates retrograde IFT) , I show that retrograde IFT is required for outer segment extension and the recycling of IFT proteins.

Broad Complex (BRC) is an ecdysone-pathway gene essential for entry into and progression through metamorphosis in D. melanogaster. Mutations of three BRC complementation groups cause numerous phenotypes, including a common suite of morphogenesis defects involving central nervous system (CNS), adult salivary glands (aSG), and male genitalia. Alternative splicing, of a protein-binding BTB-encoding exon (BTBBRC) to one of four tandemly duplicated, DNA-binding zinc-finger-encoding exons (Z1BRC, Z2BRC, Z3BRC, Z4BRC), produces four BRC isoforms. Highly conserved orthologs of BTBBRC and all four ZBRC were found in silico from Diptera, Lepidoptera, Hymenoptera and Coleoptera, indicating that BRC arose and underwent internal exon duplication before the split of holometalolous orders. Five Tramtrack subfamily members were characterized throughout Holometabola and used to root phylogenetic analyses of ZBRC exons, revealing that Z3BRC is the basal member. All four ZBRC domains, including Z4BRC which has no known essential function, are evolving in a manner consistent with selective constraint. Transgenic rescue and immunohistochemistry were used to explore how different BRC isoforms contribute to their shared tissue-morphogenesis functions at the onset of metamorphosis, when BRC is required for CNS reorganization. As predicted, the common CNS and aSG phenotypes were rescued by BRC-Z1 in rbp mutants, BRC-Z2 in br mutants, and BRC-Z3 in 2Bc mutants. However, the isoforms are required at two developmental stages, with BRC-Z2 and -Z3 required earlier than BRC-Z1. Each isoform had a unique expression pattern in the CNS, with no substantial three-way overlap among them. Z4 is strongly expressed in a novel subset of CNS neurons. The most prominent localizations of BRC-Z1, -Z2, -Z3 corresponded with glia, neuroblasts and neurons, respectively. There appears to be a switch from BRC-Z2 in proliferating cells to BRC-Z1 and BRC-Z3 in differentiating cells. The temporal-requirement and spatial-distribution data suggest that BRC-dependent CNS morphogenesis is the result of multicellular interactions among different cell types at different times. BRC-Z1-expressing glia in prepupae may mediate the final steps of CNS morphogenesis. Lastly, BRC is required for migration and programmed cell death of the ring gland, the site of ecdysone and juvenile hormone production. Therefore, BRC may function in ecdysone auto-regulation.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 107-117).