Vertebrate photoreceptors are polarized cells that consist of a specialized sensory structure termed the outer segment required for phototransduction and an inner segment that contains the cellular organelles. Proteins synthesized in the inner segment are transported to the outer segment via a connecting cilium by a process called Intraflagellar Transport (IFT). The IFT mechanism refers to the movement of a multisubunit complex along the flagellar axoneme, and mutations in some IFT components cause retinal degeneration. To better understand the role of IFT in early photoreceptor development, we studied zebrafish with mutations in genes encoding three specific subunits of the IFT particle: IFT57, IFT88 and IFT172. These mutants exhibit photoreceptor defects by five days post fertilization (dpf); however, it is not known whether outer segment formation initiates at earlier time points and then degeneration occurs or if outer segments never form at all. To understand this, we performed transmission electron microscopy to study the ultrastructure of photoreceptors at 60, 72 and 96 hours post fertilization (hpf). At 60 hpf, developing outer segments were seen in IFT57 mutant and wild type embryos, however, disorganized membranous structures were observed in IFT88 and IFT172 mutants. At 72 hpf, the number of outer segments in the IFT57 mutants was reduced by 88% when compared to wild type, indicating a defect in initiation of outer segment formation. By 96 hpf we see a reduction in both outer segment length and number in IFT57 mutants. In comparison, the IFT88 and IFT172 mutants do not grow outer segments at any time point. To complement our ultrastructural analysis, we performed immunohistochemistry to understand cell morphology and protein trafficking in these mutants. Zpr1, a marker for cone morphology, showed the presence of normal cones initially that began to degenerate at later time points. Immunohistochemistry with rhodopsin, a phototransduction protein that localizes to the outer segment, revealed that rhodopsin was mislocalized in all the three mutants by 96 hpf. Connecting cilia labeled with acetylated tubulin were highly reduced in IFT57 mutants whereas none were observed in IFT88 and IFT172 mutants. Together these data indicate that IFT57 is required for maintenance and growth of outer segments whereas IFT88 and IFT172 are required for initiating outer segment formation.

Intraflagellar transport (IFT), the key mechanism for ciliogenesis, involves large protein particles moving bi-directionally along the entire ciliary length. IFT particles contain two large protein complexes, A and B, which are constructed with proteins in a core and several peripheral proteins. Prior studies have shown that in Chlamydomonas reinhardtii, IFT46, IFT52, and IFT88 directly interact with each other and are in a subcomplex of the IFT B core. However, ift46, bld1, and ift88 mutants differ in phenotype as ift46 mutants are able to form short flagella, while the other two lack flagella completely. In this study, we investigated the functional differences of these individual IFT proteins contributing to complex B assembly, stability, and basal body localization. We found that complex B is completely disrupted in bld1 mutant, indicating an essential role of IFT52 for complex B core assembly. Ift46 mutant cells are capable of assembling a relatively intact but highly unstable complex B. In contrast, in ift88 mutant cells the complex B core still assembles and remains stable, but the peripheral proteins no longer attach to the B core. Moreover, while complex A and the anterograde IFT motor FLA10 are localized normally to the transition fibers, complex B proteins instead are accumulated at the proximal ends of the basal bodies in ift88. Taken together, these results revealed a step-wise assembly process for complex B, and showed that the complex first localizes to the proximal end of the centrioles and then translocates onto the transition fibers via an IFT88-dependent mechanism. Protein interaction analyses such as the yeast two-hybrid assay in addition to identification and characterization of novel IFT complex B mutants will reveal a more complete picture of the architecture and function of IFT complex B.

Cilia and flagella are sensory organelles, found in the majority of eukaryotic organisms that play a vital role in the general physiology, health and early development of humans. Intraflagellar transport (IFT) is tasked with building and maintaining the entire ciliary structure by facilitating the transport of axonemal precursors, trafficking of ciliary membrane proteins and turnover products. Currently, there are no complete models detailing how ciliated organisms regulate the entry and exit of IFT particles, a multi-meric adaptor complex that ferries flagellar proteins. In this thesis, I focus on small Rab-like protein IFT22, an IFT-particle integrated protein with predicted GTPase activity, as a potential regulatory component of IFT particle trafficking in Chlamydomonas. Using an artificial microRNAs strategy, I show that IFT22 regulates the available cellular pool of IFT particles and the distribution profile of the IFT particles between the cytoplasm and the flagellar compartment. Additionally, I demonstrate how the putative constitutive active mutant of IFT22 is able properly localize to the peri-basal body and enter the flagellar compartment using immunofluorescence and immunoblot analysis of flagella extracts. Finally, preliminary RNAi data suggests IFT25 the IFT particle/motor/BBSome assembly downstream of IFT22 regulation, evident from the depletion of kinesin-2 subunit FLA10, IFT-dynein-2 subunit D1bLIC and BBsome component BBS3from whole cell extracts of IFT25 knockdown transformants.

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.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21709号 / 薬科博第100号 / 新制||薬科||11(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 中山 和久, 教授 竹島 浩, 教授 土居 雅夫 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21044号 / 薬科博第87号 / 新制||薬科||9(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 中山 和久, 教授 竹島 浩, 教授 井垣 達吏 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Thesis advisor: John Wing / Thesis advisor: Stephen Wicks / Molecular biology and genetics, single nucleotide polymorphism genetic mapping, phenotypic assays including behavioral assessment, and fluorescent microscopy of GFP-tagged proteins were used to study ciliary defects in the nematode Caenorhabditis elegans. Mammalian cilia are multifunctional. Some of the physiological roles in which they are involved include sensing developmental signaling molecules and ligands as well as creating flows of mucus and cerebrospinal fluid that function as flow meters and mechanosensors. Due to the multifunctional nature of cilia, it is not surprising that many human diseases can be caused by ciliary defects. Bardet-Biedl Syndrome is a rare genetic ciliopathy characterized by retinal degeneration, polydactyly, obesity, cystic kidneys, mental retardation, and many other ailments. We have identified osm- 12/bbs-7 to be a C. elegans homologue of human BBS7, a gene known to cause Bardet-Biedl Syndrome when mutated. With the help of Michel Leroux’s group, I showed the BBS-7 protein to be localized to the base of cilia and to undergo intraflagellar transport along the ciliary axoneme. Our findings suggest that BBS- 7 plays a role in the assembly and/or functioning of the IFT complex. I also performed a mutagenesis and phenotypic screen for animals defective in the uptake of DiI into a subset of their ciliated neurons in order to identify new components involved in ciliogenesis and IFT. I describe an extended bulked segregant analysis (BSA) mapping methodology, which can save time and resources by filtering out alleles of previously known genes without performing time-consuming interval mapping. In addition, I identified one of the 11 dyefilling defective alleles from the screen to be a novel allele of dyf-3, which encodes a protein required for sensory cilia formation. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24205号 / 薬科博第158号 / 新制||薬科||17(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 中山 和久, 教授 井垣 達吏, 教授 土居 雅夫 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Primary cilia are non-motile organelles having 9+0 microtubules that project from the basal body of the cell. While the main purpose of motile cilia in mammalian cells is to move fluid or mucus over the cell surface, the purpose of primary cilia has remained elusive for the most part. Primary cilia are shortened in the kidney tubules of Tg737orpk mice, which have polycystic kidney disease due to ciliary defects. The product of the Tg737 gene is polaris, which is directly involved in a microtubule-dependent transport process called intraflagellar transport (IFT). In order to determine the importance of polaris in the development of neuronal cilia, cilium length and numerical density of cilia were quantitatively assessed in six different brain regions on postnatal days 14 and 31 in Tg737orpk mutant and wildtype mice. Our results indicate that the polaris mutation leads to shortening of cilia as well as decreased percentage of ciliated neurons in all brain regions that were quantitatively assessed. Maintainance of cilia was especially affected in the ventromedial nucleus of the hypothalamus. Furthermore, the polaris mutation curtailed cilium length more severely on postnatal day 31 than postnatal day 14. These data suggests that even after ciliogenesis, intraflagellar transport is necessary in order to maintain neuronal cilia. Regional heterogeneity in the effect of this gene mutation on neuronal cilia suggests that the functions of some brain regions might be more compromised than others.