The primary cilium is vital for the health and well being of the organism. Diseases of the cilia, referred to as ciliopathies, present with overlapping phenotypes due to a protein defect in the same organelle. Three such disorders are Nephronophthisis (NPHP), Bardet-Biedl Syndrome (BBS), and Senior-Loken Syndrome (SLS). Mutations in serologically defined colon cancer antigen 8(SDCCAG8 [MIM 613524]) have been associated with all of these disorders (BBS16, NPHP10, SLSN7). Little is known about the role of SDCCAG8 in ciliary function, and the mechanisms through which SDCCAG8 leads to ciliopathy phenotypes are potentially novel and may identify therapeutic targets for treating clia related disorders. My work aimed at elucidating these mechanisms utilizing in vitro and in vivo models to identify specific interactors of SDCCAG8. Here, we show an interaction with the multi-aminoacyl tRNA synthetase complex (MSC) and more specifically, with the aminoacyl interacting multifunctional protein 2 (AIMP2). We also determined that the interaction between these genes is dependent on the N-terminus of AIMP2 and a region in the C-terminus of SDCCAG8. Further work characterized the importance of SDCCAG8 in AIMP2 nuclear localization. Loss of SDCCAG8 results in increased AIMP2 nuclear localization and downstream upregulation of p53. We also characterized an Sdccag8 mouse model and assessed its utility as a ciliopathy model. We performed a phenotypic characterization of this model and identified a genomic deletion encompassing a neighboring gene, Akt3. Due to this finding, we suggest that individuals interested in this mouse model proceed with caution. In addition, we used this model to identify a potential modifier locus of lethality. Overall, the work presented in this thesis advances the understanding of the biology of SDCCAG8 and its role in the cell.

Meckel-Gruber syndrome (MKS) is a recessive genetic disease that is uniformly lethal in affected children due to resultant developmental defects in the kidney and brain. 13 MKS genes have been identified, and further candidate genes have been linked to this disease, all encoding unrelated proteins. Their role is believed to be in generation and compartmentalisation of the primary cilium, a microtubule-based organelle that functions in signal transduction of developmentally-crucial pathways. However, recent evidence indicates that these proteins are also likely involved in regulation of the actin cytoskeleton. Furthermore, research is beginning to uncover roles of other ciliopathy proteins in regulation of additional subcellular structures, such as the microtubule cytoskeleton, focal adhesions and the Golgi. To begin to understand the roles of the MKS proteins beyond the cilium, I examined a number of cellular features of patient fibroblasts carrying mutations in TMEM216 (MKS2) and TMEM67 (MKS3). In this thesis, I describe the temporal appearance and nature of prominent actin bundles observed in these cells, and analyse the dependency of these on the Rho/ROCK signalling pathway. Furthermore, I identify novel alterations to the microtubule cytoskeleton and organisation of the Golgi complex in MKS patient cells, and subsequently establish a temporal order of these phenotypes, demonstrating microtubule defects as the first to occur in these cells. Finally, I connect these phenotypic defects to Rho/ROCK signalling. In contrast to the prevailing view in the ciliopathy field, I believe that a diffusion barrier at the transition zone is not the primary role of MKS proteins. Instead I propose, supported by these data, that MKS protein complexes play a dual role as effectors of Rho signalling in addition to performing a structural role with particular importance in tethering the cytoskeleton to membranes. I therefore conclude that these, and other ciliopathy protein complexes, may act as important signal transduction and structural components at multiple locations throughout the cell.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第23474号 / 薬科博第144号 / 新制||薬科||16(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

The Wnt signaling network has critical roles in embryonic development and is implicated in human disease. One of the outputs of the Wnt network, called the planar cell polarity (PCP) pathway, regulates tissue polarity and directs cell migration. Core PCP components (Frizzled, Dishevelled, Prickle, Vangl, Celsr) localize asymmetrically in polarized cells and establish polarity across the tissue through protein interactions between adjacent cells. The core PCP component activate tissue-specific "effectors" which translate the signal into morphological changes. PCP is related to several disease conditions, including neural tube defects, cystic kidney disease, and cance metastasis. However, mechanisms of the PCP underlying physiological and disease-related conditions are not well understood. Here, I explore functions of the core PCP component Pk, and its relationship to disease, in the zebrafish model system. Mutations in Pk1 and Pk2 have been identified in human progressive myoclonic epilepsy patients. Pk coodinate cell movement, neuronal migration and axonal outgrowth during embryonic development. Yet, how dysfunctions of pk relates to epilepsy is unknown. Here, I show that knockdown of pk1a sensitizes the zebrafish larva to convulsant drug. To model the defects in central nervous system, I examine neurogenesis in the retina and find that both pk1a and pk2 are required for proper dendritic outgrowth in the retinal inner plexiform layer. Furthermore, I characterize the epilepsy-related mutant forms of Pk1a and Pk2. The mutant Pk1a forms show reduced ability to suppress the retinal neurogenesis defects compared to the wild-type, as well as differential ubiquitination levels. Pk2 mutant forms also show differential activities in overexpression assays and seemingly more stable proteins relative to the wild-type. Taken together, pk1a and pk2 may contribute to epilepsy by affecting neuronal patterning and thus signal processing. Another aspect of PCP function has been implicated in cilia and cilia-related disorders, also called ciliopathy. PCP effectors have been shown to modulate ciliogenesis and core PCP proteins (Vang and Dvl) regulate cilia orientation. On the other hand, cilia are not required for PCP signaling, especially asymmetric core PCP protein localization. These findings leave open the question what is the precise relationship between PCP and cilia. The Bardet Biedl Syndrome (BBS) is a type of ciliopathy that leads to obesity, retinitis pigmentosa, polydactyly, mental retardation and other symptons. A subset of BBS genes share similar knockdown phenotype in cell migration as seen in PCP knockdown embryos. Shared pehnotypes have led some to proposethat PCP and bbs genes may interact. Yet a direct relationship has yet to be established. I examine the interaction between pk2 and a central Bbs gene, bbs7. By analyzing shared phenotypes in double knockdown embryos, I find no synergistic interaction between the two, suggesting they act in distinct pathways. Bbs regulate ciliary trafficking and in zebrafish, knockdown of bbs genes leads to delayed retrograde melanosome transport. Interestingly, I find knockdown of pk2 suppresses this retrograde transport delay. Additionally, pk2 knockdown embryos show a delay in anterograde melanosome transport. These findings highlight a new role for pk2 in intracellular transport and clarifies the relationship between PCP and BBS. In summary, my work here strengthens the link between pk mutations and human epilepsy and identifies functions of pk in retinal neurogenesis and in intracellular transport. To what extent the role of neurogenesis and intracellular transport are related is worth future study. Yet, this new information provides insights into potential mechanisms of epilepsy and the relationship between PCP and BBS.

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.

Bardet-Biedl syndrome (BBS) is a pleiotropic and genetically heterogeneous disorder, and a well-known ciliopathy. Nineteen different genes have been reported for BBS, mutations in which cause characteristic phenotypes including retinal degeneration, obesity, polydactyly, renal abnormalities, hypogenitalism and cognitive impairment. Protein products of eleven BBS genes are part of two major complexes: the BBSome complex and a CCT/CTRiC/BBS complex. The CCT/CTRiC/BBS complex assists in the formation of the BBSome complex, which in turn traffics numerous receptor proteins to the cilia. However, the precise mechanism by which BBSome ciliary trafficking activity is regulated is not fully understood. In fact, a complete picture of the cellular functions of BBS proteins is still missing, and gaps remain in our understanding of the pleiotropy and heterogeneity of the disease. With the aim of bridging those gaps, this thesis project was designed to identify tissue specific cargoes of the BBSome and to characterize their BBS-related functions. To this end, we generated a transgenic LAP-BBS4 mouse, which expresses the transgene in various tissues including brain, eye, testis, heart, kidney, and adipose tissue. We found that despite tissue specific variable expression, LAP-BBS4 was able to complement the deficiency of Bbs4 and rescue all the BBS phenotypes in the Bbs4 null mice. The finding provides an encouraging prospective for gene therapy for BBS related phenotypes and potentially for other ciliopathies. We also utilized the transgenic mice to search for tissue specific BBSome cargo proteins and identified CEP131 as a novel BBSome interacting protein. Using in vitro cell culture models we show that CEP131 interacts with the BBSome through BBS4. CEP131 is not involved in BBSome assembly, but accumulation of the BBSome in cilia is enhanced upon CEP131 depletion. Our in vitro data implicate CEP131 as a negative regulator of ciliary BBSome trafficking. Finally, we show that cep131 knockdown in zebrafish embryos results in typical BBS phenotypes including Kupffer's vesicle abnormalities and melanosome transport delay. This finding confirms the association of CEP131 with the BBS pathway. Overall, the work performed for this thesis provides further insight into the regulation of BBSome ciliary trafficking and suggests CEP131 as a BBS candidate gene.

Les mutations MKS1 sont impliquées dans un groupe de ciliopathies récessives létales, telles que le syndrome de Meckel-Gruber (MKS) et le syndrome de Joubert (JBT), caractérisées par une dysplasie rénale kystique, des anomalies du système nerveux central (encéphalocèle occipitale), une polydactylie, une dysgénésie biliaire et une fibrose hépatique. MKS1 a été localisée dans la zone de transition du cil dans de nombreux types cellulaires où elle joue un rôle essentiel pour la structure et la fonction des cils, en particulier la régulation de plusieurs voies de signalisation telles que Wnt et Shh.Dans le présent travail, nous avons identifié la fonction pré-ciliaire de MKS1 dans des cellules épithéliales. Nous avons montré que la localisation subcellulaire de MKS1 varie au cours de la maturation de l’épithelium, passant du cytosol où MKS1 co-localise avec le réseau de kératine, aux jonctions cellulaires, où elle co-colocalise avec les caténines. De plus, la translocation de MKS1 des jonctions au cytosol s'est avérée être mécano-sensible, suggérant que MKS1 participe à l'homéostasie épithéliale en stabilisant les jonctions cellulaires, via la transduction des signaux mécaniques liés à la compaction de l’épithelium.L’analyse fonctionnelle a démontré que le « knockdown » de MKS1 désorganise le réseau de kératine, et déstabilise les jonctions adhérentes des cellules épithéliales en culture, avec une diminution de la β-caténine jonctionnelle et une libération de l’E-cadhérine, l’α-caténine et la vinculine dans le cytosol. De plus, la déplétion de MKS1 entraîne une diminution notable du réseau apical d’actine, ainsi que la désorganisation de la structure épithéliale et une transition partielle vers un état mésenchymateux. Ces résultats illustrent une fonction indépendante du cil de MKS1 dans l’homéostasie épithéliale, et apporte de nouvelles hypothèses quant à son rôle et celui des filaments intermédiaires dans les processus d’organogenèse des épitheliums, en particulier la tubulogenèse, qui repose à la fois sur l’équilibre de la transition épithelium/mesenchyme et la mécanotransduction des sollicitations mécaniques durant l’embryogenèseDans le but de caractériser les partenaires de MKS1, des expériences de Co-IP et d’analyses protéomiques ont permis d’identifier l’epiplakine comme un partenaire possible de MKS1. L'Epiplakine est un cytolinker capable de lier la kératine à la membrane et à l'actine ; l’interaction de MKS1 avec l’epiplakine pourrait ainsi rendre compte de la stabilisation à la fois du réseau de kératine et des jonctions cellulaires. Des analyses complémentaires de protéomique et des études fonctionnelles devront compléter ces résultats préliminaires.Finalement, ces travaux ont également permis de révéler le rôle de MKS1 dans la stabilisation des jonctions communicantes ; la déplétion de MKS1 conduisant à une diminution de la CX43 jonctionnelle et à une altération de la fonction de communication intercellulaire dans les cellules épithéliales en culture. Ces travaux, qui constituent la première mention d’une altération possible des jonctions communicantes dans ce type de maladies, devront être approfondis pour caractériser leur impact dans les processus de tubulogenèse.En conclusion, ce travail qui a permis de révéler un rôle pré-ciliaire de MKS1 dans l'homéostasie épithéliale, apporte de nouvelles hypothèses pour l’étiologie de ces maladies, jusqu’alors considérées comme essentiellement consécutives à des défauts de transduction de la signalisation ciliaire. Il propose également de nouveaux mécanismes pour rendre compte des anomalies du développement hépatique, telles que la dysgénésie des voies biliaires, et plus largement des processus de tubulogenèse impliqués dans le développement de nombreux organes. / MKS1 mutations are involved in a group of lethal recessive ciliopathies, such as Meckel-Gruber syndrome (MKS) and Joubert's syndrome (JBT), characterized by cystic renal dysplasia, central nervous system abnormalities (occipital encephalocele) , polydactyly, biliary dysgenesis and hepatic fibrosis. MKS1 has been located in the transition zone of the cilia in many cell types where it plays an essential role in the cilia structure and function, in particular in the regulation of signaling pathways such as Wnt and Shh.In the present work, we have identified the preciliary function of MKS1 in epithelial cells. We have shown that the subcellular localization of MKS1 varies during the maturation of the epithelium, from the cytosol where MKS1 co-localizes with the keratin network, to the cell junctions, where it co-localizes with the catenins. In addition, the MKS1 translocation to cytosol junctions proved to be mechano-sensitive, suggesting that MKS1 participates in epithelial homeostasis by stabilizing cell junctions, via the transduction of mechanical signals related to epithelial compaction.Functional analysis has shown that the knockdown of MKS1 disrupts the keratin network, and destabilizes the adherent junctions of epithelial cells in culture, with a decrease in the junctional β-catenin and a release of E-cadherin, the α-catenin and vinculin in the cytosol. In addition, the depletion of MKS1 results in a significant decrease in the apical actin network, as well as disorganization of the epithelial structure and a partial transition to a mesenchymal state. These results illustrate a ciliary-independent function of MKS1 in epithelial homeostasis, and provides new hypotheses regarding its role and that of intermediate filaments in epithelial organogenesis processes, in particular tubulogenesis, which is based both on the equilibrium of the epithelium / mesenchyme transition and the mechanotransduction of mechanical stresses during embryogenesisIn order to characterize MKS1 partners, Co-IP experiments and proteomic analyzes have identified epiplakin as a possible MKS1 partner. Epiplakin is a cytolinker capable of binding keratin to membrane and actin; the interaction of MKS1 with epiplakin could thus account for the stabilization of both the keratin network and cell junctions. Additional proteomic analyzes and functional studies will complement these preliminary results.Finally, this work has also revealed the role of MKS1 in the stabilization of gap junctions; the depletion of MKS1 leading to a decrease in the junctional CX43 and an alteration of the intercellular communication function in the epithelial cells in culture. This work, which constitutes the first mention of a possible alteration of gap junctions in this type of disease, will have to be further developed to characterize their impact on tubulogenesis processes.In conclusion, this work which revealed a pre-ciliary role of MKS1 in epithelial homeostasis, provides new hypotheses for the etiology of ciliopathies, previously considered as essentially consecutive to signal transduction defects. It also proposes new mechanisms to account for abnormalities of hepatic development, such as bile ducts dysgenesis, and more broadly tubulogenesis processes involved in the development of many organs.

Primary cilia are evolutionarily conserved organelles required in a number of signalling pathways influencing the development and behaviour of a diverse range of organisms. More recently, studies into a new class of human diseases known as ciliopathies have helped to shed light on the critical role of this once-ignored signalling centre. Bardet-Biedl syndrome (BBS) proteins localize to the primary cilium and participate in cilium biogenesis and function. BBS is a pleiotropic human disorder with variable severity that is suitable as a disease model for investigating the pathogenesis of a number of common ciliopathy features such as photoreceptor degeneration, renal cysts, and obesity. The C. elegans genome encodes a number of BBS proteins which undergo intraflagellar transport (IFT) at the primary cilium. Given the conservation between C. elegans and human BBS proteins, I hypothesize the existence of unidentified conserved genetic pathways related to the functions of these proteins. Using C. elegans, I characterize novel features of bbs mutants while identifying sources of genomic variation that may elucidate the variability of human BBS features. I show that C. elegans bbs mutants exhibit smaller body size, delayed development, and decreased exploration behaviour. Moreover, I identify a role for the soluble guanylate cyclases GCY-35/GCY-36 in modifying these bbs phenotypes. I conclude that BBS proteins non-cell autonomously influence a set of body cavity neurons in which GCY-35/GCY-36 function genetically upstream of a cGMP-dependent protein kinase (PKG), EGL-4, to control body size. Furthermore, the role of GCY-35/GCY-36 is unique amongst a large number of guanylate cyclases and BBS proteins may influence body size via an IFT-independent function. I explore the biological functions of EGL-4 and conclude that it may regulate body size through multiple cellular mechanisms. I also examine potential candidate genes related to cGMP production and turnover, confirming that additional cGMP-related factors can influence body size although not necessarily in body cavity neurons. In conclusion, I propose a model where BBS-expressing sensory neurons influence body size and development through cGMP-PKG signalling in body cavity neurons while functioning in parallel with additional sensory neurons (possibly BBS-independent) that use similar cGMP-PKG signalling dynamics.

Primary cilia are evolutionarily conserved organelles required in a number of signalling pathways influencing the development and behaviour of a diverse range of organisms. More recently, studies into a new class of human diseases known as ciliopathies have helped to shed light on the critical role of this once-ignored signalling centre. Bardet-Biedl syndrome (BBS) proteins localize to the primary cilium and participate in cilium biogenesis and function. BBS is a pleiotropic human disorder with variable severity that is suitable as a disease model for investigating the pathogenesis of a number of common ciliopathy features such as photoreceptor degeneration, renal cysts, and obesity. The C. elegans genome encodes a number of BBS proteins which undergo intraflagellar transport (IFT) at the primary cilium. Given the conservation between C. elegans and human BBS proteins, I hypothesize the existence of unidentified conserved genetic pathways related to the functions of these proteins. Using C. elegans, I characterize novel features of bbs mutants while identifying sources of genomic variation that may elucidate the variability of human BBS features. I show that C. elegans bbs mutants exhibit smaller body size, delayed development, and decreased exploration behaviour. Moreover, I identify a role for the soluble guanylate cyclases GCY-35/GCY-36 in modifying these bbs phenotypes. I conclude that BBS proteins non-cell autonomously influence a set of body cavity neurons in which GCY-35/GCY-36 function genetically upstream of a cGMP-dependent protein kinase (PKG), EGL-4, to control body size. Furthermore, the role of GCY-35/GCY-36 is unique amongst a large number of guanylate cyclases and BBS proteins may influence body size via an IFT-independent function. I explore the biological functions of EGL-4 and conclude that it may regulate body size through multiple cellular mechanisms. I also examine potential candidate genes related to cGMP production and turnover, confirming that additional cGMP-related factors can influence body size although not necessarily in body cavity neurons. In conclusion, I propose a model where BBS-expressing sensory neurons influence body size and development through cGMP-PKG signalling in body cavity neurons while functioning in parallel with additional sensory neurons (possibly BBS-independent) that use similar cGMP-PKG signalling dynamics.

BBSome is a protein complex crucial for trafficking of specific cargoes to the primary cilium. Although primary cilia are typically not present in cells of haematopoietic origin, such as T cells, recent research has revealed striking parallels between the primary cilium and the immunological synapse. Amongst other similarities, both structures are supposed to use the same transport machinery involving Rab8 and IFT20, the close interaction partners of BBSome. The first goal of this thesis was to investigate the role of BBSome in the biology of T cells. Using RT-qPCR, we have shown that BBSome subunits are expressed in lymphoid tissues and T cells. Studies of localization of BBSome subunits in Jurkat cell line and primary OT-I T cells revealed that the subunits have distinct localization patterns with BBS4 localizing to the centrosome and BBS1, BBS5, and BBip10 having dispersed localization. After the contact with an antigen presenting cell, BBS4 re-localizes to the immunological synapse. Mutations in BBSome encoding genes cause Bardet-Biedl syndrome (BBS), a rare ciliopathy presenting with multiorganic symptoms. The second goal of this thesis was to examine the associations between BBS and the immune system. Examination of medical records of more than 450 BBS patients revealed that autoimmune...