Thesis advisor: Junona Moroianu / In this study we investigated the hydrophobic interactions between HPV11 E7 and the FG regions of Nup62N through transfection assays with EGFP-11E7 fusion plasmids in HeLa cells and binding assays with GST-Nup62N immobilized on Glutathione-Sepharose beads. We found that EGFP-11cE7 binds to Nup62N. This suggests a possible mechanism for the nuclear import of HPV11 E7 through direct hydrophobic interactions between its carboxy-terminus and the FG region of Nup62. The interaction between HPV11 E7 and CRM1 nuclear export receptor was also examined using similar methods. Binding between these proteins suggest that nuclear export of 11E7 is mediated by CRM1 binding to its leucine-rich nuclear export signal (NES). / Thesis (BS) — Boston College, 2013. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology.

Cell division relies on many steps, precisely synchronised, to ensure the fidelity of chromosome segregation. To achieve such complex and multiple functions, cells have evolved mechanisms by which one protein can participate in numerous events on the cell life. Over the past few years, an increasing number of functions have been assigned to the proteins of the nuclear pore complex (NPC) also called nucleoporins. NPCs are large complexes studded in the nuclear envelope, which control the nucleocytoplasmic transport. It is now known that nucleoporins participate in spindle assembly, kinetochore organisation, spindle assembly checkpoint, and all processes important for genome integrity maintenance. This work demonstrates that the nucleoporin ALADIN participates in mitosis, meiosis and in cilia. In both mitosis and meiosis, ALADIN is important for proper spindle assembly. In mitosis, it was also discovered that ALADIN is a novel factor in the spatial regulation of the mitotic regulator Aurora A kinase. Without ALADIN, active Aurora A spreads from centrosomes onto spindle microtubules, which affects the distribution of a subset of microtubule regulators and slows spindle assembly and chromosome alignment. Interestingly, mutations in ALADIN causes triple A syndrome and some of the mitotic phenotypes observed after ALADIN depletion also occur in cells from triple A syndrome patients. In meiosis, ALADIN contributes to trigger the resumption of meiosis in female mouse. Impairment of ALADIN from mouse oocyte slows spindle assembly, migration and reduces oocytes ability to extrude polar bodies during meiosis I, which concomitantly affects the robustness of oocyte maturation and impairs mouse embryo development. Nucleoporins were also found at the base of the cilia, a centriole-derived organelle that participates in differentiation, migration, cell growth from development to adulthood. Here it is shown that ALADIN is also localised at the base of the cilia. With this work, new ALADIN’s functions have been identified across cell division, as well as uncovered an unexpected relation between triple A syndrome and cell division.

Background/Aims: A 33-year-old man was referred for the first time to the Division of Neurology because of the presence and progression of neurological symptoms. Dysphagia, weakness, reduced tear production, and nasal speech were present. In order to point the attention of late-onset triple A syndrome we describe this case and review the literature. Methods: Hormonal and biochemical evaluation, Schirmer test, tilt test and genetic testing for AAAS gene mutations. Results: Late-onset triple A syndrome caused by a novel homozygous missense mutation in the AAAS gene (A167V in exon 6) was diagnosed at least 17 years after symptom onset. Conclusions: The association between typical signs and symptoms of triple A syndrome should suggest the diagnosis even if they manifest in adulthood. The diagnosis should be confirmed by Schirmer test, endocrine testing (both basal and dynamic), genetic analysis, and detailed gastroenterological and neurological evaluations. Awareness of the possible late onset of the disease and of diagnosis in adulthood is still poor among clinicians, the acquaintance with the disease is more common among pediatricians. The importance of an adequate multidisciplinary clinical approach, dynamic testing for early diagnosis of adrenal insufficiency and periodical reassessment of adrenal function are emphasized. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Background/Aims: A 33-year-old man was referred for the first time to the Division of Neurology because of the presence and progression of neurological symptoms. Dysphagia, weakness, reduced tear production, and nasal speech were present. In order to point the attention of late-onset triple A syndrome we describe this case and review the literature. Methods: Hormonal and biochemical evaluation, Schirmer test, tilt test and genetic testing for AAAS gene mutations. Results: Late-onset triple A syndrome caused by a novel homozygous missense mutation in the AAAS gene (A167V in exon 6) was diagnosed at least 17 years after symptom onset. Conclusions: The association between typical signs and symptoms of triple A syndrome should suggest the diagnosis even if they manifest in adulthood. The diagnosis should be confirmed by Schirmer test, endocrine testing (both basal and dynamic), genetic analysis, and detailed gastroenterological and neurological evaluations. Awareness of the possible late onset of the disease and of diagnosis in adulthood is still poor among clinicians, the acquaintance with the disease is more common among pediatricians. The importance of an adequate multidisciplinary clinical approach, dynamic testing for early diagnosis of adrenal insufficiency and periodical reassessment of adrenal function are emphasized. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23333号 / 生博第451号 / 新制||生||60(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 永尾 雅哉, 教授 片山 高嶺, 教授 谷口 雄一 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope (NE) and composed of proteins called nucleoporins. NPCs as such control the bidirectional traffic of proteins and RNAs between the nucleus and the cytoplasm in eukaryotic cells whereas individual nucleoporins were found to be implicated in other cellular processes such as, cell division, kinetochore assembly, gene expression and cell migration. A prime example for nucleoporin functional versatility can be seen in Nup153. Nup153 is since its discovery known to be a central player in nucleocytoplasmic transport, but additionally participates directly or indirectly, for example, in gene expression and cell cycle control. In this context, it was previously shown that altered levels of Nup153 led to mitotic abnormalities, particularly in cytokinesis and in the spindle assembly checkpoint (SAC). The SAC promotes accurate chromosome separation to ensure the faithful segregation of genetic material to daughter cells. Nup153 was found to interact with the SAC protein Mad1. In the present study, we have further dissected the interaction between Nup153 and Mad1 and investigated the function of the Nup153-Mad1 complex in human cells. By using the high resolution imaging technique “in situ proximity ligation assay”, we found that Nup153 and Mad1 interact with each other exclusively in the presence of a NE, from late mitosis to prophase. By in vitro binding assays, we have confirmed the direct interaction between Nup153 and Mad1 and furthermore identified two independent Nup153-binding sites in Mad1. We have also provided some evidence that Nup153 interacts also with SUMO-modified Mad1.It was previously shown that depletion of Nup153 had no obvious effect on Mad1 and SAC activity. In the present study, we have shown by time-lapse imaging microscopy that the depletion of Mad1 led to a delayed recruitment of Nup153 at the reforming NE during anaphase in living cells, which was often accompanied by a prolongation of anaphase. Furthermore, Mad1 depletion led to alterations in the NE architecture, which were characterized by a change of the membrane curvature at the NPC-NE interface. This was followed by an expansion of the spacing between the inner and outer membranes as seen by electron microscopic and three-dimensional structured illumination investigations. This suggests an implication of Mad1 in a mechanism related to the NE reformation and stability independent of the SAC. Mad1 depletion also resulted in redistribution of the ER network and mitochondria throughout the cell as seen by fluorescence microscopy. Nup153 depletion coincided with the NE abnormalities and alteration of these organelles similar to that seen in Mad1-depleted cells. Further, by fluorescence microscopy, we have shown that Nup153 depletion, but not of Mad1, partially affected the localization of the cytoplasmic nucleoporins in human and in mouse cells and thus the NPC integrity. In conclusion, altogether, our results suggest that Nup153 is essential for NE and NPC integrity. Nup153 has likely separable roles in this context: one in post-mitotic NE reformation with Mad1 and one in interphase in NPC assembly. Nup153-Mad1 complex has a function independent of the spindle checkpoint, but important for the establishment of an intact NE architecture. / Les pores nucléaires sont des structures enchâssées dans l’enveloppe nucléaire et composées de protéines appelées les nucléoporines. Ces pores nucléaires contrôlent le trafic bidirectionnel des protéines et des ARNs entre le noyau et le cytoplasme dans les cellules eucaryotes tandis que les nucléoporines individuelles sont également impliquées dans d’autres processus cellulaires tels que la division cellulaire, l’assemblage des kinétochores, l’expression génétique et la migration cellulaire. Un exemple primordial de la versatilité fonctionnelle des nucléoporines peut être observé à travers Nup153. Depuis sa découverte, Nup153 est connue pour être un élément clé dans le transport nucléo-cytoplasmique, mais il a également été démontré qu’elle participait directement ou indirectement à l’expression génétique et au contrôle du cycle cellulaire. Dans ce contexte, nous avons montrés précédemment que des niveaux altérés de Nup153 menaient à des anomalies mitotiques, particulièrement en cytokinèse et dans le point de contrôle de l’assemblage du fuseau mitotique (SAC). Le SAC assure la ségrégation correcte du matériel génétique entre les cellules filles. Il a été montré que Nup153 interagit avec la protéine du SAC Mad1. Dans cette étude, nous avons utilisé une technique d’imagerie de haute résolution, « in situ proximity ligation assay » pour disséquer davantage l’interaction entre Nup153 et Mad1 dans les cellules humaines. Nous avons montré que ces deux protéines interagissent exclusivement au niveau de l’enveloppe nucléaire, depuis les dernières phases de la mitose jusqu’à la prophase. Par des expériences d’interaction in vitro, nous avons également identifiés sur Mad1 deux sites de liaison indépendants pour Nup153. Nous avons également fourni des indications que Nup153 interagit aussi avec une forme SUMOylée de Mad1. La déplétion de Mad1 menait à un recrutement tardif de Nup153 au niveau de l’enveloppe nucléaire en cours de reformation en anaphase dans les cellules vivantes et à des altérations de l’architecture de l’enveloppe nucléaire, caractérisées par un changement de la courbure membranaire au niveau de l’interface pore nucléaire-enveloppe nucléaire. Suite à cela, une expansion de l’espace entre les membranes nucléaires internes et externes a été observée par microscopie électronique. Ceci suggère une implication de Mad1 dans un mécanisme lié à la stabilité de l’enveloppe nucléaire indépendant du SAC. La déplétion de Mad1 résultait également en une redistribution du RE et des mitochondries à travers la cellule. La déplétion de Nup153 coïncidait avec des anomalies similaires au niveau de l’enveloppe nucléaire et des organelles. De plus, la déplétion de Nup153 affectait partiellement la localisation des nucléoporines cytoplasmiques, contrairement à la déplétion de Mad1. Ensemble, nos résultats suggèrent que Nup153 est essentielle pour l’intégrité des pores nucléaires et de l’enveloppe nucléaire. Nup153 semble avoir deux rôles, un au niveau de la formation de l’enveloppe nucléaire en fin de mitose, en complexe avec Mad1 et un autre rôle au niveau de l’assemblage des pores nucléaires. Le complexe Nup153-Mad1 a une fonction indépendante du SAC, mais importante pour l’établissement d’une enveloppe nucléaire intacte. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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Chez les trypanosomatidés, on observe un double cycle cellulaire : karyokinèse et cytodiérèse sont synchronisées mais indépendantes. La membrane nucléaire persiste au cours de la mitose dite fermée. Les modalités de réplication de l'ADN et de sa régulation, de même que plusieurs étapes de la ségrégation des chromosomes, restent non élucidées : le kinétochore est composé de protéines très atypiques (KKTs), et il existe un déficit du nombre de kinétochores par rapport au nombre de chromosomes. D'autre part, la constitution des pôles du fuseau mitotique associés à la membrane nucléaire est totalement inconnue. Les nucléoporines sont des protéines conservées au cours de l'évolution, principalement impliquées dans la constitution des pores nucléaires et le trafic entre le noyau et le cytoplasme, mais également de plus en plus considérées comme des acteurs importants de la dynamique chromatinienne. En utilisant des vecteurs d'expression protéique sous forme fusionnée à la GFP, nous avons déterminé la localisation subcellulaire de 15 nucléoporines chez Leishmania major. Si la plupart de ces nucléoporines se localisent à la membrane nucléaire, plusieurs d'entre elles ont des localisations secondaires qui peuvent être prédominantes. Ainsi la nucléoporine Mlp2 est préférentiellement localisée au niveau du kinétochore et, en fin de mitose, à l'extrémité du fuseau mitotique chez les deux parasites Leishmania major et Trypanosoma brucei. En accord avec la localisation de TbMlp2 au kinétochore chez T. brucei, où les centromères sont identifiés, nous avons fréquemment détecté TbMlp2 à proximité des séquences centromériques, elles-mêmes détectées par FISH en périphérie du nucléole. Egalement grâce à la technique de FISH, nous montrons que l'inhibition de l'expression de TbMlp2 par ARN-interférence perturbe la distribution des chromosomes au cours de la mitose, conduisant à une aneuploïdie. Paradoxalement, cette inhibition n'a aucun effet sur la croissance des cellules. Nous présentons également le cas singulier de Mlp1, dont la localisation chez T. brucei dépend du site d'intégration choisi. Cette localisation sera discutée à la lumière des phénotypes observés lors de l'inhibition de son expression. / Trypanosomatid parasites exhibit two independent though coordinated (nuclear and mitochondrial) cell cycles, and a closed mitosis, of which many constituents and processes are unknown. In particular, most steps of the chromosome segregation remain elusive: the kinetochore is composed of atypical proteins called KKT and the number of kinetochores is deficient in relation to the number of chromosomes. Moreover, the constitution of the nuclear membrane-associated mitotic spindle poles is unknown. Nucleoporins are evolutionary conserved proteins mainly involved in the constitution of the nuclear pores and trafficking between the nucleus and cytoplasm, but are also increasingly viewed as main actors in chromatin dynamics. Using GFP-fused proteins, we determined the cellular localization of the 15 nucleoporins in Leishmania major. If most of these nucleoporins localized at the nuclear membrane, some of them exhibited secondary locations which are predominant in a few cases. Thus, the nucleoporin Mlp2 localized preferentially at the kinetochore and, at the end of mitosis, at the mitotic spindle poles in both parasites Leishmania major and Trypanosoma brucei. Consistent with the localisation of TbMlp2 to the kinetochore in T. brucei, where centromeres are identified, TbMlp2 was frequently detected in the vicinity of the centromeric sequences in the periphery of the nucleolus. The use of FISH allowed us to show that RNAi knockdowns of TbMlp2 disturbed the distribution of chromosomes during mitosis, leading to aneuploidy. Paradoxically RNAi knockdowns of TbMlp2 had no effect on cell growth. We will also present the singular case of Mlp1 whose location is dependent on the integration site in T. brucei. This location will be discussed in the light of the phenotypes observed after inhibition of its expression.

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