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Lin, Junyan. "Assembly and function of cytosolic nuclear pore complexes." Electronic Thesis or Diss., Strasbourg, 2024. http://www.theses.fr/2024STRAJ037.
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Les complexes des pores nucléaires (CPN) sont d'énormes assemblages protéiques intégrés dans l'enveloppe nucléaire (EN). Ils servent de structures pour le transport bidirectionnel. Essentiels, ils permettent le maintien de l'équilibre entre le noyau et le cytoplasme. Au-delà de leur résidence dans l'EN, les CPN se trouvent également dans des feuillets du réticulum endoplasmique (RE) empilés connus sous le nom de lamelles annulaires (LA). Cependant, la fonction et les voies régissant la biogenèse des LA restent énigmatiques. Notre investigation dans les cellules de mammifères révèle un mécanisme où la formation des LA résulte de la fusion de CPN cytosoliques préassemblés. Le mouvement des CPN cytosoliques est intimement lié à la dynamique du RE, car ils migrent vers l'EN et s'y intègrent en début d'interphase en conditions de croissance normales, ce processus est médié par les microtubules. RanBP2 (Nup358), un constituant des filaments cytoplasmiques des CPN, apparaît comme nécessaire et suffisant pour la formation des LA dans le cytoplasme. Mécaniquement, les répétitions FG dans le N-terminus de RanBP2 jouent un rôle crucial en orchestrant l'état d'oligomérisation des unités de l'anneau extérieur des CPN, connues sous le nom de complexes Y. Notre étude élucide un processus d'assemblage crucial pour nourrir l'EN, assurant la fonctionnalité des pores nucléaires et soulignant l'importance des CPN cytosoliques dans l'homéostasie cellulaire des mammifères<br>Nuclear pore complexes (NPCs), huge protein assemblies built into the nuclear envelope (NE), serve as pivotal structures for bidirectional transport, maintaining the equilibrium between the nucleus and cytoplasm. Beyond their residence within the NE, NPCs are also found in stacked cytoplasmic membranes known as annulate lamellae (AL). However, the function and pathways governing the biogenesis of AL remain enigmatic. Our investigation in mammalian cells unveils a mechanism wherein AL formation arises through the fusion of pre-assembled cytosolic NPCs. The movement of cytosolic NPCs is intricately linked to the dynamics of the endoplasmic reticulum (ER), as they migrate towards and integrate into NE during early interphase under normal growth conditions, a process mediated by microtubules. RanBP2 (Nup358), a constituent of the NPC cytoplasmic filaments, emerges as necessary and sufficient for AL formation in the cytoplasm. Mechanistically, the FG repeats in the N-terminus of RanBP2 play a pivotal role by orchestrating the oligomerization state of the NPC outer ring units, known as Y-complexes. Our study elucidates an assembly process crucial for NE nourishment, ensuring the functionality of nuclear pores and underscoring the significance of cytosolic NPCs in mammalian cellular homeostasis
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Walther, Tobias. "The role of Peripheral Nuclear Pore Complex (NPC) structures in nuclear transport and NPC architecture." Diss., lmu, 2002. http://nbn-resolving.de/urn:nbn:de:bvb:19-4945.
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Kelich, Joseph M. "Single-Molecule Studies on Nuclear Pore Complex Structure and Function." Diss., Temple University Libraries, 2018. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/511772.
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Biology<br>Ph.D.<br>Nuclear pore complexes (NPCs) are large macromolecular gateways embedded in the nuclear envelope of Eukaryotic cells that serve to regulate bi-directional trafficking of particles to and from the nucleus. NPCs have been described as creating a selectively permeable barrier mediating the nuclear export of key endogenous cargoes such as mRNA, and pre-ribosomal subunits as well as allow for the nuclear import of nuclear proteins and some viral particles. Remarkably, other particles that are not qualified for nucleocytoplasmic transport are repelled from the NPC, unable to translocate. The NPC is made up of over 30 unique proteins, each present in multiples of eight copies. The two primary protein components of the NPC can be simplified as scaffold nucleoporins which form the main structure of the NPC and the phenylalanine-glycine (FG) motif containing nucleoporins (FG-Nups) which anchor to the scaffold and together create the permeability barrier within the pore. Advances in fluorescence microscopy techniques including single-molecule and super-resolution microscopy have made it possible to label and visualize the dynamic components of the NPC as well as track the rapid nucleocytoplasmic transport process of importing and exporting cargoes. The focus of this dissertation will be on live cell fluorescence microscopy application in probing the dynamic components of the NPC as well as tracking the processes of nucleocytoplasmic transport.<br>Temple University--Theses
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Lolodi, Ogheneochukome. "Kinetic analysis of karyopherin-mediated transport through the nuclear pore complex." 京都大学 (Kyoto University), 2016. http://hdl.handle.net/2433/215696.
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Authors are permitted to post the MBoC PDF of their articles (and/or supplemental material) on their personal websites or in an online institutional repository provided there appears always the proper citation of the manuscript in MBoC and a link to the original publication of the manuscript in MBoC (http://www.molbiolcell.org/site/misc/ifora.xhtml)<br>Kyoto University (京都大学)<br>0048<br>新制・課程博士<br>博士(生命科学)<br>甲第19869号<br>生博第350号<br>新制||生||46(附属図書館)<br>32905<br>京都大学大学院生命科学研究科統合生命科学専攻<br>(主査)教授 河内 孝之, 教授 藤田 尚志, 教授 永尾 雅哉<br>学位規則第4条第1項該当
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Liu, Hui-Lin. "Analyses of mitotic nuclear pore complex dynamics in Aspergillus nidulans." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1243862963.
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Xu, Xianfeng. "Two sides of the plant nuclear pore complex and a potential link between Ran GTPase and plant cell division." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1190050471.
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Markossian, Sarine W. "Nup2 and a Newly Discovered Nuclear Pore Complex Protein, NupA, Function at Mitotic Chromatin Controlled by the NIMA Kinase." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1306851345.
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Anderson, Daniel J. "Dynamics of nuclear envelope and nuclear pore complex formation." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p3336561.
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Thesis (Ph. D.)--University of California, San Diego, 2008.<br>Title from first page of PDF file (viewed December 16, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 127-145).
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Onischenko, Evgeny. "Disassembly and reassembly of the nuclear pore complex /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-929-7/.
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Osmanovic, D. "Polymer theory applied to the nuclear pore complex." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1451621/.
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Physically interesting behaviour can arise when soft matter is confined to nanoscale dimensions. A highly relevant biological example of such a phenomenon is the Nuclear Pore Complex (NPC), found perforating the Nuclear Envelope of all eukaryotic cells. In the central conduit of the NPC, of 30-60 nm diameter, a disordered arrangement of proteins regulates all macromolecular transport between the nucleus and the cytoplasm. Its selectivity for larger macromolecules relies on changes in a permeability barrier that is formed by these unstructured proteins, induced by interactions of these proteins with molecules called Nuclear Transport Receptors (NTRs), which can chaperone larger macromolecules through the NPC. The exact mechanism for the transport selectivity is unknown. To model these unstructured proteins in the nanoscale channel of the NPC, a density functional theory approach is developed that treats the proteins as interacting polymers. This new method is tested against Monte Carlo to show its validity. A detailed comparison between this model system and those previously proposed in the literature is provided. In a parameter range relevant for the NPC, the system shows bimodal behaviour The polymers can alternate between two condensed states: An open state, in which this condensation takes place at the channel wall, and a closed state in which it occurs at the channel centre. We then extend this model by including explicitly the effect of Nuclear Transport Receptors on the conformations of the polymers. The model takes into account the finite size of the transport receptors relative to the NPC diameter. Mapping the polymer and transport receptor behaviour over a set of physiologically relevant parameters gives different structural scenarios for the various hypothesized transport mechanisms. Further to this, the transport rates for each parameter set can be obtained, showing whether such parameters are consistent with experimental evidence. In addition to this, we study the effect of relaxing some of the assumptions of our model, specifically by looking at azimuthal symmetry breaking effects in two dimensions. We also compare our model to experimental results measuring the thickness of planar polymer brushes comprised of NPC proteins to further justify parameter choices.
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Talamas, Jessica Arielle. "Cell cycle dependent differences in nuclear pore complex assembly." Diss., [La Jolla] : University of California, San Diego, 2010. http://wwwlib.umi.com/cr/ucsd/fullcit?p3403712.
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Thesis (Ph. D.)--University of California, San Diego, 2010.<br>Title from first page of PDF file (viewed June 1, 2010). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (leaves 90-98).
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Stanley, George. "Probing the transport barrier of the nuclear pore complex." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10061418/.
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The nuclear pore complex (NPC) is the selective gateway through which macromolecules must pass when entering or exiting the nucleus. It is a cog in the gene expression pathway, an entrance to the nucleus exploited by viruses, and a highly-tuned nanoscale filter. The NPC is a large proteinaceous assembly with a central channel occluded by natively disordered proteins, known as FG-nucleoporins (or FG-nups). These FG-nups, along with a family of soluble proteins (known as nuclear transport receptors, or NTRs), form the selective transport barrier. Although much is known about the transport cycle and the necessity of NTRs for chaperoning cargo molecules through the NPC, the mechanism by which NTRs and NTR•cargo complexes translocate the selective transport barrier is not well understood. How can intrinsically disordered FG-nups and soluble NTRs form a transport barrier that is selective, ATP-free, and fast? In this thesis, high-resolution atomic force microscopy (AFM) and a new, fast force-spectroscopy technique (PeakForce QNM) are used to provide a structural and nanomechanical analysis of individual NPCs. This data highlights the structural diversity and complexity at the nuclear envelope, showing the interplay between the lamina network, actin filaments, and the NPCs. It reveals the dynamic behaviour of NPC scaffolds and displays pores of varying sizes. Of functional importance, the NPC central channel shows large structural diversity (in both its mechanical properties and topography), supporting the notion that FG-nup cohesiveness is in a range that facilitates collective rearrangements at little energetic cost. Furthermore, various NTRs are shown to interact in qualitatively different ways with the FG-nups, with particularly strong binding of importin-β. Next, a method for analysing the dynamics of reconstituted FG-nups inside mimetic NPCs is presented - with the results highlighting the surprisingly slow time-scale for collective rearrangement of FG-nup morphologies in the pore geometry. When this analysis is applied to the real NPC, however, no dynamic movement of FG-nups is detected. Finally, preliminary results from AFM imaging experiments of large cargoes (in this case, the hepatitis B virus capsid) translocating the NPC, are presented. This thesis supports the notion that FG-nup cohesiveness is tuned such that the energetics of stable FG-nup morphologies lie near transition states, thereby allowing the collective rearrangement of FG-nups at little energetic cost. Furthermore, it suggests that NTRs with several FG-nup binding sites (such as importin-β) are an intrinsic component of the transport barrier.
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Suresh, Subbulakshmi. "Nup2: A multifunctional player in nuclear transport and mitotic nuclear pore complex inheritance." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1480153558155228.
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Quintana, Star-Lena Jaramillo. "Discrete 3D Model of Molecular Diffusion Through the Nuclear Pore Complex." Master's thesis, Temple University Libraries, 2015. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/343686.
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Mathematics<br>M.S.<br>Nuclear pore complexes (NPCs) are passageways that exist within the nuclear envelope (NE) of a eukaryotic cell. Molecular cargo travel through the passageways to either import to the nucleus or export to the cytoplasm of the cell. Efficient export of certain cargo is necessary for maintained health of a cell, and hence, the organism. Traditional methods of observing NPCs lack resolution great enough for scientists to study the many interactions that take place inside of the complex. A discrete 3D model of the molecular diffusion was built to understand how cargo moves through the NPCs and how to improve import and export efficiency of particular molecules. The basis of the model is a Langevin equation that was customized to the environment of the central channel of a NPC. The model incorporated not only the Brownian motion of the molecules, but also the geometry of the channel, the diffusion coefficient for molecules in the fluid of the central channel, and a potential energy (PE) function to describe drifting affects by the dense layers of phenylalanine-glycine (FG) repeats located in the channel and a concentration of transport receptors located on either ends of the NPC. The model simulated the movement of spherical molecules through the NPC and kept track of their location during their transport. The model showed that the cargo’s movement has a distinct dependence on the PE function. The model can be further, and easily, manipulated and used for more comparisons to experimentally determined export efficiency for different cargo.<br>Temple University--Theses
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Bestembayeva, A. "Biophysical properties of the transport barrier in the nuclear pore complex." Thesis, University College London (University of London), 2016. http://discovery.ucl.ac.uk/1474430/.
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The Nuclear Pore Complex (NPC) is a large protein structure found in eukaryotic cells, perforating the nuclear envelope. It mediates bidirectional selective transport between the nucleus and the cytoplasm. The NPC contains a permeability barrier consisting of unstructured nuclear pore proteins. The structure of the permeability barrier is not well defined. As a consequence, various models have been proposed for its structure and functionality. Typically, these models consider the unstructured nuclear pore proteins as weakly or strongly interacting polymers: In the first case nuclear pore proteins protrude from the pore creating an entropic barrier; in the second case they may form a meshwork occupying the central channel, resembling a hydrogel. In this thesis, I measure the nanomechanical properties of this barrier in intact NPCs, and compare them to the properties expected for entropic brushes and gel-like materials. To this end, I carried out nanometre-scale force spectroscopy measurements using Atomic Force Microscopy (AFM). Prior to the measurements the pores were treated with reagents that activated the transport process, thus flushing out the pores to ensure that I was probing the barrier itself instead of cargo stuck in transit. I carried out Laser Scanning Confocal Microscopy experiments to verify this procedure, as well as to measure transport properties of the pores in isolated nuclei. For comparison, I also measure nanomechanical properties of artificial polymer brushes, and set the first steps towards creating protein-coated solid-state nanopores as a reductionist model system for the NPC. My results indicate that the proteins in the NPC form a condensed network, more closely resembling a hydrogel than a brush dominated by entropic interactions.
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Mossaid, Ikram. "The nuclear pore protein Nup153: Dissecting its role in nuclear envelope and nuclear pore complex architecture and its interaction with the spindle assembly checkpoint protein Mad1." Doctoral thesis, Universite Libre de Bruxelles, 2016. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/234375.
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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.<br>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.<br>Doctorat en Sciences<br>info:eu-repo/semantics/nonPublished
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Kelley, Kotaro. "Structural and biochemical characterization of nuclear pore complex structural scaffold sub-complexes." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/113466.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2017<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references.<br>The nuclear pore complex (NPC) is a large, modular protein assembly that regulates nucleocytoplasmic transport in all eukaryotes. The ~60-120 MDa NPC is a modular assembly of multiple copies of ~30 distinct proteins that are arranged into biochemically distinct sub-complexes. We believe that the structural characterization of the NPC is essential for understanding its transport mechanisms and various pathologies and human diseases associated with deletions or mutations of constituents. To obtain detailed structural information of the NPC, techniques that span several resolution ranges are necessary due to its large size and complexity. For instance, recent progress in the structural characterization of the overall architecture of the NPC by cryo-electron tomography(cryo-ET) to ~23Å resolution has revealed its size, shape, and course arrangement, but lacks distinguishable protein-protein boundaries and secondary structural details.<br>Although the entire NPC is not amenable to high resolution X-ray crystallography, we complement the cryo-ET reconstructions with a divide and conquer approach by obtaining high resolution X-ray crystal structures of individual subcomplexes. By taking advantage of the modular nature of the NPC, we can dock subcomplexes into the cryo-ET reconstructions to identify their location within the NPC. This composite structure will bridge the meso resolution cryo-ET reconstructions of the entire NPC and the incomplete but high resolution X-ray crystal structure of individual subcomplexes. As a first step towards understanding the detailed organization of the NPC, our goal is to solve the high resolution structures of the two principal structural scaffold subcomplexes, the Y and Nic96 complexes. In this study, we present the high resolution composite X-ray crystal structure of the Y complex.<br>Docking the composite model into previously solved random conical tilt(RCT) and tomographic reconstructions of negatively stained samples of the Y complex shows overall consistency between the three methods, yet we highlight important structural differences that constrain possible arrangements of multiple Y complexes within the NPC. By docking the composite model into the cryo-ET reconstructions of the entire NPC, we propose an arrangement of multiple Y complexes that is consistent with our composite structure. In addition, progress on structurally characterizing the Nic96 complex will be presented. Preliminary results suggest that Nup1 92 and Nic96 form a flexible, yet semi-ordered interface. Future directions for characterizing the rest of the Nic96 complex, including current challenges and suggestions will be discussed.<br>by Kotaro Kelley.<br>Ph. D.<br>Ph.D. Massachusetts Institute of Technology, Department of Biology
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Hamed, Mohamed [Verfasser]. "A nuclear export sequence in Nup214 promotes its targeting to the nuclear pore complex / Mohamed Hamed." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2020. http://d-nb.info/1220504505/34.
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Hase, Manuela. "Molecular and ultrastructural analysis of Tpr, a nuclear pore complex-attached coiled-coil protein /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-525-5/.
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Loeb, Jonathan David Joshua. "Molecular characterization of components of the nuclear pore complex and the nuclear import system in Saccharomyces cerevisiae." Thesis, Massachusetts Institute of Technology, 1995. http://hdl.handle.net/1721.1/33532.
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Sachdev, Ruchika [Verfasser], and Ralf-Peter [Akademischer Betreuer] Jansen. "Functional Characterization of the Nup93 Complex in Nuclear Pore Complex Assembly / Ruchika Sachdev ; Betreuer: Ralf-Peter Jansen." Tübingen : Universitätsbibliothek Tübingen, 2013. http://d-nb.info/1162844442/34.
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Zhang, Wanzhen. "Redox-dependent regulation of molecular crowding barrier in the nuclear pore." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263794.
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Zimmerli, Christian Eugen [Verfasser], and Ed [Akademischer Betreuer] Hurt. "In cellulo architecture of the nuclear pore complex / Christian Eugen Zimmerli ; Betreuer: Ed Hurt." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/1233867547/34.
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Chalhoub, Antonious. "The Amyotrophic Lateral Sclerosis 8 Mutant VAPB-P56S Causes a Nuclear Envelope and Nuclear Pore Defect." Thèse, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/23191.
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A P56S mutation in the VAPB MSP domain is linked to adult-onset amyotrophic lateral sclerosis 8. The objective of this study is to characterize the functional role of VAPB in transport of NE and NPC proteins from the ER to the NE. Over-expression of VAPB-P56S blocked the transport of nucleoporins (Nups) and NE proteins, resulting in their sequestration in dilated cytoplasmic membranes. Simultaneous overexpression of the FFAT motif (two phenylalanines in an acidic track) antagonizes mutant VAPB effects and restores transport to the NE. VAPB function is required for transport to the NE because knockdown of endogenous VAPB recapitulates this phenotype. Moreover, the compartment in which Nups and NE proteins are sequestered and retained was identified as ER-Golgi intermediate compartment (ERGIC). Moreover, a defect in the transport of NE and NPC proteins attenuates nucleocytoplasmic shuttling of the glucocorticoid receptor (GR). Further, VAPB-P56S which is only soluble in SDS was solubilized in the Triton-X-100 fraction similar to VAPB-WT upon co-transfection with the FFAT motif suggesting that FFAT interacts with the insoluble VAPB-P56S protein changing its biophysical properties.
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Nordeen, Sarah Ann. "A nanobody suite for yeast scaffold nucleoporins provides details of the Y complex structure and nuclear pore complex assembly." Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/127138.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, May, 2020<br>Cataloged from the official PDF of thesis.<br>Includes bibliographical references.<br>Nuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups, that can be classified into three categories: 1. Stably associated scaffolding nups, 2. Peripheral nups, and 3. Phenylalanine-glycine (FG) repeat containing nups that form the permeability barrier of the NPC. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 complex. Working in S. cerevisiae to study the assembly of these two essential subcomplexes, we developed a suite of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. The nanobodies bind their targets specifically and with high affinity, albeit with varying kinetics. We mapped the epitope of eight members of the nanobody library via crystal structures of nup-nanobody co-complexes.<br>Nuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups, that can be classified into three categories: 1. Stably associated scaffolding nups, 2. Peripheral nups, and 3. Phenylalanine-glycine (FG) repeat containing nups that form the permeability barrier of the NPC. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 complex. Working in S. cerevisiae to study the assembly of these two essential subcomplexes, we developed a suite of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. The nanobodies bind their targets specifically and with high affinity, albeit with varying kinetics. We mapped the epitope of eight members of the nanobody library via crystal structures of nup-nanobody co-complexes.<br>In two cases, the nanobodies facilitated the crystallization of novel nup structures, namely the full-length Nup84-Nup133 [alpha]-helical domain structure and the Nup133 [beta]-propeller domain structure. Together these two structures completely characterize the S. cerevisiae Y complex molecular assembly. Further, the Nup133 [beta]-propeller domain contains a structurally conserved amphipathic lipid packing sensor (ALPS) motif thought to anchor the Y complex to the nuclear envelope, which we confirmed by liposome interaction studies. An additional nanobody facilitated the structure of Nic96 at an improved resolution, revealing previously missing helices. In addition to the utility of these nanobodies for in vitro characterization of NPC assemblies, we also show that expression of nanobody-fluorescent protein fusions reveals details of the NPC assembly in their native, in vivo environment, and possibly of NPC heterogeneity within the nuclear envelope.<br>Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.<br>by Sarah Ann Nordeen.<br>Ph. D.<br>Ph.D. Massachusetts Institute of Technology, Department of Biology
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Orjalo, Arturo V. "Functional analysis of the Nup107-160 complex, the major subunit of the vertebrate nuclear pore /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2005. http://wwwlib.umi.com/cr/ucsd/fullcit?p3190168.
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Partridge, James R. (James Robert). "Biophysical and structural characterization of components from the nuclear pore complex and the ubiquitin pathway." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/57994.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (p. 136-151).<br>Formation of an endomembrane system in the eukaryotic cell is a hallmark of biological evolution. One such system is the nuclear envelope (NE), composed of an inner and outer membrane, used to form a nucleus and enclose the cell's genome. Access to the nucleus from the cytoplasm is mediated by a massive macromolecular machine called the nuclear pore complex (NPC). The NPC resides as a circular opening embedded in the NE and is composed of only -30 proteins that assemble with octagonal symmetry as biochemically defined subcomplexes to form the NPC. One such subcomplex is the Nspl / Nup62 complex, composed of three proteins and stabilized by coiled-coil interactions. Here we reconstitute a tetrameric assembly between the Nspl-complex and a fourth nucleoporin (Nup) Nic96. Nic96 harbors a 20 kDa coiled-coil domain at the N-terminus followed by a 65 kDa stacked helical domain. The coiled-coil domain of the Nspl -complex and the N-terminus of Nic96 combine to form a tetrameric assembly, integrated into the NPC lattice scaffold via the stacked helical domain of Nic96. We characterized the coiled-coil assembly with size exclusion chromatography and analytical ultracentrifugation. Deletion experiments and point mutations, directed by hydrophobic cluster analysis, were used to map connecting helices between members of the protein assembly. Although the core of the NPC is a rigid scaffold built for structural integrity, the NPC as a whole is a dynamic macromolecular machine. Protein transport is regulated by the small G protein Ran. Ran interacts with the NPC of metazoa via two asymmetrically localized components, Nupl53 at the nuclear face and Nup358 at the cytoplasmic face. Both Nups contain distinct RANBP2 type zinc finger (ZnF) domains. We present crystallographic data detailing the interaction between Nup1 53-ZnFs and RanGDP. A crystal-engineering approach led to well-diffracting crystals so that all ZnF-Ran complex structures are refined to high resolution. Each of the four zinc finger modules of Nup1 53 binds one Ran molecule in largely independent fashion. Nupl53-ZnFs bind RanGDP with higher affinity than RanGTP, however the modest difference suggests that this may not be physiologically meaningful. ZnFs may be used to concentrate Ran at the NPC to facilitate nucleocytoplasmic transport. In a separate study we present a structural analysis of the HECT domain from the E3 ubiquitin ligase HUWE1 and with biophysical data we show that an N-terminal helix stabilizes the HECT domain. This element modulates activity, as measured by self-ubiquitination induced in the absence of this helix, distinct from its effects on Ub conjugation of substrate Mcl-1. Such subtle structural elements in this domain potentially regulate the variable substrate specificity displayed by all HECT domain type, E3 ubiquitin ligases.<br>by James R. Partridge.<br>Ph.D.
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Brohawn, Stephen Graf. "Structural elucidation of a common architecture of the nuclear pore complex and COPIl vesicle coats." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/58396.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (p. 156-169).<br>Nuclear pore complexes (NPCs) are massive protein assemblies that perforate the nuclear envelope and form the exclusive passageway for nucleocytoplasmic transport. NPCs play critical roles in molecular transport and a myriad of other cellular processes. Elucidation of the structure of the NPC is thus expected to provide important insight into cell biology. In this thesis, I investigate the structure of a key subcomplex of the NPC and discuss the evolutionary relationship between the NPC and COPIl vesicle coats it illustrates. The NPC is a modular assembly, with a stable structural scaffold supporting dynamically attached components. The structural scaffold is constructed from multiple copies of the Y-shaped complex and the Nic96 complex. We solved the crystal structure of the heterodimeric Nup85-Sehl module that forms a short arm in the Y complex. Nup85 is found to contain a conserved fold, the ancestral coatomer element 1 (ACE1), also present in three other components of the NPC and in the COPI vesicle coat, providing structural evidence of coevolution from a common ancestor. Sec3l ACE1 units interact to form edge elements in the COPI lattice. Using structural knowledge of this edge element, we identified corresponding interactions between ACE1 proteins Nup84 and Nup145C in the NPC. We solved the crystal structure of the heterotrimeric Nup84-Nupl 45C-Secl 3 module that forms the top of the long arm in the Y complex. The heterotypic ACE1 interaction of Nup145C and Nup84 is analogous to the homotypic Sec31 edge element interaction in the COPIl coat. From these and other structures, we assemble a near complete structural model of the Y complex. Further, based on the demonstrated relationship with the COPIl coatomer lattice, we propose a lattice model for the entire NPC scaffold. The common architectural principles of the edge elements in the NPC and COPI lead us to predict that Y complexes will be arranged as struts in the NPC lattice. In this manner, Nup84-Nup145C edge elements are arranged parallel to the transport axis to stabilize the positively curved nuclear envelope. From a lattice model of the NPC follow hypotheses for how other components are integrated into and function within the NPC.<br>by Stephen Graf Brohawn.<br>Ph.D.
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Khalaf, Bouchra. "Beyond the Nuclear Pore Complex, Nup358 Clusters at the Axon Initial Segment of Cultured Neurons." Doctoral thesis, Università degli studi di Trento, 2018. https://hdl.handle.net/11572/368993.
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Nup358 is the largest nucleoporin of around 358 kDa molecular weight that localizes on the cytoplasmic face of the nuclear pore complex (NPC). It takes part in the overall activity of the NPC mediating the transport of nucleic acids and proteins between the nucleus and the cytoplasm. However, due to its multi-domain configuration, Nup358 has a more pleiotropic function in several cellular mechanisms such as mediating the stability of microtubules and axon specification. Since little is known about the non-conventional role of Nup358 in neuronal polarity, my PhD thesis was focused on characterizing the subcellular distribution and expression pattern of Nup358 protein in cultured neurons. My results show Nup358 present at the nuclear rim of neuronal cells, associated with NPCs, also in the cytoplasm having a spotted pattern along the neuronal processes. Interestingly, Nup358 was remarkably clustered at the axon initial segment (AIS) of mature neurons and dependent on a prior recruitment of the master AIS scaffold, Ankyrin-G (AnkG), to this specific region. Of the distinct domains present in Nup358 protein, the N-terminal region was found to be crucial for its localization at the AIS. Further, changes in Nup358 protein expression were monitored during neuronal development. Indeed, I detected the presence of a shorter isoform of Nup358 that was increasing as neurons develop whereas the full-length protein had an opposite decreasing trend. To gain knowledge about the functional role of Nup358 in neurons, I investigated its protein expression/distribution in response to an increasing or decreasing neuronal activity with specific drug treatments. Surprisingly, Nup358 protein expression was reduced following the stimulation or the depolarization of neurons, mediated by calcium influx and NMDA receptors. Overall, my results show that Nup358 has a unique subcellular distribution in neurons, being enriched at the AIS at advanced stages of development, therefore, suggest an involvement of Nup358 protein in maintaining and modulating neuronal polarity and activity.
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Khalaf, Bouchra. "Beyond the Nuclear Pore Complex, Nup358 Clusters at the Axon Initial Segment of Cultured Neurons." Doctoral thesis, University of Trento, 2018. http://eprints-phd.biblio.unitn.it/2971/3/Thesis_BKhalaf.pdf.
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Nup358 is the largest nucleoporin of around 358 kDa molecular weight that localizes on the cytoplasmic face of the nuclear pore complex (NPC). It takes part in the overall activity of the NPC mediating the transport of nucleic acids and proteins between the nucleus and the cytoplasm. However, due to its multi-domain configuration, Nup358 has a more pleiotropic function in several cellular mechanisms such as mediating the stability of microtubules and axon specification. Since little is known about the non-conventional role of Nup358 in neuronal polarity, my PhD thesis was focused on characterizing the subcellular distribution and expression pattern of Nup358 protein in cultured neurons. My results show Nup358 present at the nuclear rim of neuronal cells, associated with NPCs, also in the cytoplasm having a spotted pattern along the neuronal processes. Interestingly, Nup358 was remarkably clustered at the axon initial segment (AIS) of mature neurons and dependent on a prior recruitment of the master AIS scaffold, Ankyrin-G (AnkG), to this specific region. Of the distinct domains present in Nup358 protein, the N-terminal region was found to be crucial for its localization at the AIS. Further, changes in Nup358 protein expression were monitored during neuronal development. Indeed, I detected the presence of a shorter isoform of Nup358 that was increasing as neurons develop whereas the full-length protein had an opposite decreasing trend. To gain knowledge about the functional role of Nup358 in neurons, I investigated its protein expression/distribution in response to an increasing or decreasing neuronal activity with specific drug treatments. Surprisingly, Nup358 protein expression was reduced following the stimulation or the depolarization of neurons, mediated by calcium influx and NMDA receptors. Overall, my results show that Nup358 has a unique subcellular distribution in neurons, being enriched at the AIS at advanced stages of development, therefore, suggest an involvement of Nup358 protein in maintaining and modulating neuronal polarity and activity.
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Enninga, Jost. "Studies on the characterization and regulation of two major nuclear pore complex proteins Nup98 and Nup96." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972566570.
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Tabarraei, Alijan. "Studies on the interaction of HSV-1 multifunctional protein ICP27 with the nuclear pore complex proteins." Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/750/.
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Herpes simplex virus (HSV-1) ICP27 is a multifunctional immediate early (IE) protein, required for expression of several early and late genes and acts at transcriptional and posttranscriptional levels. ICP27 contains both a nuclear localization signal and a nuclear export signal and shuttles between the nucleus and the cytoplasm. It interacts directly with various cellular proteins including REF and cellular mRNA export receptor TAP to export viral mRNAs. However other transport mechanisms may also be employed. All transport across the nuclear envelope must occur via the nuclear pore complex, which consists of proteins called nucleoporins (NUPs). NUPs act to regulate the transport cargos by interaction with mRNA bound proteins and transport receptors, karyopherins. To further explore the transport requirements for ICP27 and viral mRNA export, we investigated its direct interaction with nucleoporins and show that ICP27 interacts directly with nucleoporins tested, in vitro and in wild type and mutant virus infected cells and from RNase I treated and untreated cell extracts indicating that this interaction is not mediated via RNA. Implications of this interaction is discussed in this thesis.
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Capra, T. "THE DNA DAMAGE CHECKPOINT PRESERVES REPLICATION FORK INTEGRITY BY REGULATING TRANSCRIBED GENES ASSOCIATION TO THE NUCLEAR PORE COMPLEX." Doctoral thesis, Università degli Studi di Milano, 2010. http://hdl.handle.net/2434/150161.
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Genome instability, defined as the occurrence and amplification of mutations and chromosomal rearrangements, is a hallmark of neoplasic transformation and cancer onset. Cells have evolved DNA damage checkpoint processes that act in response to replicative stress induced by genotoxic agents. In Saccharomyces cerevisiae the DNA damage checkpoint requires the essential- Rad53 kinase. Checkpoint-defective cells accumulate gross chromosomal rearrangements and loose viability following exposure to hydroxyurea (HU), mainly due to their failure to stabilize stalled replication forks. In this work I show that mutations in THO/TREX and TREX2 genes, that encode factors required for mRNA transcription, export and association of transcribed genes to the nuclear envelope (Gene gating), rescue the viability of rad53 checkpoint deficient mutants treated with low concentrations of HU. This suppression is not affected by the RNAse H overexpression, suggesting that is not related to R-loop formation, transcription defects or hyper-recombination. By 2D gels and BrdU-ChIP analyses, I found that SAC3 (TREX2) deletion suppresses replication fork progression defects and collapse in rad53 cells treated with HU. Ablation of genes encoding Nuclear Pore Complex inner basket factors, that are required for gene gating, also suppresses the HU-sensitivity ofcheckpoint mutants. Moreover I found that upon replication stress, transcribed gene dissociate from the nuclear envelope in a Rad53-dependent manner. Based on these results, I propose that in cells experiencing replication stress the DNA damage checkpoint stabilizes replication forks by releasing the topological constraints imposed by NPC-associated genes.
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Olsson, Magnus. "Nuclear pore membrane glycoprotein 210 as a new marker for epithelial cells." Doctoral thesis, Uppsala University, Department of Cell and Molecular Biology, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3265.
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<p>Epithelial cell polarisation is a prerequisite for the branching morphogenesis in several organs. Differential screening techniques were used to identify genes, which are upregulated during induction of epithelium in early kidney development. This investigation revealed two separate genes, Nuclear localising protein 1 (Nulp1), a previously undescribed gene with sequence characteristics of the basic helix-loop-helix transcription factor family, and glycoprotein 210 (gp210, POM210), an integral membrane protein constituent of the nuclear pore complex (NPC). Of these, gp210 was found to be upreglated during conversion of mesenchyme to epithelium. </p><p>The nuclear envelope, which demarcates the nuclear region in the eukaryotic cell, consists of an inner and an outer membrane that are fused at the locations for NPCs. These large macromolecular assemblages are tube like structures connecting the cytoplasmic and nuclear compartments of the cell. NPCs serve as the only conduits for exchange of molecular information between these cellular rooms. Electron microscopy techniques have revealed detailed information about the NPC architecture. A number of proteins (nucleoporins) have been characterised and embodied as components of the NPC structure. Active, energy dependent nucleocytoplasmic transport of RNAs and proteins is mediated by a group of soluble receptor proteins, collectively termed karyopherins. </p><p>Gp210 has been suggested to be important for nuclear pore formation. Nevertheless, our analyses showed a limited expression pattern of gp210, with its mRNA and protein largely confined to epithelial cells in the mouse embryo. Furthermore, in several cell lines, gp210 was undetectable. The expression pattern of gp210 was not synchronised with some other nucleoporins, indicating NPC heterogeneity. Characterisation of the structure of the human gp210 gene, including its promoter region, gave insight about possible cell-type specific gene regulatory mechanisms. </p><p>Regulation of molecular traffic between the nucleus and the cytoplasm leads to transcriptional control. Cell specific configuration of the NPC structure, due to diffential expression of gp210, could be involved in this control. Gp210 could be of importance for the development of epithelial cell polarisation.</p>
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Monette, Anne. "The remodelling of the nuclear pore complex by human immunodeficiency virus type 1: proteomic analysis and biological significance." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=96805.
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Human immunodeficiency virus type 1 (HIV-1) commandeers host proteins and cellular machineries to its advantage at every step of its replication cycle. This work initially shows that HIV-1 infection causes enhanced expression of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and promotes its cytoplasmic retention; and that this is dependent on the nuclear export of the unspliced viral genomic RNA (vRNA) and to alterations in the abundance and localization of nucleoporin p62 (Nup62). HnRNP A1 and the vRNA remain colocalized in the cytoplasm where hnRNP A1 acts as an internal ribosomal entry site (IRES) trans-acting factor (ITAF) to up-regulate IRES-mediated translation initiation of the HIV-1 vRNA to ensure abundant expression of viral structural proteins in cells infected with HIV-1. To define which components of the nuclear pore complex (NPC) are responsible for alterations in cellular Nup62 expression levels, nuclear envelopes (NEs) were isolated from mock- or HIV-1-infected T-cells for a comparative mass spectrometry study. The level of compositional and abundance changes to NE-associated proteins by the infection was surprisingly extensive, where it caused a large decrease in the presence of anchoring, scaffolding and core Nups. Immunoelectron microscopy analysis reveals that the loss of Nups from HIV-1 infection is not accompanied by changes to the general structure of NEs or NPC, and immunogold labelling shows a scattering of Nups into and across the cytoplasm, and their localization at assembling and budding viruses. Purification of cell free viruses reveals that Nup62 is encapsidated, suggesting that it is not simply ejected from the nuclear envelopes of HIV-1 infected cells, but rather may play an important role during HIV-1 replication.<br>Le virus de l'immunodéficience humaine type 1 (VIH-1) contrôle les protéines et la machinerie de l'hôte à chaque étape de son cycle de réplication. Ce projet a débuté suite à l'observation que l'infection par le VIH-1 augmente l'expression de la protéine ribonucléoprotéique nucléaire hétérogène A1 (hnRNP A1) et cause sa rétention cytoplasmique. Ceci est dépendant de l'exportation nucléaire de l'ARN viral et de modifications de l'abondance et de la localisation de la nucléoporine p62 (Nup62). Dans le cytoplasme, hnRNP A1 sert de facteur facilitant le recrutement des ribosomes sur des sites appelés IRES ('Internal Ribosomal Entry Site') pour garantir l'expression abondante de protéines structurelles virales. Donc, pour définir quels composants du complexe du pore nucléaire (CPN) sont responsables des modifications de Nup62, nous avons isolé les enveloppes nucléaires (ENs) de lymphocytes T infectées pour une étude de spectrométrie de masse comparative. Celle-ci a démontré d'importantes variations dans la composition et la représentation des protéines de l'EN suite à l'infection, entre autres une diminution de l'abondance des Nucléoporines (Nups) qui ancrent et échafaudent le CPN ainsi que celles du noyau. Ensuite, l'analyse ultrastructurelle par microscopie immuno-électronique a révélé que la perte des Nups causée par l'infection n'est pas accompagnée par des changements au niveau de la structure générale des ENs ou CPNs. Nous avons également observé une dispersion des Nups dans et à travers le cytoplasme ainsi que leur présence dans les virus bourgeonnants. La purification de virus libres révèle que Nup62 est incorporée dans ceux-ci, suggérant qu'elle n'est pas simplement éjectée des ENs par l'infection mais plutôt qu'elle joue un rôle important pour la réplication du VIH-1.
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Farr, Julia Christina [Verfasser], and Berenike [Akademischer Betreuer] Maier. "Structural and functional analysis of the nuclear pore complex in Saccharomyces cerevisiae / Julia Christina Farr ; Betreuer: Berenike Maier." Münster : Universitäts- und Landesbibliothek Münster, 2009. http://d-nb.info/1140917676/34.
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Vollmer, Benjamin [Verfasser], and Wolfram [Akademischer Betreuer] Antonin. "The membrane interaction of Nup53 and its implication in nuclear pore complex assembly / Benjamin Vollmer ; Betreuer: Wolfram Antonin." Tübingen : Universitätsbibliothek Tübingen, 2015. http://d-nb.info/1163321087/34.
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Bangs, Peter Lawrence. "Cloning, Characterization and Functional Analysis of TPR, an Oncogene-Activating Protein of the Nuclear Pore Complex: A Dissertation." eScholarship@UMMS, 1998. http://escholarship.umassmed.edu/gsbs_diss/146.
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A monoclonal antibody, mAb 203.37, raised against purified nuclear matrix proteins identified a single ~270 kDa protein that localized to the nuclear envelope. Double-label immunofluorescent microscopy using differential permeabilization protocols showed that this protein was present exclusively on the nucleoplasmic side of the nuclear envelope and that it co-localized with components of the nuclear pore complex. The nucleotide sequence of clones isolated using mAb 203.37 identified this protein as Tpr, a protein previously shown to be involved in oncogenic fusions with a number of protein kinases. Sequence analysis showed Tpr to be a 2348 amino acid protein with a predicted molecular weight of 265 kDa protein and a bipartite structure consisting of an ~1600 amino acid N-terminal domain that is almost entirely an α-helical coiled-coil followed by a highly acidic non-coiled carboxy-terminus. Ectopic expression of epitope-tagged Tpr constructs revealed two functional domains for Tpr: a nuclear pore complex binding domain and a nuclear localization sequence. The amino-terminus of Tpr, the portion of the protein shown to activate protein kinase oncogenes, did not localize to the nuclear pore complex indicating that the transforming activity of Tpr-protein kinase chimeras did not involve interactions with the nuclear pore complex. Ectopic expression of Tpr and a number of Tpr constructs resulted in the accumulation of poly (A)+ RNA in the nuclear interior but did not effect the import of a reporter protein into the nucleus indicating a role for Tpr in the export of mRNA from the nucleus.
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Al-Haboubi, Teiba. "Characterisation of the molecular links between the nuclear pore complex and the nuclear lamins and reconstitution of the xenopus oocyte lamin assembly in vitro /." [S.l.] : [s.n.], 2009. http://edoc.unibas.ch/diss/DissB_8778.
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Apelt, Luise Karin [Verfasser]. "Investigation of the binary protein-protein interactions of the yeast and the human nuclear pore complex / Luise Karin Apelt." Berlin : Freie Universität Berlin, 2015. http://d-nb.info/1073868710/34.
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Sarma, Ashapurna. "A Single Molecule Study of Calcium Effect on Nuclear Transport." Bowling Green State University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1282326584.
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De, Magistris Paola [Verfasser], and Wolfram [Akademischer Betreuer] Antonin. "Functional characterization of the nucleoporins Nup50 and Nup155 in postmitotic nuclear pore complex assembly / Paola De Magistris ; Betreuer: Wolfram Antonin." Tübingen : Universitätsbibliothek Tübingen, 2019. http://d-nb.info/1198859067/34.
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Collins, Patrick. "The Characterisation of Putative Nuclear Pore-Anchoring Proteins in Arabidopsis thaliana." Thesis, University of Canterbury. Biological Sciences, 2013. http://hdl.handle.net/10092/8885.
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The nuclear pore complex (NPC) is perhaps the largest protein complex in the eukaryotic cell, and controls the movement of molecules across the nuclear envelope. The NPC is composed of up to 30 proteins termed nucleoporins (Nups), each grouped in different sub-complexes. The transmembrane ring sub-complex is composed of Nups responsible for anchoring the NPC to the nuclear envelope. Bioinformatic analysis has traced all major sub-complexes of the NPC back to the last eukaryotic common ancestor, meaning that the nuclear pore structure and function is conserved amongst all eukaryotes. In this study Arabidopsis T-DNA knockout lines for these genes were investigated to characterise gene function. Differences in plant growth and development were observed for the ndc1 knockout line compared to wild-type but gp210 plants showed no phenotypic differences. The double knockout line gp210 ndc1 was generated through crosses to observe plant response to the knockout of two anchoring-Nup genes. No synergistic affect from this double knockout was observed, suggesting that more, as yet unidentified Nups function the transmembrane ring in plants. The sensitivity to nuclear export inhibitor leptomycin B (LMB) was tested also for knockout lines, although growth sensitivity to the drug was not observed. Nucleocytoplasmic transport of knockout lines was measured in cells transformed by particle bombardment. To express fluorescent protein constructs actively transported through the NPC, localisation of protein determined the nucleocytoplasmic transport of the cell. The ndc1single knockout and the double knockout gp210 ndc1 exhibited decreased nuclear export. Further experiments in determining NDC1 localisation and identification of other Nups in the transmembrane ring sub-complex would bring a more comprehensive understanding to the plant NPC.
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Floch, Aurélie. "Mécanismes d'adressage de Pom33, protéine transmembranaire associée aux pores nucléaires chez la levure Saccharomyces cerevisiae levure Saccharomyces cerevisiae." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA112182.
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Chez les eucaryotes, les pores nucléaires (NPCs), ancrés dans l’enveloppe nucléaire (EN), régulent les échanges nucléocytoplasmiques. Ces complexes, très conservés, sont composés d’une trentaine de protéines appelées nucléoporines (Nups) présentes en multiples copies au sein de chaque NPC. Chez la levure S. cerevisiae, seules quatre Nups, dont la protéine Pom33, possèdent des domaines transmembranaires. Une étude réalisée en amont de ce projet a permis de caractériser Pom33 et de montrer que le mutant pom33∆ est viable et ne présente pas de défaut apparent de transport nucléocytoplasmique mais se caractérise par un défaut de distribution des NPCs. Pom33 joue également un rôle dans l’assemblage des pores nucléaires au sein de l’EN (biogenèse de novo des NPCs). POM33 appartient à une famille de gènes très conservés. Il possède un paralogue chez S. cerevisiae, PER33, qui code pour une protéine localisée majoritairement au réticulum endoplasmique et minoritairement aux NPCs et qui n’est pas impliquée dans la biogenèse des NPCs. Chez les mammifères, il n’existe qu’un homologue de Pom33/Per33, TMEM33. Dans le cadre de ce doctorat, nous nous sommes demandés quels étaient les déterminants contribuant à l’adressage spécifique de Pom33 au niveau des NPCs et à sa fonction dans la biogenèse de ces structures. La purification de Pom33-ProtA, suivie de spectrométrie de masse, nous a permis d’identifier un nouveau partenaire de Pom33, le facteur d’import Kap123. Des approches in vitro ont montré une interaction directe entre Kap123 et le domaine C-terminal (CTD) de Pom33, qui est perturbée en présence de RanGTP. Par ailleurs, des prédictions in silico ont révélé la présence dans ce domaine CTD de deux hélices amphipathiques, conservées chez l’humain. Des analyses par dichroïsme circulaire et flottaisons ont confirmé la capacité du CTD à s’organiser en hélice en présence de membranes lipidiques et à interagir préférentiellement avec les membranes très courbées. L’expression d’une version mutée de Pom33-CTD, incapable de se lier aux membranes et couplée à la GFP, a révélé la capacité de ce domaine à agir comme un NLS, importé spécifiquement dans le noyau par Kap123. Alors que la délétion du domaine CTD affecte l’adressage de Pom33 aux NPCs et provoque un défaut de distribution des NPCs, la mutation des résidus basiques impliqués dans l’interaction avec Kap123 ou des résidus permettant sa liaison aux membranes lipidiques ne récapitule pas ce phénotype. En revanche, la perte combinée de ces deux déterminants affecte l’adressage de Pom33 aux NPCs et provoque un défaut de distribution des NPCs ainsi qu'une interaction génétique avec le mutant nup133∆, impliqué dans la biogenèse de novo des NPCs. Les résultats obtenus lors de cette étude indiquent donc que l’adressage de Pom33 est un mécanisme actif et multifactoriel, qui met en jeu au moins deux déterminants dans son domaine CTD. Ces données indiquent également un rôle de ce domaine dans la biogenèse de novo des NPCs, qui pourrait néanmoins n’être qu’un effet indirect de son rôle dans l’adressage de Pom33 aux NPCs. Au cours de cette étude, nous avons également mis en évidence d’autres partenaires potentiels de Pom33, en particulier Myo2, une localisation de Pom33 au niveau du bourgeon lors de la division et une interaction génétique entre POM33 et KAP123. Ces observations préliminaires ouvrent de nouvelles pistes de réflexion quant au rôle de Pom33 lors de la division cellulaire<br>In eukaryotic cells, nucleocytoplasmic exchanges take place through the nuclear pores complexes (NPCs). These conserved macromolecular assemblies are embedded in the nuclear envelope (NE) and composed of ~30 distinct proteins called nucleoporins (Nups), each presents in multiple copies. In the budding yeast Sacharomyces cerevisiae, there are only four transmembrane Nups, including Pom33. A previous study leds to the characterization of Pom33 and revealed that pom33∆ mutant cells, although viable and without apparent alteration in nucleocytoplasmic transport, display NPCs distribution defect. Pom33 also contributes to the biogenesis of NPCs into the intact NE (de novo biogenesis). Pom33 is highly conserved among species and has a paralogue in S. cerevisiae, Per33, which can associate with NPCs but is mainly localized at the endoplasmic reticulum (ER) and NE. Unlike Pom33, Per33 is not involved in NPCs distribution and biogenesis. In mammalian cells, there is a unique homologue of Pom33/Per33, named TMEM33. In the context of this thesis, we aimed to identify the determinants involved in the specific targeting of Pom33 to NPCs and in its function in pore biogenesis. To characterize these determinants, we first performed affinity-purification experiments followed by mass spectrometry analyses. This identified a novel Pom33 partner, the nuclear import factor Kap123. In vitro experiments revealed a direct interaction between Pom33 C-terminal domain (CTD) and Kap123 that involves positively-charged residues within Pom33-CTD and is altered in the presence of Ran-GTP. Moreover, in silico analyses predicted the presence of two evolutionarily-conserved amphipathic ~-helices within Pom33-CTD. Circular dichroism studies and liposome co-floatation assays confirmed that this CTD domain is able to fold into ~-helices in the presence of liposomes and revealed its preferential binding to highly curved lipid membranes. When expressed in yeast, under conditions abolishing Pom33-CTD membrane association, Pom33-CTD behaves as a Kap123-dependent nuclear localization domain. While deletion of Pom33 C-terminal domain (Pom33-∆CTD-GFP) impairs Pom33 NPC targeting and stability and leads to a NPC distribution phenotype, mutants affecting either Kap123 binding or the amphipathic properties of the ~-helices do not display any detectable defect. However, combined impairment of lipid and Kap123 binding affects Pom33 targeting to NPCs and leads to an altered NPC distribution and a genetic interaction with the deletion of NUP133, a gene coding for a nucleoporin involved in NPCs biogenesis. Together, these results indicate that Pom33 targeting to NPCs is an active and multifactorial process that requires at least two determinants within its CTD. They also suggest a role of Pom33-CTD in the de novo NPCs biogenesis process, which could however only be an indirect consequence of its requirement for Pom33 targeting to NPCs. Our mass spectrometry analysis also identified other partners of Pom33, in particular Myo2, a molecular motor required for the cell cycle-regulated transport of various organelles and proteins and for correct alignment of the spindle during mitosis. Our studies also revealed a specific localization of Pom33 at the bud tip during mitosis and a genetic interaction between POM33 and KAP123. Taken together, these preliminary observations open new perspectives regarding additional functions of Pom33 during cell division
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Matreyek, Kenneth Anzai. "HIV-1 capsid engages nucleoporin NUP153 to promote viral nuclear entry." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11210.
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Lentiviruses can infect non-dividing cells, and various cellular nuclear transport proteins provide crucial functions for lentiviral nuclear entry and integration. Genome-wide small interfering RNA screens previously identified nuclear pore complex component nucleoporin 153 (NUP153) as being important for infection by human immunodeficiency virus type 1 (HIV-1). We found that HIV-1 infection of NUP153 depleted cells resulted in normal levels of reverse transcription, a moderate reduction of 2-long terminal repeat circles, and a relatively large reduction in integrated proviruses, consistent with a role for NUP153 during nuclear entry of the HIV-1 pre-integration complex. We ascertained the capsid (CA) to be the major viral determinant for NUP153 dependence during infection, and accordingly observed a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG)-repeat enriched NUP153 C-terminal domain (NUP153C). NUP153C fused to the effector domains of the rhesus Trim5alpha restriction factor (Trim-NUP153C) potently restricted HIV-1, providing an intracellular readout for the NUP153C-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV) bound NUP153C under these conditions, results that correlated with direct binding between purified recombinant proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 function during infection. Mutagenesis experiments identified NUP153C and CA residues important for binding, and different FG motifs within NUP153C mediated binding to HIV-1 versus EIAV CA proteins. HIV-1 CA binding mapped to residues that line a common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74) inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6) protein, with Asn57 (Asp58 in EIAV) playing a particularly important role. PF74 and CPSF6 each competed with NUP153C for binding to HIV-1 CA, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153C expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicated that the NUP153C-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. We propose that HIV-1 CA binds NUP153 FG motifs to affect viral nuclear import, serving as a novel example of viral hijacking of a fundamental cellular process.
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Jimenez, Sabinina Vilma [Verfasser], and Ed [Akademischer Betreuer] Hurt. "Analysis of the Architecture of the Nuclear Pore Complex by 3D super-resolution fluorescence microscopy / Vilma Jimenez Sabinina ; Betreuer: Ed Hurt." Heidelberg : Universitätsbibliothek Heidelberg, 2019. http://d-nb.info/1201346363/34.
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Eisenhardt, Nathalie [Verfasser], and Wolfram [Akademischer Betreuer] Antonin. "Functional characterisation of the interplay between Ndc1, Nup53 and Nup155 in nuclear pore complex biogenesis / Nathalie Eisenhardt ; Akademischer Betreuer: Wolfram Antonin." Tübingen : Universitätsbibliothek Tübingen, 2014. http://d-nb.info/1163239968/34.
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Böhmer, Thomas. "Functional and structural dissection of Nup107 and Nup133, two members of the Nup107-160 subcomplex, linchpin of the vertebrate nuclear pore complex." [S.l. : s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=975117866.
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Marelli, Marcello. "The yeast nucleoporin Nup53p provides a specific binding site for the karyopherin Kap121p and has a role in nuclear pore complex assembly." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0013/NQ59631.pdf.
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Mackmull, Marie-Therese [Verfasser], and Darren [Akademischer Betreuer] Gilmour. "The landscape of the nucleocytoplasmic transport system and cell-type specific variations of the nuclear pore complex / Marie-Therese Mackmull ; Betreuer: Darren Gilmour." Heidelberg : Universitätsbibliothek Heidelberg, 2017. http://d-nb.info/1180985680/34.
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