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1
Rout, M. P., and G. Blobel. "Isolation of the yeast nuclear pore complex." Journal of Cell Biology 123, no. 4 (1993): 771–83. http://dx.doi.org/10.1083/jcb.123.4.771.
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Nuclear pore complexes (NPCs) have been isolated from the yeast Saccharomyces. Negative stain electron microscopy of the isolated NPCs and subsequent image reconstruction revealed the octagonal symmetry and many of the ultrastructural features characteristic of vertebrate NPCs. The overall dimensions of the yeast NPC, both in its isolated form as well as in situ, are smaller than its vertebrate counterpart. However, the diameter of the central structures are similar. The isolated yeast NPC has a sedimentation coefficient of approximately 310 S and an M(r) of approximately 66 MD. It retains all but one of the eight known NPC proteins. In addition it contains as many as 80 uncharacterized proteins that are candidate NPC proteins.
2
Hampoelz, Bernhard, Amparo Andres-Pons, Panagiotis Kastritis, and Martin Beck. "Structure and Assembly of the Nuclear Pore Complex." Annual Review of Biophysics 48, no. 1 (2019): 515–36. http://dx.doi.org/10.1146/annurev-biophys-052118-115308.
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Nuclear pore complexes (NPCs) mediate nucleocytoplasmic exchange. They are exceptionally large protein complexes that fuse the inner and outer nuclear membranes to form channels across the nuclear envelope. About 30 different protein components, termed nucleoporins, assemble in multiple copies into an intricate cylindrical architecture. Here, we review our current knowledge of the structure of nucleoporins and how those come together in situ. We delineate architectural principles on several hierarchical organization levels, including isoforms, posttranslational modifications, nucleoporins, and higher-order oligomerization of nucleoporin subcomplexes. We discuss how cells exploit this modularity to faithfully assemble NPCs.
3
Reichelt, R., A. Holzenburg, E. L. Buhle, M. Jarnik, A. Engel, and U. Aebi. "Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components." Journal of Cell Biology 110, no. 4 (1990): 883–94. http://dx.doi.org/10.1083/jcb.110.4.883.
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Nuclear pore complexes (NPCs) prepared from Xenopus laevis oocyte nuclear envelopes were studied in "intact" form (i.e., unexposed to detergent) and after detergent treatment by a combination of conventional transmission electron microscopy (CTEM) and quantitative scanning transmission electron microscopy (STEM). In correlation-averaged CTEM pictures of negatively stained intact NPCs and of distinct NPC components (i.e., "rings," "spoke" complexes, and "plug-spoke" complexes), several fine structural features arranged with octagonal symmetry about a central axis could reproducibly be identified. STEM micrographs of unstained/freeze-dried intact NPCs as well as of their components yielded comparable but less distinct features. Mass determination by STEM revealed the following molecular masses: intact NPC with plug, 124 +/- 11 MD; intact NPC without plug, 112 +/- 11 MD; heavy ring, 32 +/- 5 MD; light ring, 21 +/- 4 MD; plug-spoke complex, 66 +/- 8 MD; and spoke complex, 52 +/- 3 MD. Based on these combined CTEM and STEM data, a three-dimensional model of the NPC exhibiting eightfold centrosymmetry about an axis perpendicular to the plane of the nuclear envelope but asymmetric along this axis is proposed. This structural polarity of the NPC across the nuclear envelope is in accord with its well-documented functional polarity facilitating mediated nucleocytoplasmic exchange of molecules and particles.
4
Lyman, Susan K., and Larry Gerace. "Nuclear pore complexes: dynamics in unexpected places." Journal of Cell Biology 154, no. 1 (2001): 17–20. http://dx.doi.org/10.1083/jcb.200106071.
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In vivo studies on the dynamics of the nuclear pore complex (NPC) in yeast suggested that NPCs are highly mobile in the nuclear envelope. However, new evidence indicates that in mammalian cells NPCs are stably attached to a flexible lamina framework, but a peripheral component can exchange rapidly with an intranuclear pool.
5
Allen, T. D., J. M. Cronshaw, S. Bagley, E. Kiseleva, and M. W. Goldberg. "The nuclear pore complex: mediator of translocation between nucleus and cytoplasm." Journal of Cell Science 113, no. 10 (2000): 1651–59. http://dx.doi.org/10.1242/jcs.113.10.1651.
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The enclosure of nuclear contents in eukaryotes means that cells require sites in the boundary that mediate exchange of material between nucleus and cytoplasm. These sites, termed nuclear pore complexes (NPCs), number 100–200 in yeast, a few thousand in mammalian cells and approximately 50 million in the giant nuclei of amphibian oocytes. NPCs are large (125 MDa) macromolecular complexes that comprise 50–100 different proteins in vertebrates. In spite of their size and complex structure, NPCs undergo complete breakdown and reformation at cell division. Transport through NPCs can be rapid (estimated at several hundred molecules/pore/second) and accommodates both passive diffusion of relatively small molecules, and active transport of complexes up to several megadaltons in molecular mass. Each pore can facilitate both import and export. The two processes apparently involve multiple pathways for different cargoes, and their transport signals, transport receptors and adapters, and the molecules (and their regulators) that underpin the transport mechanisms. Over the past few years there has been an increasing interest in the pore complex: structural studies have been followed by elucidation of the biochemical aspects of nuclear import, and subsequent investigations into nuclear export. The current challenge is to understand the interactions between the structural elements of the pore complex and the mechanisms that drive the physical processes of translocation through it.
6
Makio, Tadashi, Leslie H. Stanton, Cheng-Chao Lin, David S. Goldfarb, Karsten Weis, and Richard W. Wozniak. "The nucleoporins Nup170p and Nup157p are essential for nuclear pore complex assembly." Journal of Cell Biology 185, no. 3 (2009): 459–73. http://dx.doi.org/10.1083/jcb.200810029.
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We have established that two homologous nucleoporins, Nup170p and Nup157p, play an essential role in the formation of nuclear pore complexes (NPCs) in Saccharomyces cerevisiae. By regulating their synthesis, we showed that the loss of these nucleoporins triggers a decrease in NPCs caused by a halt in new NPC assembly. Preexisting NPCs are ultimately lost by dilution as cells grow, causing the inhibition of nuclear transport and the loss of viability. Significantly, the loss of Nup170p/Nup157p had distinct effects on the assembly of different architectural components of the NPC. Nucleoporins (nups) positioned on the cytoplasmic face of the NPC rapidly accumulated in cytoplasmic foci. These nup complexes could be recruited into new NPCs after reinitiation of Nup170p synthesis, and may represent a physiological intermediate. Loss of Nup170p/Nup157p also caused core and nucleoplasmically positioned nups to accumulate in NPC-like structures adjacent to the inner nuclear membrane, which suggests that these nucleoporins are required for formation of the pore membrane and the incorporation of cytoplasmic nups into forming NPCs.
7
Akey, C. W. "Interactions and structure of the nuclear pore complex revealed by cryo-electron microscopy." Journal of Cell Biology 109, no. 3 (1989): 955–70. http://dx.doi.org/10.1083/jcb.109.3.955.
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Nuclear pore complexes (NPCs) play a central role in mediating nucleocytoplasmic transport and exchange processes in eukaryotic cells. The arrangement and interactions of NPCs within amphibian nuclear envelopes have been studied using cryo-electron microscopy of unfixed and frozen hydrated specimens. The nuclear lamina in Necturus forms an orthogonal network with crossover distances which vary between 1,600 and 4,000 A and which may be related to the basic filament repeat of lamins. Furthermore, the NPCs are attached randomly within the confines of the lamin network, presumably by their nucleoplasmic rings. Image analysis of edge-on and en face projections of detergent-extracted NPCs has been combined with data on the coaxial thin rings to provide a quantitative evaluation of the triple ring model of NPC architecture proposed previously (Unwin, P. N. T., and R. Milligan. 1982. J. Cell Biol. 93:63-75). Additional details of the complex have been visualized including an intimate association of the inner spoke domains as an inner spoke ring, extensive domains within the spokes and coaxial thin rings, and interestingly, a central channel-like feature. Membrane-associated NPCs and detergent-extracted NPCs both possess peripherally located radial arms resulting in an effective diameter of approximately 1,450-1,500 A. In projection, the radial arms possess approximate mirror symmetry suggesting that they originate from both sides of the assembly. Moreover, membrane-associated NPCs are asymmetric at most radii and right-handed as viewed from the cytoplasm; detergent-extracted NPCs appear to be symmetric and have approximately 822 symmetry. Taken together, the data suggests that the framework of membrane-associated NPCs is perturbed from a symmetrical configuration, either during isolation of nuclei or by interactions with the lamina and the nuclear envelope in vivo. However, detergent extraction of nuclei appears to result in a more symmetrical alignment of components in apposing halves of the assembly.
8
Stavru, Fabrizia, Gitte Nautrup-Pedersen, Volker C. Cordes, and Dirk Görlich. "Nuclear pore complex assembly and maintenance in POM121- and gp210-deficient cells." Journal of Cell Biology 173, no. 4 (2006): 477–83. http://dx.doi.org/10.1083/jcb.200601002.
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So far, POM121 and gp210 are the only known anchoring sites of vertebrate nuclear pore complexes (NPCs) within the lipid bilayer of the nuclear envelope (NE) and, thus, are excellent candidates for initiating the NPC assembly process. Indeed, we demonstrate that POM121 can recruit several nucleoporins, such as Nup62 or Nup358, to ectopic assembly sites. It thus appears to act as a nucleation site for the assembly of NPC substructures. Nonetheless, we observed functional NPCs and intact NEs in severely POM121-depleted cells. Double knockdowns of gp210 and POM121 in HeLa cells, as well as depletion of POM121 from human fibroblasts, which do not express gp210, further suggest that NPCs can assemble or at least persist in a POM121- and gp210-free form. This points to extensive redundancies in protein–protein interactions within NPCs and suggests that vertebrate NPCs contain additional membrane-integral nucleoporins for anchorage within the lipid bilayer of the NE. In Stavru et al. (on p. 509 of this issue), we describe such an additional transmembrane nucleoporin as the metazoan orthologue of yeast Ndc1p.
9
Dultz, Elisa, Matthias Wojtynek, Ohad Medalia, and Evgeny Onischenko. "The Nuclear Pore Complex: Birth, Life, and Death of a Cellular Behemoth." Cells 11, no. 9 (2022): 1456. http://dx.doi.org/10.3390/cells11091456.
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Nuclear pore complexes (NPCs) are the only transport channels that cross the nuclear envelope. Constructed from ~500–1000 nucleoporin proteins each, they are among the largest macromolecular assemblies in eukaryotic cells. Thanks to advances in structural analysis approaches, the construction principles and architecture of the NPC have recently been revealed at submolecular resolution. Although the overall structure and inventory of nucleoporins are conserved, NPCs exhibit significant compositional and functional plasticity even within single cells and surprising variability in their assembly pathways. Once assembled, NPCs remain seemingly unexchangeable in post-mitotic cells. There are a number of as yet unresolved questions about how the versatility of NPC assembly and composition is established, how cells monitor the functional state of NPCs or how they could be renewed. Here, we review current progress in our understanding of the key aspects of NPC architecture and lifecycle.
10
Dultz, Elisa, Matthias Wojtynek, Ohad Medalia, and Evgeny Onischenko. "The Nuclear Pore Complex: Birth, Life, and Death of a Cellular Behemoth." Cells 11, no. 9 (2022): 1456. http://dx.doi.org/10.3390/cells11091456.
Pełny tekst źródłaStreszczenie:
Nuclear pore complexes (NPCs) are the only transport channels that cross the nuclear envelope. Constructed from ~500–1000 nucleoporin proteins each, they are among the largest macromolecular assemblies in eukaryotic cells. Thanks to advances in structural analysis approaches, the construction principles and architecture of the NPC have recently been revealed at submolecular resolution. Although the overall structure and inventory of nucleoporins are conserved, NPCs exhibit significant compositional and functional plasticity even within single cells and surprising variability in their assembly pathways. Once assembled, NPCs remain seemingly unexchangeable in post-mitotic cells. There are a number of as yet unresolved questions about how the versatility of NPC assembly and composition is established, how cells monitor the functional state of NPCs or how they could be renewed. Here, we review current progress in our understanding of the key aspects of NPC architecture and lifecycle.
11
Dultz, Elisa, Matthias Wojtynek, Ohad Medalia, and Evgeny Onischenko. "The Nuclear Pore Complex: Birth, Life, and Death of a Cellular Behemoth." Cells 11, no. 9 (2022): 1456. http://dx.doi.org/10.3390/cells11091456.
Pełny tekst źródłaStreszczenie:
Nuclear pore complexes (NPCs) are the only transport channels that cross the nuclear envelope. Constructed from ~500–1000 nucleoporin proteins each, they are among the largest macromolecular assemblies in eukaryotic cells. Thanks to advances in structural analysis approaches, the construction principles and architecture of the NPC have recently been revealed at submolecular resolution. Although the overall structure and inventory of nucleoporins are conserved, NPCs exhibit significant compositional and functional plasticity even within single cells and surprising variability in their assembly pathways. Once assembled, NPCs remain seemingly unexchangeable in post-mitotic cells. There are a number of as yet unresolved questions about how the versatility of NPC assembly and composition is established, how cells monitor the functional state of NPCs or how they could be renewed. Here, we review current progress in our understanding of the key aspects of NPC architecture and lifecycle.
12
Suresh, Subbulakshmi, Sarine Markossian, Aysha H. Osmani, and Stephen A. Osmani. "Mitotic nuclear pore complex segregation involves Nup2 in Aspergillus nidulans." Journal of Cell Biology 216, no. 9 (2017): 2813–26. http://dx.doi.org/10.1083/jcb.201610019.
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Transport through nuclear pore complexes (NPCs) during interphase is facilitated by the nucleoporin Nup2 via its importin α– and Ran-binding domains. However, Aspergillus nidulans and vertebrate Nup2 also locate to chromatin during mitosis, suggestive of mitotic functions. In this study, we report that Nup2 is required for mitotic NPC inheritance in A. nidulans. Interestingly, the role of Nup2 during mitotic NPC segregation is independent of its importin α– and Ran-binding domains but relies on a central targeting domain that is necessary for localization and viability. To test whether mitotic chromatin-associated Nup2 might function to bridge NPCs with chromatin during segregation, we provided an artificial link between NPCs and chromatin via Nup133 and histone H1. Using this approach, we bypassed the requirement of Nup2 for NPC segregation. This indicates that A. nidulans cells ensure accurate mitotic NPC segregation to daughter nuclei by linking mitotic DNA and NPC segregation via the mitotic specific chromatin association of Nup2.
13
Winey, Mark, Defne Yarar, Thomas H. Giddings, and David N. Mastronarde. "Nuclear Pore Complex Number and Distribution throughout theSaccharomyces cerevisiaeCell Cycle by Three-Dimensional Reconstruction from Electron Micrographs of Nuclear Envelopes." Molecular Biology of the Cell 8, no. 11 (1997): 2119–32. http://dx.doi.org/10.1091/mbc.8.11.2119.
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The number of nuclear pore complexes (NPCs) in individual nuclei of the yeast Saccharomyces cerevisiae was determined by computer-aided reconstruction of entire nuclei from electron micrographs of serially sectioned cells. Nuclei of 32 haploid cells at various points in the cell cycle were modeled and found to contain between 65 and 182 NPCs. Morphological markers, such as cell shape and nuclear shape, were used to determine the cell cycle stage of the cell being examined. NPC number was correlated with cell cycle stage to reveal that the number of NPCs increases steadily, beginning in G1-phase, suggesting that NPC assembly occurs continuously throughout the cell cycle. However, the accumulation of nuclear envelope observed during the cell cycle, indicated by nuclear surface area, is not continuous at the same rate, such that the density of NPCs per unit area of nuclear envelope peaks in apparent S-phase cells. Analysis of the nuclear envelope reconstructions also revealed no preferred NPC-to-NPC distance. However, NPCs were found in large clusters over regions of the nuclear envelope. Interestingly, clusters of NPCs were most pronounced in early mitotic nuclei and were found to be associated with the spindle pole bodies, but the functional significance of this association is unknown.
14
Lusk, C. Patrick, Taras Makhnevych, Marcello Marelli, John D. Aitchison, and Richard W. Wozniak. "Karyopherins in nuclear pore biogenesis." Journal of Cell Biology 159, no. 2 (2002): 267–78. http://dx.doi.org/10.1083/jcb.200203079.
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The mechanisms that govern the assembly of nuclear pore complexes (NPCs) remain largely unknown. Here, we have established a role for karyopherins in this process. We show that the yeast karyopherin Kap121p functions in the targeting and assembly of the nucleoporin Nup53p into NPCs by recognizing a nuclear localization signal (NLS) in Nup53p. This karyopherin-mediated function can also be performed by the Kap95p–Kap60p complex if the Kap121p-binding domain of Nup53p is replaced by a classical NLS, suggesting a more general role for karyopherins in NPC assembly. At the NPC, neighboring nucleoporins bind to two regions in Nup53p. One nucleoporin, Nup170p, associates with a region of Nup53p that overlaps with the Kap121p binding site and we show that they compete for binding to Nup53p. We propose that once targeted to the NPC, dissociation of the Kap121p–Nup53p complex is driven by the interaction of Nup53p with Nup170p. At the NPC, Nup53p exists in two separate complexes, one of which is capable of interacting with Kap121p and another that is bound to Nup170p. We propose that fluctuations between these two states drive the binding and release of Kap121p from Nup53p, thus facilitating Kap121p's movement through the NPC.
15
Walther, Tobias C., Helen S. Pickersgill, Volker C. Cordes, et al. "The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import." Journal of Cell Biology 158, no. 1 (2002): 63–77. http://dx.doi.org/10.1083/jcb.200202088.
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The nuclear pore complex (NPC) mediates bidirectional macromolecular traffic between the nucleus and cytoplasm in eukaryotic cells. Eight filaments project from the NPC into the cytoplasm and are proposed to function in nuclear import. We investigated the localization and function of two nucleoporins on the cytoplasmic face of the NPC, CAN/Nup214 and RanBP2/Nup358. Consistent with previous data, RanBP2 was localized at the cytoplasmic filaments. In contrast, CAN was localized near the cytoplasmic coaxial ring. Unexpectedly, extensive blocking of RanBP2 with gold-conjugated antibodies failed to inhibit nuclear import. Therefore, RanBP2-deficient NPCs were generated by in vitro nuclear assembly in RanBP2-depleted Xenopus egg extracts. NPCs were formed that lacked cytoplasmic filaments, but that retained CAN. These nuclei efficiently imported nuclear localization sequence (NLS) or M9 substrates. NPCs lacking CAN retained RanBP2 and cytoplasmic filaments, and showed a minor NLS import defect. NPCs deficient in both CAN and RanBP2 displayed no cytoplasmic filaments and had a strikingly immature cytoplasmic appearance. However, they showed only a slight reduction in NLS-mediated import, no change in M9-mediated import, and were normal in growth and DNA replication. We conclude that RanBP2 is the major nucleoporin component of the cytoplasmic filaments of the NPC, and that these filaments do not have an essential role in importin α/β– or transportin-dependent import.
16
Chugani, D. C., L. H. Rome, and N. L. Kedersha. "Evidence that vault ribonucleoprotein particles localize to the nuclear pore complex." Journal of Cell Science 106, no. 1 (1993): 23–29. http://dx.doi.org/10.1242/jcs.106.1.23.
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Vaults are cytoplasmic ribonucleoprotein organelles that are highly conserved among diverse eukaryotic species. Their mass (12.9 MDa), diameter (26-35 nm) and shape (two halves, each with eightfold radial symmetry) have recently been determined and are similar to those ascribed to the central plug (or transporter) of the nuclear pore complex (NPC). The size and eightfold symmetry of the vault particle make it conducive to interacting physically in a complementary manner with NPCs. The present study demonstrates that vaults specifically associate with nuclei by both immunoblotting and immunofluorescence. Immunogold EM confirmed that vaults associate with the nuclear envelope in tissue sections and with NPCs of isolated nuclei.
17
Fahrenkrog, Birthe, Eduard C. Hurt, Ueli Aebi, and Nelly Panté. "Molecular Architecture of the Yeast Nuclear Pore Complex: Localization of Nsp1p Subcomplexes." Journal of Cell Biology 143, no. 3 (1998): 577–88. http://dx.doi.org/10.1083/jcb.143.3.577.
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The nuclear pore complex (NPC), a supramolecular assembly of ∼100 different proteins (nucleoporins), mediates bidirectional transport of molecules between the cytoplasm and the cell nucleus. Extensive structural studies have revealed the three- dimensional (3D) architecture of Xenopus NPCs, and eight of the ∼12 cloned and characterized vertebrate nucleoporins have been localized within the NPC. Thanks to the power of yeast genetics, 30 yeast nucleoporins have recently been cloned and characterized at the molecular level. However, the localization of these nucleoporins within the 3D structure of the NPC has remain elusive, mainly due to limitations of preparing yeast cells for electron microscopy (EM). We have developed a new protocol for preparing yeast cells for EM that yielded structurally well-preserved yeast NPCs. A direct comparison of yeast and Xenopus NPCs revealed that the NPC structure is evolutionarily conserved, although yeast NPCs are 15% smaller in their linear dimensions. With this preparation protocol and yeast strains expressing nucleoporins tagged with protein A, we have localized Nsp1p and its interacting partners Nup49p, Nup57p, Nup82p, and Nic96p by immuno-EM. Accordingly, Nsp1p resides in three distinct subcomplexes which are located at the entry and exit of the central gated channel and at the terminal ring of the nuclear basket.
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Quimby, B. Booth, Alexei Arnaoutov, and Mary Dasso. "Ran GTPase Regulates Mad2 Localization to the Nuclear Pore Complex." Eukaryotic Cell 4, no. 2 (2005): 274–80. http://dx.doi.org/10.1128/ec.4.2.274-280.2005.
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ABSTRACT In yeast and mammalian cells, the spindle assembly checkpoint proteins Mad1p and Mad2p localize to the nuclear pore complex (NPC) during interphase. Deletion of MAD1 or MAD2 did not affect steady-state nucleocytoplasmic distribution of a classical nuclear localization signal-containing reporter, a nuclear export signal-containing reporter, or Ran localization. We utilized cells with conditional mutations in the yeast Ran GTPase pathway to examine the relationship between Ran and targeting of checkpoint regulators to the NPC. Mutations that disrupt the concentration of Ran in the nucleus displaced Mad2p but not Mad1p from the NPC. The displacement of Mad2p in M-phase cells was correlated with activation of the spindle checkpoint. Our observations demonstrate that Mad2p localization at NPCs is sensitive to nuclear levels of Ran and suggest that release of Mad2p from NPCs is closely linked with spindle assembly checkpoint activation in yeast. This is the first evidence indicating that Ran affects the localization of Mad2p to the NPC.
19
Yang, Q., J.-F. Ménétret, I. V. Akey, K. Plath, T. A. Rapoport, and C. W. Akey. "Structural Studies of Translocation Channels: The Nuclear Pore Complex and the Translocon." Microscopy and Microanalysis 4, S2 (1998): 960–61. http://dx.doi.org/10.1017/s1431927600024922.
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Protein translocation plays a critical role in the targeting of both soluble and membrane proteins to their correct intra- and inter-cellular compartments. We are studying the 3D architecture of two rather different translocation machines, the Nuclear Pore Complex (NPC) and the ribosome-Sec61p complex (translocon), with the aim of understanding their physical mechanisms of gating and transport. Towards this end, we are using single particle electron cryomicroscopy and 3D reconstruction of frozen hydrated specimens to obtain interpretable maps that are biologically relevant.Previous work suggested that a central channel complex (termed the transporter) is present in vertebrate NPCs. Based on classification studies of the transporter and STEM images of Chironomus NPCs caught translocating large mRNPs, we have hypothesized that the transport mechanism utilizes a double iris-like gating mechanism, in which oppositely facing gates located at either end of the transporter open asynchronously. Recently, we have extended our studies to yeast NPCs and shown that this organelle is markedly smaller than its vertebrate cousin.
20
Hamed, Mohamed, and Wolfram Antonin. "Dunking into the Lipid Bilayer: How Direct Membrane Binding of Nucleoporins Can Contribute to Nuclear Pore Complex Structure and Assembly." Cells 10, no. 12 (2021): 3601. http://dx.doi.org/10.3390/cells10123601.
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Nuclear pore complexes (NPCs) mediate the selective and highly efficient transport between the cytoplasm and the nucleus. They are embedded in the two membrane structure of the nuclear envelope at sites where these two membranes are fused to pores. A few transmembrane proteins are an integral part of NPCs and thought to anchor these complexes in the nuclear envelope. In addition, a number of nucleoporins without membrane spanning domains interact with the pore membrane. Here we review our current knowledge of how these proteins interact with the membrane and how this interaction can contribute to NPC assembly, stability and function as well as shaping of the pore membrane.
21
Cohen, Merav, Naomi Feinstein, Katherine L. Wilson, and Yosef Gruenbaum. "Nuclear Pore Protein gp210 Is Essential for Viability in HeLa Cells and Caenorhabditis elegans." Molecular Biology of the Cell 14, no. 10 (2003): 4230–37. http://dx.doi.org/10.1091/mbc.e03-04-0260.
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Gp210 is an evolutionarily conserved membrane protein of the nuclear pore complex (NPC). We studied the phenotypes produced by RNAi-induced downregulation of gp210 in both human (HeLa) cells and Caenorhabditis elegans embryos. HeLa cell viability requires Gp210 activity. The dying cells accumulated clustered NPCs and aberrant membrane structures at the nuclear envelope, suggesting that gp210 is required directly or indirectly for nuclear pore formation and dilation as well as the anchoring or structural integrity of mature NPCs. Essential roles for gp210 were confirmed in C. elegans, where RNAi-induced reduction of gp210 caused embryonic lethality. The nuclear envelopes of embryos with reduced gp210 also had aberrant nuclear membrane structures and clustered NPCs, confirming that gp210 plays critical roles at the nuclear membrane through mechanisms that are conserved from nematodes to humans.
22
Varberg, Joseph M., Jay R. Unruh, Andrew J. Bestul, Azqa A. Khan, and Sue L. Jaspersen. "Quantitative analysis of nuclear pore complex organization in Schizosaccharomyces pombe." Life Science Alliance 5, no. 7 (2022): e202201423. http://dx.doi.org/10.26508/lsa.202201423.
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The number, distribution, and composition of nuclear pore complexes (NPCs) in the nuclear envelope varies between cell types and changes during cellular differentiation and in disease. To understand how NPC density and organization are controlled, we analyzed the NPC number and distribution in the fission yeast Schizosaccharomyces pombe using structured illumination microscopy. The small size of yeast nuclei, genetic features of fungi, and our robust image analysis pipeline allowed us to study NPCs in intact nuclei under multiple conditions. Our data revealed that NPC density is maintained across a wide range of nuclear sizes. Regions of reduced NPC density are observed over the nucleolus and surrounding the spindle pole body (SPB). Lem2-mediated tethering of the centromeres to the SPB is required to maintain NPC exclusion near SPBs. These findings provide a quantitative understanding of NPC number and distribution in S. pombe and show that interactions between the centromere and the nuclear envelope influences local NPC distribution.
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Popken, Petra, Ali Ghavami, Patrick R. Onck, Bert Poolman, and Liesbeth M. Veenhoff. "Size-dependent leak of soluble and membrane proteins through the yeast nuclear pore complex." Molecular Biology of the Cell 26, no. 7 (2015): 1386–94. http://dx.doi.org/10.1091/mbc.e14-07-1175.
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Nuclear pore complexes (NPCs) allow selective import and export while forming a barrier for untargeted proteins. Using fluorescence microscopy, we measured in vivo the permeability of the Saccharomyces cerevisiae NPC for multidomain proteins of different sizes and found that soluble proteins of 150 kDa and membrane proteins with an extralumenal domain of 90 kDa were still partly localized in the nucleus on a time scale of hours. The NPCs thus form only a weak barrier for the majority of yeast proteins, given their monomeric size. Using FGΔ-mutant strains, we showed that specific combinations of Nups, especially with Nup100, but not the total mass of FG-nups per pore, were important for forming the barrier. Models of the disordered phase of wild-type and mutant NPCs were generated using a one bead per amino acid molecular dynamics model. The permeability measurements correlated with the density predictions from coarse-grained molecular dynamics simulations in the center of the NPC. The combined in vivo and computational approach provides a framework for elucidating the structural and functional properties of the permeability barrier of nuclear pore complexes.
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Terry, Laura J., and Susan R. Wente. "Nuclear mRNA export requires specific FG nucleoporins for translocation through the nuclear pore complex." Journal of Cell Biology 178, no. 7 (2007): 1121–32. http://dx.doi.org/10.1083/jcb.200704174.
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Trafficking of nucleic acids and large proteins through nuclear pore complexes (NPCs) requires interactions with NPC proteins that harbor FG (phenylalanine-glycine) repeat domains. Specialized transport receptors that recognize cargo and bind FG domains facilitate these interactions. Whether different transport receptors utilize preferential FG domains in intact NPCs is not fully resolved. In this study, we use a large-scale deletion strategy in Saccharomyces cerevisiae to generate a new set of more minimal pore (mmp) mutants that lack specific FG domains. A comparison of messenger RNA (mRNA) export versus protein import reveals unique subsets of mmp mutants with functional defects in specific transport receptors. Thus, multiple functionally independent NPC translocation routes exist for different transport receptors. Our global analysis of the FG domain requirements in mRNA export also finds a requirement for two NPC substructures—one on the nuclear NPC face and one in the NPC central core. These results pinpoint distinct steps in the mRNA export mechanism that regulate NPC translocation efficiency.
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Wente, S. R., and G. Blobel. "A temperature-sensitive NUP116 null mutant forms a nuclear envelope seal over the yeast nuclear pore complex thereby blocking nucleocytoplasmic traffic." Journal of Cell Biology 123, no. 2 (1993): 275–84. http://dx.doi.org/10.1083/jcb.123.2.275.
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NUP116 encodes a 116-kD yeast nuclear pore complex (NPC) protein that is not essential but its deletion (nup116 delta) slows cell growth at 23 degrees C and is lethal at 37 degrees C (Wente, S. R., M. P. Rout, and G. Blobel. 1992. J. Cell Biol. 119:705-723). Electron microscopic analysis of nup116 delta cells shifted to growth at 37 degrees C revealed striking perturbations of the nuclear envelope: a double membrane seal that was continuous with the inner and outer nuclear membranes had formed over the cytoplasmic face of the NPCs. Electron-dense material was observed accumulating between the cytoplasmic face of these NPCs and the membrane seal, resulting in "herniations" of the nuclear envelope around individual NPCs. In situ hybridization with poly(dT) probes showed the accumulation of polyadenylated RNA in the nuclei of arrested nup116 delta cells, sometimes in the form of punctate patches at the nuclear periphery. This is consistent with the electron microscopically observed accumulation of electron-dense material within the nuclear envelope herniations. We propose that nup116 delta NPCs remain competent for export, but that the formation of the membrane seals over the NPCs blocks nucleocytoplasmic traffic.
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Chadrin, Anne, Barbara Hess, Mabel San Roman, et al. "Pom33, a novel transmembrane nucleoporin required for proper nuclear pore complex distribution." Journal of Cell Biology 189, no. 5 (2010): 795–811. http://dx.doi.org/10.1083/jcb.200910043.
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The biogenesis of nuclear pore complexes (NPCs) represents a paradigm for the assembly of high-complexity macromolecular structures. So far, only three integral pore membrane proteins are known to function redundantly in NPC anchoring within the nuclear envelope. Here, we describe the identification and functional characterization of Pom33, a novel transmembrane protein dynamically associated with budding yeast NPCs. Pom33 becomes critical for yeast viability in the absence of a functional Nup84 complex or Ndc1 interaction network, which are two core NPC subcomplexes, and associates with the reticulon Rtn1. Moreover, POM33 loss of function impairs NPC distribution, a readout for a subset of genes required for pore biogenesis, including members of the Nup84 complex and RTN1. Consistently, we show that Pom33 is required for normal NPC density in the daughter nucleus and for proper NPC biogenesis and/or stability in the absence of Nup170. We hypothesize that, by modifying or stabilizing the nuclear envelope–NPC interface, Pom33 may contribute to proper distribution and/or efficient assembly of nuclear pores.
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Mitic, Kristina, Marianne Grafe, Petros Batsios, and Irene Meyer. "Partial Disassembly of the Nuclear Pore Complex Proteins during Semi-Closed Mitosis in Dictyostelium discoideum." Cells 11, no. 3 (2022): 407. http://dx.doi.org/10.3390/cells11030407.
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Dictyostelium cells undergo a semi-closed mitosis, during which the nuclear envelope (NE) persists; however, free diffusion between the cytoplasm and the nucleus takes place. To permit the formation of the mitotic spindle, the nuclear envelope must be permeabilized in order to allow diffusion of tubulin dimers and spindle assembly factors into the nucleus. In Aspergillus, free diffusion of proteins between the cytoplasm and the nucleus is achieved by a partial disassembly of the nuclear pore complexes (NPCs) prior to spindle assembly. In order to determine whether this is also the case in Dictyostelium, we analysed components of the NPC by immunofluorescence microscopy and live cell imaging and studied their behaviour during interphase and mitosis. We observed that the NPCs are absent from the contact area of the nucleoli and that some nucleoporins also localize to the centrosome and the spindle poles. In addition, we could show that, during mitosis, the central FG protein NUP62, two inner ring components and Gle1 depart from the NPCs, while all other tested NUPs remained at the NE. This leads to the conclusion that indeed a partial disassembly of the NPCs takes place, which contributes to permeabilisation of the NE during semi-closed mitosis.
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Lau, Corine K., Thomas H. Giddings, and Mark Winey. "A Novel Allele of Saccharomyces cerevisiae NDC1 Reveals a Potential Role for the Spindle Pole Body Component Ndc1p in Nuclear Pore Assembly." Eukaryotic Cell 3, no. 2 (2004): 447–58. http://dx.doi.org/10.1128/ec.3.2.447-458.2004.
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ABSTRACT Both the spindle pole body (SPB) and the nuclear pore complex (NPC) are essential organelles embedded in the nuclear envelope throughout the life cycle of the budding yeast Saccharomyces cerevisiae. However, the mechanism by which these two multisubunit structures are inserted into the nuclear envelope during their biogenesis is not well understood. We have previously shown that Ndc1p is the only known integral membrane protein that localizes to both the SPBs and the NPCs and is required for SPB duplication. For this study, we generated a novel temperature-sensitive (ts) allele of NDC1 to investigate the role of Ndc1p at the NPCs. Yeast cells carrying this allele (ndc1-39) failed to insert the SPB into the nuclear envelope at the restrictive temperature. Importantly, the double mutation of ndc1-39 and NPC assembly mutant nic96-1 resulted in cells with enhanced growth defects. While nuclear protein import and NPC distribution in the nuclear envelope were unaffected, ndc1-39 mutants failed to properly incorporate the nucleoporin Nup49p into NPCs. These results provide evidence that Ndc1p is required for NPC assembly in addition to its role in SPB duplication. We postulate that Ndc1p is crucial for the biogenesis of both the SPBs and the NPCs at the step of insertion into the nuclear envelope.
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Orlova, А. V., S. G. Georgieva, and D. V. Kopytova. "Assembly and Disassembly of Nuclear Pore Complex: a View from Structural Side." Молекулярная биология 57, no. 4 (2023): 573–86. http://dx.doi.org/10.31857/s0026898423040171.
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Nucleocytoplasmic exchange in the cell occurs through the nuclear pore complexes (NPCs). NPCs are large multiprotein complexes with octagonal symmetry about their axis and imperfect mirror symmetry about a plane parallel with the nuclear envelop (NE). NPC fuses the inner and outer nuclear membranes and opens up а channel between nucleus and cytoplasm. NPC is built of nucleoporins. Each nucleoporin occurs in at least eight copies per NPC. Inside the NPC forms a permeability barrier by which NPC can ensure fast and selectable transport of molecules from one side of nuclear membrane to another. NPC architecture is based on hierarchical principle of organization. Nucleoporins are integrated into complexes that oligomerizes into bigger octomeric high-order structures. These structures are the main components of NPC. In the first part of this work the main attention is paid to NPC structure and nucleoporins’ properties. The second part is dedicated to mechanisms of NPC assembly and disassembly at different stages of cell cycle.
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Marelli, Marcello, David J. Dilworth, Richard W. Wozniak, and John D. Aitchison. "The dynamics of karyopherin-mediated nuclear transport." Biochemistry and Cell Biology 79, no. 5 (2001): 603–12. http://dx.doi.org/10.1139/o01-149.
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The regulated exchange of proteins and nucleic acids between the nucleus and cytoplasm demands a complex interplay between nuclear pore complexes (NPCs), which provide conduits in the nuclear envelope, and mobile transport receptors (or karyopherins, also known as importins/exportins) that bind and mediate the translocation of cargoes through the NPCs. Biochemical characterization of individual karyopherins has led to the identification of many of their cargoes and to the elucidation of the mechanisms by which they mediate transport. Likewise, the characterization of numerous NPC-associated components, in combination with structural studies of NPCs, have begun to address the possible mechanisms that drive nucleocytoplasmic transport, and the role that different nucleoporins play in the transport process. Some recent studies indicate that several NPC-associated factors, previously thought to be stable components of the NPC, dynamically interact with both nuclear and cytoplasmic aspects of the NPC. The mobility of these components challenges our conventional view of the NPC as the stationary phase of transport. These components and their potiential roles in nucleo-cytoplasmic transport are discussed.Key words: Nucleocytoplasmic transport, nuclear pore complex, nucleoporin, karyopherin, Nup2p.
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Rexach, Michael. "Piecing together nuclear pore complex assembly during interphase." Journal of Cell Biology 185, no. 3 (2009): 377–79. http://dx.doi.org/10.1083/jcb.200904022.
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All nucleocytoplasmic traffic of macromolecules occurs through nuclear pore complexes (NPCs), which function as stents in the nuclear envelope to keep nuclear pores open but gated. Three studies in this issue (Flemming, D., P. Sarges, P. Stelter, A. Hellwig, B. Böttcher, and E. Hurt. 2009. J. Cell Biol. 185:387–395; Makio, T., L.H. Stanton, C.-C. Lin, D.S. Goldfarb, K. Weis, and R.W. Wozniak. 2009. J. Cell Biol. 185:459–491; Onishchenko, E., L.H. Stanton, A.S. Madrid, T. Kieselbach, and K. Weis. 2009. J. Cell Biol. 185:475–491) further our understanding of the NPC assembly process by reporting what happens when the supply lines of key proteins that provide a foundation for building these marvelous supramolecular structures are disrupted.
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Theerthagiri, Gandhi, Nathalie Eisenhardt, Heinz Schwarz, and Wolfram Antonin. "The nucleoporin Nup188 controls passage of membrane proteins across the nuclear pore complex." Journal of Cell Biology 189, no. 7 (2010): 1129–42. http://dx.doi.org/10.1083/jcb.200912045.
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All transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). Despite their enormous size, ∼60 MD in vertebrates, they are comprised of only ∼30 distinct proteins (nucleoporins or Nups), many of which form subcomplexes that act as building blocks for NPC assembly. One of these evolutionarily conserved subcomplexes, the Nup93 complex, is a major structural component linking the NPC to the membranes of the NE. Using in vitro nuclear assembly assays, we show that two components of the Nup93 complex, Nup188 and Nup205, are dispensable for NPC formation. However, nuclei lacking Nup188 increase in size by several fold compared with wild type. We demonstrate that this phenotype is caused by an accelerated translocation of integral membrane proteins through NPCs, suggesting that Nup188 confines the passage of membrane proteins and is thus crucial for the homeostasis of the different nuclear membranes.
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Dange, Thomas, David Grünwald, Antje Grünwald, Reiner Peters, and Ulrich Kubitscheck. "Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study." Journal of Cell Biology 183, no. 1 (2008): 77–86. http://dx.doi.org/10.1083/jcb.200806173.
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All molecular traffic between nucleus and cytoplasm occurs via the nuclear pore complex (NPC) within the nuclear envelope. In this study we analyzed the interactions of the nuclear transport receptors kapα2, kapβ1, kapβ1ΔN44, and kapβ2, and the model transport substrate, BSA-NLS, with NPCs to determine binding sites and kinetics using single-molecule microscopy in living cells. Recombinant transport receptors and BSA-NLS were fluorescently labeled by AlexaFluor 488, and microinjected into the cytoplasm of living HeLa cells expressing POM121-GFP as a nuclear pore marker. After bleaching the dominant GFP fluorescence the interactions of the microinjected molecules could be studied using video microscopy with a time resolution of 5 ms, achieving a colocalization precision of 30 nm. These measurements allowed defining the interaction sites with the NPCs with an unprecedented precision, and the comparison of the interaction kinetics with previous in vitro measurements revealed new insights into the translocation mechanism.
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Kiseleva, Elena, Sandra Rutherford, Laura M. Cotter, Terence D. Allen, and Martin W. Goldberg. "Steps of nuclear pore complex disassembly and reassembly during mitosis in earlyDrosophilaembryos." Journal of Cell Science 114, no. 20 (2001): 3607–18. http://dx.doi.org/10.1242/jcs.114.20.3607.
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The mechanisms of nuclear pore complex (NPC) assembly and disassembly during mitosis in vivo are not well defined. To address this and to identify the steps of the NPC disassembly and assembly, we investigated Drosophila embryo nuclear structure at the syncytial stage of early development using field emission scanning electron microscopy (FESEM), a high resolution surface imaging technique, and transmission electron microscopy. Nuclear division in syncytial embryos is characterized by semi-closed mitosis, during which the nuclear membranes are ruptured only at the polar regions and are arranged into an inner double membrane surrounded by an additional ‘spindle envelope’. FESEM analysis of the steps of this process as viewed on the surface of the dividing nucleus confirm our previous in vitro model for the assembly of the NPCs via a series of structural intermediates, showing for the first time a temporal progression from one intermediate to the next. Nascent NPCs initially appear to form at the site of fusion between the mitotic nuclear envelope and the overlying spindle membrane. A model for NPC disassembly is offered that starts with the release of the central transporter and the removal of the cytoplasmic ring subunits before the star ring.
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Kann, Michael, Beate Sodeik, Angelika Vlachou, Wolfram H. Gerlich, and Ari Helenius. "Phosphorylation-dependent Binding of Hepatitis B Virus Core Particles to the Nuclear Pore Complex." Journal of Cell Biology 145, no. 1 (1999): 45–55. http://dx.doi.org/10.1083/jcb.145.1.45.
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Although many viruses replicate in the nucleus, little is known about the processes involved in the nuclear import of viral genomes. We show here that in vitro generated core particles of human hepatitis B virus bind to nuclear pore complexes (NPCs) in digitonin-permeabilized mammalian cells. This only occurred if the cores contained phosphorylated core proteins. Binding was inhibited by wheat germ agglutinin, by antinuclear pore complex antibodies, and by peptides corresponding either to classical nuclear localization signals (NLS) or to COOH-terminal sequences of the core protein. Binding was dependent on the nuclear transport factors importins (karyopherins) α and β. The results suggested that phosphorylation induces exposure of NLS in the COOH-terminal portion of the core protein that allows core binding to the NPCs by the importin- (karyopherin-) mediated pathway. Thus, phosphorylation of the core protein emerged as an important step in the viral replication cycle necessary for transport of the viral genome to the nucleus.
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Gaik, Monika, Dirk Flemming, Alexander von Appen, et al. "Structural basis for assembly and function of the Nup82 complex in the nuclear pore scaffold." Journal of Cell Biology 208, no. 3 (2015): 283–97. http://dx.doi.org/10.1083/jcb.201411003.
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Nuclear pore complexes (NPCs) are huge assemblies formed from ∼30 different nucleoporins, typically organized in subcomplexes. One module, the conserved Nup82 complex at the cytoplasmic face of NPCs, is crucial to terminate mRNA export. To gain insight into the structure, assembly, and function of the cytoplasmic pore filaments, we reconstituted in yeast the Nup82–Nup159–Nsp1–Dyn2 complex, which was suitable for biochemical, biophysical, and electron microscopy analyses. Our integrative approach revealed that the yeast Nup82 complex forms an unusual asymmetric structure with a dimeric array of subunits. Based on all these data, we developed a three-dimensional structural model of the Nup82 complex that depicts how this module might be anchored to the NPC scaffold and concomitantly can interact with the soluble nucleocytoplasmic transport machinery.
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Mitic, Kristina, Irene Meyer, Ralph Gräf, and Marianne Grafe. "Temporal Changes in Nuclear Envelope Permeability during Semi-Closed Mitosis in Dictyostelium Amoebae." Cells 12, no. 10 (2023): 1380. http://dx.doi.org/10.3390/cells12101380.
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The Amoebozoan Dictyostelium discoideum exhibits a semi-closed mitosis in which the nuclear membranes remain intact but become permeabilized to allow tubulin and spindle assembly factors to access the nuclear interior. Previous work indicated that this is accomplished at least by partial disassembly of nuclear pore complexes (NPCs). Further contributions by the insertion process of the duplicating, formerly cytosolic, centrosome into the nuclear envelope and nuclear envelope fenestrations forming around the central spindle during karyokinesis were discussed. We studied the behavior of several Dictyostelium nuclear envelope, centrosomal, and nuclear pore complex (NPC) components tagged with fluorescence markers together with a nuclear permeabilization marker (NLS-TdTomato) by live-cell imaging. We could show that permeabilization of the nuclear envelope during mitosis occurs in synchrony with centrosome insertion into the nuclear envelope and partial disassembly of nuclear pore complexes. Furthermore, centrosome duplication takes place after its insertion into the nuclear envelope and after initiation of permeabilization. Restoration of nuclear envelope integrity usually occurs long after re-assembly of NPCs and cytokinesis has taken place and is accompanied by a concentration of endosomal sorting complex required for transport (ESCRT) components at both sites of nuclear envelope fenestration (centrosome and central spindle).
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Makarov, Alexandr A., Norma E. Padilla-Mejia, and Mark C. Field. "Evolution and diversification of the nuclear pore complex." Biochemical Society Transactions 49, no. 4 (2021): 1601–19. http://dx.doi.org/10.1042/bst20200570.
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The nuclear pore complex (NPC) is responsible for transport between the cytoplasm and nucleoplasm and one of the more intricate structures of eukaryotic cells. Typically composed of over 300 polypeptides, the NPC shares evolutionary origins with endo-membrane and intraflagellar transport system complexes. The modern NPC was fully established by the time of the last eukaryotic common ancestor and, hence, prior to eukaryote diversification. Despite the complexity, the NPC structure is surprisingly flexible with considerable variation between lineages. Here, we review diversification of the NPC in major taxa in view of recent advances in genomic and structural characterisation of plant, protist and nucleomorph NPCs and discuss the implications for NPC evolution. Furthermore, we highlight these changes in the context of mRNA export and consider how this process may have influenced NPC diversity. We reveal the NPC as a platform for continual evolution and adaptation.
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Zhang, Wanlu, Annett Neuner, Diana Rüthnick, et al. "Brr6 and Brl1 locate to nuclear pore complex assembly sites to promote their biogenesis." Journal of Cell Biology 217, no. 3 (2018): 877–94. http://dx.doi.org/10.1083/jcb.201706024.
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The paralogous Brr6 and Brl1 are conserved integral membrane proteins of the nuclear envelope (NE) with an unclear role in nuclear pore complex (NPC) biogenesis. Here, we analyzed double-degron mutants of Brr6/Brl1 to understand this function. Depletion of Brr6 and Brl1 caused defects in NPC biogenesis, whereas the already assembled NPCs remained unaffected. This NPC biogenesis defect was not accompanied by a change in lipid composition. However, Brl1 interacted with Ndc1 and Nup188 by immunoprecipitation, and with transmembrane and outer and inner ring NPC components by split yellow fluorescent protein analysis, indicating a direct role in NPC biogenesis. Consistently, we found that Brr6 and Brl1 associated with a subpopulation of NPCs and emerging NPC assembly sites. Moreover, BRL1 overexpression affected NE morphology without a change in lipid composition and completely suppressed the nuclear pore biogenesis defect of nup116Δ and gle2Δ cells. We propose that Brr6 and Brl1 transiently associate with NPC assembly sites where they promote NPC biogenesis.
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Hawryluk-Gara, Lisa A., Melpomeni Platani, Rachel Santarella, Richard W. Wozniak, and Iain W. Mattaj. "Nup53 Is Required for Nuclear Envelope and Nuclear Pore Complex Assembly." Molecular Biology of the Cell 19, no. 4 (2008): 1753–62. http://dx.doi.org/10.1091/mbc.e07-08-0820.
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Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53–Nup155 complex plays a critical role in the processes of NPC and NE assembly.
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Allen, T. D., E. V. Kiseleva, and M. W. Goldberg. "Internal organisation of the nuclear pore complex by surface imaging with field emission in lens SEM (FEISEM)." Proceedings, annual meeting, Electron Microscopy Society of America 54 (August 11, 1996): 822–23. http://dx.doi.org/10.1017/s0424820100166579.
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We have been working towards a 3 dimensional structural understanding of the Nuclear Pore Complex (NPC) with a view to investigating structural alterations associated with the molecular mechanism of transport across the nuclear envelope. FEISEM allows direct visualisation of changes in individual NPCs which will complement information from TEM 3D reconstructions. FEISEM has produced significant new information on the more peripheral elements of the NPC, most notably the nuclear pore basket or ‘fishtrap’ and the nuclear envelope lattice. NPC baskets have been recognised in both avian and insect species as well as amphibia and are likely to be evolutionarily conserved. In the salivary gland nuclei of Chironomus different configurations of basket structure have been recognised undergoing interaction with Balbiani Ring particles during mRNA export. (Fig 1).The ability to observe and directly compare large numbers of NPCs on isolated NEs from either the cytoplasmic or nucleoplasmicface, at equivalent biological resolution to TEM has enabled us to access internal NPC organisation by surface imaging.
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Kuersten, Scott, Gert-Jan Arts, Tobias C. Walther, Ludwig Englmeier, and Iain W. Mattaj. "Steady-State Nuclear Localization of Exportin-t Involves RanGTP Binding and Two Distinct Nuclear Pore Complex Interaction Domains." Molecular and Cellular Biology 22, no. 16 (2002): 5708–20. http://dx.doi.org/10.1128/mcb.22.16.5708-5720.2002.
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ABSTRACT Vertebrate tRNA export receptor exportin-t (Xpo-t) binds to RanGTP and mature tRNAs cooperatively to form a nuclear export complex. Xpo-t shuttles bidirectionally through nuclear pore complexes (NPCs) but is mainly nuclear at steady state. The steady-state distribution of Xpo-t is shown to depend on its interaction with RanGTP. Two distinct Xpo-t NPC interaction domains that bind differentially to peripherally localized nucleoporins in vitro are identified. The N terminus binds to both Nup153 and RanBP2/Nup358 in a RanGTP-dependent manner, while the C terminus binds to CAN/Nup214 independently of Ran. We propose that these interactions increase the concentration of tRNA export complexes and of empty Xpo-t in the vicinity of NPCs and thus increase the efficiency of the Xpo-t transport cycle.
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Stanley, George J., Ariberto Fassati, and Bart W. Hoogenboom. "Atomic force microscopy reveals structural variability amongst nuclear pore complexes." Life Science Alliance 1, no. 4 (2018): e201800142. http://dx.doi.org/10.26508/lsa.201800142.
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The nuclear pore complex (NPC) is a proteinaceous assembly that regulates macromolecular transport into and out of the nucleus. Although the structure of its scaffold is being revealed in increasing detail, its transport functionality depends upon an assembly of intrinsically disordered proteins (called FG-Nups) anchored inside the pore's central channel, which have hitherto eluded structural characterization. Here, using high-resolution atomic force microscopy, we provide a structural and nanomechanical analysis of individual NPCs. Our data highlight 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, supporting the notion that FG-Nup cohesiveness is in a range that facilitates collective rearrangements at little energetic cost. Finally, different nuclear transport receptors are shown to interact in qualitatively different ways with the FG-Nups, with particularly strong binding of importin-β.
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Shevelyov, Yuri Y. "The Role of Nucleoporin Elys in Nuclear Pore Complex Assembly and Regulation of Genome Architecture." International Journal of Molecular Sciences 21, no. 24 (2020): 9475. http://dx.doi.org/10.3390/ijms21249475.
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For a long time, the nuclear lamina was thought to be the sole scaffold for the attachment of chromosomes to the nuclear envelope (NE) in metazoans. However, accumulating evidence indicates that nuclear pore complexes (NPCs) comprised of nucleoporins (Nups) participate in this process as well. One of the Nups, Elys, initiates NPC reassembly at the end of mitosis. Elys directly binds the decondensing chromatin and interacts with the Nup107–160 subcomplex of NPCs, thus serving as a seeding point for the subsequent recruitment of other NPC subcomplexes and connecting chromatin with the re-forming NE. Recent studies also uncovered the important functions of Elys during interphase where it interacts with chromatin and affects its compactness. Therefore, Elys seems to be one of the key Nups regulating chromatin organization. This review summarizes the current state of our knowledge about the participation of Elys in the post-mitotic NPC reassembly as well as the role that Elys and other Nups play in the maintenance of genome architecture.
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Sachdev, Ruchika, Cornelia Sieverding, Matthias Flötenmeyer, and Wolfram Antonin. "The C-terminal domain of Nup93 is essential for assembly of the structural backbone of nuclear pore complexes." Molecular Biology of the Cell 23, no. 4 (2012): 740–49. http://dx.doi.org/10.1091/mbc.e11-09-0761.
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Nuclear pore complexes (NPCs) are large macromolecular assemblies that control all transport across the nuclear envelope. They are formed by about 30 nucleoporins (Nups), which can be roughly categorized into those forming the structural skeleton of the pore and those creating the central channel and thus providing the transport and gating properties of the NPC. Here we show that the conserved nucleoporin Nup93 is essential for NPC assembly and connects both portions of the NPC. Although the C-terminal domain of the protein is necessary and sufficient for the assembly of a minimal structural backbone, full-length Nup93 is required for the additional recruitment of the Nup62 complex and the establishment of transport-competent NPCs.
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Lin, Daniel H., and André Hoelz. "The Structure of the Nuclear Pore Complex (An Update)." Annual Review of Biochemistry 88, no. 1 (2019): 725–83. http://dx.doi.org/10.1146/annurev-biochem-062917-011901.
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The nuclear pore complex (NPC) serves as the sole bidirectional gateway of macromolecules in and out of the nucleus. Owing to its size and complexity (∼1,000 protein subunits, ∼110 MDa in humans), the NPC has remained one of the foremost challenges for structure determination. Structural studies have now provided atomic-resolution crystal structures of most nucleoporins. The acquisition of these structures, combined with biochemical reconstitution experiments, cross-linking mass spectrometry, and cryo–electron tomography, has facilitated the determination of the near-atomic overall architecture of the symmetric core of the human, fungal, and algal NPCs. Here, we discuss the insights gained from these new advances and outstanding issues regarding NPC structure and function. The powerful combination of bottom-up and top-down approaches toward determining the structure of the NPC offers a paradigm for uncovering the architectures of other complex biological machines to near-atomic resolution.
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Allen, T. D., G. R. Bcnnion, S. A. Rutherford, E. Kiscleva, and M. W. Goldberg. "FEISEM, Form and Function in the Nuclear Pore Complex." Microscopy and Microanalysis 4, S2 (1998): 958–59. http://dx.doi.org/10.1017/s1431927600024910.
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Recent initiatives have resulted in a considerable increase in our understanding of the structure of the nuclear pore complex (NPC). The biochemical factors involved in both import and export have been rapidly characterised, with steady progress in the molecular dissection of the structural elements of the NPC, which is a unit of considerable molecular architecture (MW 125 kD), comprising an estimated 50- 100 different proteins. Despite this progress, the crucial molecular interactions involved in the mechanics of transport through the central transporter of the NPC remain unclear. NPC structure in Diptera, fish, (Fig 1) amphibians, birds and mammals shows a high degree of evolutionary conservation. 3D reconstructions of isolated yeast NPCs, show that the core structure is very similar to ‘higher’ organisms, but peripheral structures may be considerably reduced in structural complexity (1).Individual NPC components have been accessed in FEISEM by a variety of methods, including proteolysis,
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Shulga, Nataliya, and David S. Goldfarb. "Binding Dynamics of Structural Nucleoporins Govern Nuclear Pore Complex Permeability and May Mediate Channel Gating." Molecular and Cellular Biology 23, no. 2 (2003): 534–42. http://dx.doi.org/10.1128/mcb.23.2.534-542.2003.
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ABSTRACT The nuclear pore complex (NPC) is a permeable sieve that can dilate to facilitate the bidirectional translocation of a wide size range of receptor-cargo complexes. The binding of receptors to FG nucleoporin docking sites triggers channel gating by an unknown mechanism. Previously, we used deoxyglucose and chilling treatments to implicate Nup170p and Nup188p in the control of NPC sieving in Saccharomyces cerevisiae. Here, we report that aliphatic alcohols increase the permeability of wild-type and nup170Δ NPCs. In conjunction with increases in permeability, aliphatic alcohols, deoxyglucose, and chilling trigger the reversible dissociation of several nucleoporins from nup170Δ NPCs. These results are consistent with the hypothesis that NPC gating occurs when molecular latches composed of FG repeats and structural nucleoporins dissociate.
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Galy, Vincent, Iain W. Mattaj, and Peter Askjaer. "Caenorhabditis elegans Nucleoporins Nup93 and Nup205 Determine the Limit of Nuclear Pore Complex Size Exclusion In Vivo." Molecular Biology of the Cell 14, no. 12 (2003): 5104–15. http://dx.doi.org/10.1091/mbc.e03-04-0237.
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Nuclear pore complexes (NPCs) span the nuclear envelope and mediate communication between the nucleus and the cytoplasm. To obtain insight into the structure and function of NPCs of multicellular organisms, we have initiated an extensive analysis of Caenorhabditis elegans nucleoporins. Of 20 assigned C. elegans nucleoporin genes, 17 were found to be essential for embryonic development either alone or in combination. In several cases, depletion of nucleoporins by RNAi caused severe defects in nuclear appearance. More specifically, the C. elegans homologs of vertebrate Nup93 and Nup205 were each found to be required for normal NPC distribution in the nuclear envelope in vivo. Depletion of Nup93 or Nup205 caused a failure in nuclear exclusion of nonnuclear macromolecules of ∼70 kDa without preventing active nuclear protein import or the assembly of the nuclear envelope. The defects in NPC exclusion were accompanied by abnormal chromatin condensation and early embryonic arrest. Thus, the contribution to NPC structure of Nup93 and Nup205 is essential for establishment of normal NPC function and for cell viability.
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Shimi, Takeshi, and Hiroshi Kimura. "A mosaic of old and young nucleoporins." Journal of Cell Biology 218, no. 2 (2019): 385–86. http://dx.doi.org/10.1083/jcb.201811170.
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Some nucleoporins, the nuclear pore complex (NPC) components, have exceptionally long lifetimes. In this issue, Toyama et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201809123) report that NPCs are maintained by a slow piecemeal replacement of NPC components in dividing and terminally differentiated cells and by whole-pore exchange in quiescent cells.