scholarly journals TPX2 is required for postmitotic nuclear assembly in cell-free Xenopus laevis egg extracts

2006 ◽  
Vol 173 (5) ◽  
pp. 685-694 ◽  
Author(s):  
Lori L. O'Brien ◽  
Christiane Wiese
Keyword(s):  
Xenopus Laevis ◽  
Nuclear Envelope ◽  
Mitotic Spindle ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Assembly ◽  
Egg Extracts ◽  
Mitotic Spindle Assembly ◽  
Pore Complexes

Cell division in many metazoa is accompanied by the disassembly of the nuclear envelope and the assembly of the mitotic spindle. These dramatic structural rearrangements are reversed after mitosis, when the mitotic spindle is dismantled and the nuclear envelope reassembles. The targeting protein for XKlp2 (TPX2) plays important roles in mitotic spindle assembly. We report that TPX2 depletion from nuclear assembly extracts prepared from Xenopus laevis eggs results in the formation of nuclei that are only about one fifth the size of control nuclei. TPX2-depleted nuclei assemble nuclear envelopes, nuclear pore complexes, and a lamina, and they perform nuclear-specific functions, including DNA replication. We show that TPX2 interacts with lamina-associated polypeptide 2 (LAP2), a protein known to be required for nuclear assembly in interphase extracts and in vitro. LAP2 localization is disrupted in TPX2-depleted nuclei, suggesting that the interaction between TPX2 and LAP2 is required for postmitotic nuclear reformation.

scholarly journals Transportin Regulates Major Mitotic Assembly Events: From Spindle to Nuclear Pore Assembly

2009 ◽  
Vol 20 (18) ◽  
pp. 4043-4058 ◽  
Author(s):  
Corine K. Lau ◽  
Valerie A. Delmar ◽  
Rene C. Chan ◽  
Quang Phung ◽  
Cyril Bernis ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Mitotic Chromosomes ◽  
Nuclear Assembly ◽  
Importin Beta ◽  
Egg Extracts ◽  
Multiple Cell ◽  
Pore Complexes ◽  
Nuclear Pore Assembly

Mitosis in higher eukaryotes is marked by the sequential assembly of two massive structures: the mitotic spindle and the nucleus. Nuclear assembly itself requires the precise formation of both nuclear membranes and nuclear pore complexes. Previously, importin alpha/beta and RanGTP were shown to act as dueling regulators to ensure that these assembly processes occur only in the vicinity of the mitotic chromosomes. We now find that the distantly related karyopherin, transportin, negatively regulates nuclear envelope fusion and nuclear pore assembly in Xenopus egg extracts. We show that transportin—and importin beta—initiate their regulation as early as the first known step of nuclear pore assembly: recruitment of the critical pore-targeting nucleoporin ELYS/MEL-28 to chromatin. Indeed, each karyopherin can interact directly with ELYS. We further define the nucleoporin subunit targets for transportin and importin beta and find them to be largely the same: ELYS, the Nup107/160 complex, Nup53, and the FG nucleoporins. Equally importantly, we find that transportin negatively regulates mitotic spindle assembly. These negative regulatory events are counteracted by RanGTP. We conclude that the interplay of the two negative regulators, transportin and importin beta, along with the positive regulator RanGTP, allows precise choreography of multiple cell cycle assembly events.

scholarly journals The Three Fungal Transmembrane Nuclear Pore Complex Proteins of Aspergillus nidulans Are Dispensable in the Presence of an Intact An-Nup84-120 Complex

2009 ◽  
Vol 20 (2) ◽  
pp. 616-630 ◽  
Author(s):  
Hui-Lin Liu ◽  
Colin P.C. De Souza ◽  
Aysha H. Osmani ◽  
Stephen A. Osmani
Keyword(s):  
High Temperature ◽  
Nuclear Envelope ◽  
Aspergillus Nidulans ◽  
Nuclear Pore Complex ◽  
Mitotic Spindle ◽  
Spindle Pole ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Spindle Pole Bodies ◽  
Pore Complexes

In Aspergillus nidulans nuclear pore complexes (NPCs) undergo partial mitotic disassembly such that 12 NPC proteins (Nups) form a core structure anchored across the nuclear envelope (NE). To investigate how the NPC core is maintained, we affinity purified the major core An-Nup84-120 complex and identified two new fungal Nups, An-Nup37 and An-ELYS, previously thought to be vertebrate specific. During mitosis the An-Nup84-120 complex locates to the NE and spindle pole bodies but, unlike vertebrate cells, does not concentrate at kinetochores. We find that mutants lacking individual An-Nup84-120 components are sensitive to the membrane destabilizer benzyl alcohol (BA) and high temperature. Although such mutants display no defects in mitotic spindle formation, they undergo mitotic specific disassembly of the NPC core and transient aggregation of the mitotic NE, suggesting the An-Nup84-120 complex might function with membrane. Supporting this, we show cells devoid of all known fungal transmembrane Nups (An-Ndc1, An-Pom152, and An-Pom34) are viable but that An-ndc1 deletion combined with deletion of individual An-Nup84-120 components is either lethal or causes sensitivity to treatments expected to destabilize membrane. Therefore, the An-Nup84-120 complex performs roles, perhaps at the NPC membrane as proposed previously, that become essential without the An-Ndc1 transmembrane Nup.

Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent ‘envelope smoothing’ event

1997 ◽  
Vol 110 (13) ◽  
pp. 1489-1502 ◽  
Author(s):  
C. Wiese ◽  
M.W. Goldberg ◽  
T.D. Allen ◽  
K.L. Wilson
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Transmission Electron ◽  
Egg Extracts ◽  
Nuclear Envelope Assembly ◽  
Attachment Proteins ◽  
Scanning Em ◽  
Pore Complexes ◽  
Xenopus Egg Extracts

We analyzed the pathway of nuclear envelope assembly in Xenopus egg extracts using field emission in-lens scanning electron microscopy. The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail. The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fully enclosed by membranes. Confirming previous transmission electron microscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying (‘rough’) vesicles, many of which bound directly to chromatin, and ‘smooth’ vesicles, which appeared to associate primarily with other nuclear vesicles or membrane patches. The presence of ribosomes, an outer nuclear membrane marker, on many chromatin-binding vesicles suggested that chromatin-attachment proteins integral to the inner membrane were present on vesicles that also carried markers of the outer membrane and endoplasmic reticulum. Chromatin-associated vesicles also carried pore membrane proteins, since pore complexes formed when these vesicles were incubated with cytosol. A change in nuclear envelope morphology termed ‘envelope smoothing’ occurred 5–15 minutes after enclosure. Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-binding nucleoporins, and therefore unable to form functional pore complexes, remained wrinkled, suggesting that ‘smoothing’ required active nuclear transport. Lamins accumulated with time when nuclei were enclosed and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes. Very low levels of lamins were detected on nuclear intermediates whose surfaces were substantially covered with patches of pore-complex-containing envelope, suggesting that pore complexes might be functional before enclosure.

scholarly journals The nucleoporin Nup188 controls passage of membrane proteins across the nuclear pore complex

2010 ◽  
Vol 189 (7) ◽  
pp. 1129-1142 ◽  
Author(s):  
Gandhi Theerthagiri ◽  
Nathalie Eisenhardt ◽  
Heinz Schwarz ◽  
Wolfram Antonin
Keyword(s):  
Membrane Proteins ◽  
Building Blocks ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Wild Type ◽  
Nuclear Assembly ◽  
Evolutionarily Conserved ◽  
Major Structural Component ◽  
Pore Complexes

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.

scholarly journals In cell architecture of the nuclear pore complex and snapshots of its turnover

2020 ◽  
Author(s):  
Matteo Allegretti ◽  
Christian E. Zimmerli ◽  
Vasileios Rantos ◽  
Florian Wilfling ◽  
Paolo Ronchi ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Mrna Export ◽  
Light And Electron Microscopy ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Formidable Challenge ◽  
Cellular Context ◽  
Pore Complexes

SummaryNuclear pore complexes (NPCs) mediate exchange across the nuclear envelope. They consist of hundreds of proteins called nucleoporins (Nups) that assemble in multiple copies to fuse the inner and outer nuclear membranes. Elucidating the molecular function and architecture of NPCs imposes a formidable challenge and requires the convergence of in vitro and in situ approaches. How exactly NPC architecture accommodates processes such as mRNA export or NPC assembly and turnover inside of cells remains poorly understood. Here we combine integrated in situ structural biology, correlative light and electron microscopy with yeast genetics to structurally analyze NPCs within the native context of Saccharomyces cerevisiae cells under conditions of starvation and exponential growth. We find an unanticipated in situ layout of nucleoporins with respect to overall dimensions and conformation of the NPC scaffold that could not have been predicted from previous in vitro analysis. Particularly striking is the configuration of the Nup159 complex, which appears critical to spatially accommodate not only mRNA export but also NPC turnover by selective autophagy. We capture structural snapshots of NPC turnover, revealing that it occurs through nuclear envelope herniae and NPC-containing nuclear vesicles. Our study provides the basis for understanding the various membrane remodeling events that happen at the interface of the nuclear envelope with the autophagy apparatus and emphasizes the need of investigating macromolecular complexes in their cellular context.

scholarly journals The nucleoporin Nup50 activates the Ran guanyl-nucleotide exchange factor RCC1 to promote mitotic NPC assembly

2021 ◽  
Author(s):  
Guillaume Holzer ◽  
Paola De Magistris ◽  
Cathrin Gramminger ◽  
Ruchika Sachdev ◽  
Adriana Magalska ◽  
...  
Keyword(s):  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor ◽  
Egg Extracts ◽  
Importin Α ◽  
Pore Complexes ◽  
Xenopus Egg Extracts

During mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport-competent nucleus. We show here that Nup50 plays a crucial role in NPC assembly that is independent of its well-established function in nuclear transport. RNAi-mediated downregulation in cells or immunodepletion of the protein in Xenopus egg extracts interferes with NPC assembly. We define a conserved central region of 46 residues in Nup50 that is crucial for Nup153 and MEL28/ELYS binding, and NPC interaction. Surprisingly, neither NPC interaction nor binding of Nup50 to importin α, β, the GTPase Ran or chromatin is crucial for its function in the assembly process. Instead, we discovered that an N-terminal fragment of Nup50 can stimulate the Ran guanine exchange factor RCC1 and NPC assembly, indicating that Nup50 acts via the Ran system in mitotic NPC reformation. In support of this conclusion, Nup50 mutants defective in RCC1 binding and stimulation cannot replace the wild type protein in in vitro NPC assembly assays.

scholarly journals Nuclear Envelope Breakdown Is Coordinated by Both Nup358/RanBP2 and Nup153, Two Nucleoporins with Zinc Finger Modules

2006 ◽  
Vol 17 (2) ◽  
pp. 760-769 ◽  
Author(s):  
Amy J. Prunuske ◽  
Jin Liu ◽  
Suzanne Elgort ◽  
Jomon Joseph ◽  
Mary Dasso ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Zinc Finger ◽  
Nuclear Lamina ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Zinc Finger Domain ◽  
Membrane Remodeling ◽  
Nuclear Envelope Breakdown ◽  
Pore Complexes

When higher eukaryotic cells transition into mitosis, the nuclear envelope, nuclear pore complexes, and nuclear lamina are coordinately disassembled. The COPI coatomer complex, which plays a major role in membrane remodeling at the Golgi, has been implicated in the process of nuclear envelope breakdown and requires interactions at the nuclear pore complex for recruitment to this new site of action at mitosis. Nup153, a resident of the nuclear pore basket, was found to be involved in COPI recruitment, but the molecular nature of the interface between COPI and the nuclear pore has not been fully elucidated. To better understand what occurs at the nuclear pore at this juncture, we have probed the role of the nucleoporin Nup358/RanBP2. Nup358 contains a repetitive zinc finger domain with overall organization similar to a region within Nup153 that is critical to COPI association, yet inspection of these two zinc finger domains reveals features that also clearly distinguish them. Here, we found that the Nup358 zinc finger domain, but not a zinc finger domain from an unrelated protein, binds to COPI and dominantly inhibits progression of nuclear envelope breakdown in an assay that robustly recapitulates this process in vitro. Moreover, the Nup358 zinc finger domain interferes with COPI recruitment to the nuclear rim. Consistent with a role for this pore protein in coordinating nuclear envelope breakdown, Nup358-specific antibodies impair nuclear disassembly. Significantly, targeting either Nup153 or Nup358 for inhibition perturbs nuclear envelope breakdown, supporting a model in which these nucleoporins play nonredundant roles, perhaps contributing to COPI recruitment platforms on both the nuclear and cytoplasmic faces of the pore. We found that an individual zinc finger is the minimal interface for COPI association, although tandem zinc fingers are optimal. These results provide new information about the critical components of nuclear membrane remodeling and lay the foundation for a better understanding of how this process is regulated.

scholarly journals Involvement of the Lamin Rod Domain in Heterotypic Lamin Interactions Important for Nuclear Organization

2001 ◽  
Vol 153 (3) ◽  
pp. 479-490 ◽  
Author(s):  
Eric C. Schirmer ◽  
Tinglu Guan ◽  
Larry Gerace
Keyword(s):  
Nuclear Envelope ◽  
Cultured Cells ◽  
Nuclear Lamina ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Binding Studies ◽  
Lamin B1 ◽  
Vitro Binding ◽  
Pore Complexes

The nuclear lamina is a meshwork of intermediate-type filament proteins (lamins) that lines the inner nuclear membrane. The lamina is proposed to be an important determinant of nuclear structure, but there has been little direct testing of this idea. To investigate lamina functions, we have characterized a novel lamin B1 mutant lacking the middle ∼4/5 of its α-helical rod domain. Though retaining only 10 heptads of the rod, this mutant assembles into intermediate filament-like structures in vitro. When expressed in cultured cells, it concentrates in patches at the nuclear envelope. Concurrently, endogenous lamins shift from a uniform to a patchy distribution and lose their complete colocalization, and nuclei become highly lobulated. In vitro binding studies suggest that the internal rod region is important for heterotypic associations of lamin B1, which in turn are required for proper organization of the lamina. Accompanying the changes in lamina structure induced by expression of the mutant, nuclear pore complexes and integral membrane proteins of the inner membrane cluster, principally at the patches of endogenous lamins. Considered together, these data indicate that lamins play a major role in organizing other proteins in the nuclear envelope and in determining nuclear shape.

scholarly journals Nup53 Is Required for Nuclear Envelope and Nuclear Pore Complex Assembly

2008 ◽  
Vol 19 (4) ◽  
pp. 1753-1762 ◽  
Author(s):  
Lisa A. Hawryluk-Gara ◽  
Melpomeni Platani ◽  
Rachel Santarella ◽  
Richard W. Wozniak ◽  
Iain W. Mattaj
Keyword(s):  
Nuclear Envelope ◽  
Critical Role ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Egg Extracts ◽  
Multiple Copies ◽  
Xenopus Egg ◽  
Pore Complexes ◽  
Xenopus Egg Extracts

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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