scholarly journals A distinct vesicle population targets membranes and pore complexes to the nuclear envelope in Xenopus eggs.

1991 ◽  
Vol 112 (4) ◽  
pp. 545-556 ◽  
Author(s):  
G P Vigers ◽  
M J Lohka
Keyword(s):  
Nuclear Envelope ◽  
Gradient Centrifugation ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Envelope Assembly ◽  
Pore Complexes ◽  
Envelope Membranes ◽  
Alpha Glucosidase ◽  
Soluble Components

Extracts from Xenopus eggs capable of nuclear envelope assembly in vitro were fractionated by differential and density gradient centrifugation. Nuclear envelope assembly was found to require soluble components in the cytosol and two distinct particulate fractions, which we have called nuclear envelope precursor fractions A and B (NEP-A and NEP-B). Both NEP-A and NEP-B are sensitive to treatments with trypsin, sodium carbonate, and detergents, but can be distinguished from each other by their sensitivities to high salt and N-ethylmaleimide and by their levels of alpha-glucosidase activity. Vesicles in NEP-B bind to chromatin, whereas those in NEP-A do not. NEP-B may therefore be involved in the targeting of membranes to the surface of the chromatin, whereas NEP-A may provide a pool of vesicles that contributes many of the nuclear envelope membranes. NEP-B may also play a role in the assembly of nuclear pore complexes because the density of nuclear pores in the resulting envelope is dependent on the ratio of NEP-B to NEP-A in the reconstituted extract.

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 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 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 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 Nuclear envelope assembly defects link mitotic errors to chromothripsis

2018 ◽  
Author(s):  
Shiwei Liu ◽  
Mijung Kwon ◽  
Mark Mannino ◽  
Nachen Yang ◽  
Alexey Khodjakov ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Cytoplasmic Dna ◽  
Cancer Genomes ◽  
Nuclear Envelope Assembly ◽  
Spindle Disassembly ◽  
Physiological Impact ◽  
Pore Complexes ◽  
Regulatory Controls

Defects in the architecture or integrity of the nuclear envelope (NE) are associated with a variety of human diseases1. Micronuclei, one common nuclear aberration, are an origin for chromothripsis2,3, a catastrophic mutational process commonly observed in cancer genomes and other contexts4-6. Micronuclei have a defective NE, with the extensive chromosome fragmentation that generates chromothripsis occurring after abrupt, spontaneous loss of NE integrity7. After NE disruption, the exposed cytoplasmic DNA can additionally initiate proinflammatory signaling linked to senescence, metastasis, and the immune clearance of tumor cells8. Despite its broad physiological impact, the basis for the nuclear envelope fragility of micronuclei is unknown. Here we demonstrate that micronuclei undergo markedly defective NE assembly: Only “core” NE proteins9,10 assemble efficiently on lagging chromosomes whereas “non-core” NE proteins9,10, including nuclear pore complexes (NPCs), fail to properly assemble. Consequently, micronuclei have impaired nuclear import, and key nuclear proteins required to maintain the integrity of the NE and the genome fail to accumulate normally. We show that densely bundled spindle microtubules inhibit non-core NE assembly, leading to an irreversible NE assembly defect. Accordingly, experimental manipulations that position missegregated chromosomes away from the spindle correct defective NE assembly, prevent spontaneous NE disruption, and suppress DNA damage in micronuclei. Our findings indicate that chromosome segregation and NE assembly are only loosely coordinated through the timing of mitotic spindle disassembly. The absence of precise regulatory controls can explain why errors during mitotic exit are frequent, and a major trigger for catastrophic genome rearrangements5,6.

scholarly journals High-precision mapping of nuclear pore-chromatin interactions reveals new principles of genome organization at the nuclear envelope

2021 ◽  
Author(s):  
Martin W Hetzer ◽  
swati tyagi ◽  
Fei Chen ◽  
Jialiang Huang
Keyword(s):  
Nuclear Envelope ◽  
Genome Organization ◽  
Structural Proteins ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Dna Interactions ◽  
Chromatin Interactions ◽  
Genome Wide ◽  
Pore Complexes

The role of nuclear pore complexes (NPCs) in genome organization remains poorly characterized due to technical limitations in probing genome-wide protein-DNA interactions that are specific to the nuclear envelope. Here, we developed a sensitive method, NPC-DamID, which combines in vitro reconstitution of nuclear import and DamID technology. This fixation-free method specifically identifies genomic DNA interactions at the NPCs in intact nuclei. We found that NPCs are preferentially associated with common and hierarchically arranged super-enhancers (SEs) across multiple cells and tissue types. We also uncovered phase-separated condensates at NPCs that compartmentalize and concentrate transcriptional coactivators and structural proteins at SE-regulated genes. Our results support the idea that NPCs are sites of anchoring SE regulatory hubs through their interaction with CTCF and also maintains the conformation of SEs through interaction with structural proteins of SEs like Med1 and PolII.

Nuclear Envelope Dynamics During Mitosis

1988 ◽  
Vol 46 ◽  
pp. 224-225
Author(s):  
Brian Burke
Keyword(s):  
Nuclear Envelope ◽  
Membrane Vesicles ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Animal Cells ◽  
Interphase Chromosome ◽  
Lamin B ◽  
Chromosome Architecture ◽  
Complex Membrane ◽  
Pore Complexes

The nuclear envelope is a complex membrane structure that forms the boundary of the nuclear compartment in eukaryotes. It regulates the passage of macromolecules between the two compartments and may be important for organizing interphase chromosome architecture. In interphase animal cells it forms a remarkably stable structure consisting of a double membrane ouerlying a protein meshwork or lamina and penetrated by nuclear pore complexes. The latter form the channels for nucleocytoplasmic exchange of macromolecules, At the onset of mitosis, however, it rapidly disassembles, the membranes fragment to yield small vesicles and the lamina, which is composed of predominantly three polypeptides, lamins R, B and C (MW approx. 74, 68 and 65 kDa respectiuely), breaks down. Lamins B and C are dispersed as monomers throughout the mitotic cytoplasm, while lamin B remains associated with the nuclear membrane vesicles.

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.

scholarly journals In Vivo Dynamics of Nuclear Pore Complexes in Yeast

1997 ◽  
Vol 136 (6) ◽  
pp. 1185-1199 ◽  
Author(s):  
Mirella Bucci ◽  
Susan R. Wente
Keyword(s):  
Nuclear Envelope ◽  
Recipient Cell ◽  
Nucleocytoplasmic Transport ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Central Location ◽  
Nuclear Membranes ◽  
Mutant Strains ◽  
Pore Complexes

While much is known about the role of nuclear pore complexes (NPCs) in nucleocytoplasmic transport, the mechanism of NPC assembly into pores formed through the double lipid bilayer of the nuclear envelope is not well defined. To investigate the dynamics of NPCs, we developed a live-cell assay in the yeast Saccharomyces cerevisiae. The nucleoporin Nup49p was fused to the green fluorescent protein (GFP) of Aequorea victoria and expressed in nup49 null haploid yeast cells. When the GFP–Nup49p donor cell was mated with a recipient cell harboring only unlabeled Nup49p, the nuclei fused as a consequence of the normal mating process. By monitoring the distribution of the GFP–Nup49p, we could assess whether NPCs were able to move from the donor section of the nuclear envelope to that of the recipient nucleus. We observed that fluorescent NPCs moved and encircled the entire nucleus within 25 min after fusion. When assays were done in mutant kar1-1 strains, where nuclear fusion does not occur, GFP–Nup49p appearance in the recipient nucleus occurred at a very slow rate, presumably due to new NPC biogenesis or to exchange of GFP– Nup49p into existing recipient NPCs. Interestingly, in a number of existing mutant strains, NPCs are clustered together at permissive growth temperatures. This has been explained with two different hypotheses: by movement of NPCs through the double nuclear membranes with subsequent clustering at a central location; or, alternatively, by assembly of all NPCs at a central location (such as the spindle pole body) with NPCs in mutant cells unable to move away from this point. Using the GFP–Nup49p system with a mutant in the NPCassociated factor Gle2p that exhibits formation of NPC clusters only at 37°C, it was possible to distinguish between these two models for NPC dynamics. GFP– Nup49p-labeled NPCs, assembled at 23°C, moved into clusters when the cells were shifted to growth at 37°C. These results indicate that NPCs can move through the double nuclear membranes and, moreover, can do so to form NPC clusters in mutant strains. Such clusters may result by releasing NPCs from a nuclear tether, or by disappearance of a protein that normally prevents pore aggregation. This system represents a novel approach for identifying regulators of NPC assembly and movement in the future.

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