Mlp-dependent anchorage and stabilization of a desumoylating enzyme is required to prevent clonal lethality

Myosin-like proteins 1 and 2 (Mlp1 and Mlp2) form filaments attached to the nucleoplasmic side of the nuclear pore complexes via interaction with the nucleoporin Nup60. Here, we show that Mlps and Nup60, but not several other nucleoporins, are required to localize and stabilize a desumoylating enzyme Ulp1. Moreover, like Mlps, Ulp1 exhibits a unique asymmetric distribution on the nuclear envelope. Consistent with a role in regulating Ulp1, removal of either or both MLPs affects the SUMO conjugate pattern. We also show that deleting MLPs or the localization domains of Ulp1 results in DNA damage sensitivity and clonal lethality, the latter of which is caused by increased levels of 2-micron circle DNA. Epistatic and dosage suppression analyses further demonstrate that Mlps function upstream of Ulp1 in 2-micron circle maintenance and the damage response. Together, our results reveal that Mlps play important roles in regulating Ulp1 and subsequently affect sumoylation stasis, growth, and DNA repair.

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CDK1 and PLK1 co-ordinate the disassembly and re-assembly of the Nuclear Envelope in vertebrate mitosis

10.1101/185660 ◽

2017 ◽

Author(s):

Ines J de Castro ◽

Raquel Sales Gil ◽

Lorena Ligammari ◽

Maria Laura Di Giacinto ◽

Paola Vagnarelli

Keyword(s):

Dna Repair ◽

Cell Division ◽

Nuclear Envelope ◽

Genetic Instability ◽

Nuclear Pore ◽

Lamin A ◽

Nuclear Pore Complexes ◽

Regulatory Pathways ◽

Mitotic Entry ◽

Pore Complexes

ABSTRACTMicronuclei (MN) arise from chromosomes or fragments that fail to be incorporated into the primary nucleus after cell division. These structures are a major source of genetic instability caused by DNA repair and replication defects coupled to aberrant Nuclear Envelope (NE). These problems ultimately lead to a spectrum of chromosome rearrangements called chromothripsis, a phenomenon that is a hallmark of several cancers. Despite its importance, the molecular mechanism at the origin of this instability is still not understood. Here we show that lagging chromatin, although it can efficiently assemble Lamin A/C, always fails to recruit Nuclear Pore Complexes (NPCs) proteins and that Polo-Like Kinase (PLK1) negatively regulates the NPC assembly. We also provide evidence for the requirement of PLK1 activity for the disassembly of NPCs, but not Lamina (A/C), at mitotic entry. Altogether this study reveals the existence of independent regulatory pathways for Lamin A/C and NPC reorganization during mitosis where Lamin A targeting to the chromatin is controlled by CDK1 activity (a clock-based model) while the NPC loading is also spatially monitored by PLK1.

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Nuclear Envelope Dynamics During Mitosis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100103188 ◽

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e08-06-0628 ◽

2009 ◽

Vol 20 (2) ◽

pp. 616-630 ◽

Cited By ~ 73

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.

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In Vivo Dynamics of Nuclear Pore Complexes in Yeast

The Journal of Cell Biology ◽

10.1083/jcb.136.6.1185 ◽

1997 ◽

Vol 136 (6) ◽

pp. 1185-1199 ◽

Cited By ~ 82

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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Phosphorylation-dependent mitotic SUMOylation drives nuclear envelope–chromatin interactions

The Journal of Cell Biology ◽

10.1083/jcb.202103036 ◽

2021 ◽

Vol 220 (12) ◽

Cited By ~ 1

Author(s):

Christopher Ptak ◽

Natasha O. Saik ◽

Ashwini Premashankar ◽

Diego L. Lapetina ◽

John D. Aitchison ◽

...

Keyword(s):

Gene Expression ◽

Nuclear Envelope ◽

Spatial Organization ◽

G1 Phase ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Chromatin Binding ◽

Chromatin Interactions ◽

Sumo Ligase ◽

Pore Complexes

In eukaryotes, chromatin binding to the inner nuclear membrane (INM) and nuclear pore complexes (NPCs) contributes to spatial organization of the genome and epigenetic programs important for gene expression. In mitosis, chromatin–nuclear envelope (NE) interactions are lost and then formed again as sister chromosomes segregate to postmitotic nuclei. Investigating these processes in S. cerevisiae, we identified temporally and spatially controlled phosphorylation-dependent SUMOylation events that positively regulate postmetaphase chromatin association with the NE. Our work establishes a phosphorylation-mediated targeting mechanism of the SUMO ligase Siz2 to the INM during mitosis, where Siz2 binds to and SUMOylates the VAP protein Scs2. The recruitment of Siz2 through Scs2 is further responsible for a wave of SUMOylation along the INM that supports the assembly and anchorage of subtelomeric chromatin at the INM and localization of an active gene (INO1) to NPCs during the later stages of mitosis and into G1-phase.

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The preparation and ultrastructure of avian erythrocyte nuclear envelope enclosed by the plasma membrane

Journal of Cell Science ◽

10.1242/jcs.34.1.81 ◽

1978 ◽

Vol 34 (1) ◽

pp. 81-90

Author(s):

J.R. Harris

Keyword(s):

Plasma Membrane ◽

Nuclear Envelope ◽

Ammonium Molybdate ◽

Negative Staining ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Thin Sectioning ◽

Avian Erythrocyte ◽

Pore Complexes ◽

Low Ionic Strength

A procedure is described for the preparation of avian erythrocyte nuclear envelope ghosts which remain enclosed by the ellipsoid plasma membrane. Haemoglobin-free nucleated chicken erythrocyte ghosts are treated in a low ionic strength buffer plus heparin which brings about decondensation of the chromatin. This is followed by solubilization of the chromatin by digestion with pancreatic deoxyribonuclease-1. When studied by light microscopy using either phase-contrast or Nomarski interference optics, the ellipsoid plasma membrane is clearly seen to remain with the collapsed nuclear envelope trapped inside. This interpretation is supported by negative-staining electron microscopy using ammonium molybdate, which in addition reveals the presence of the nuclear pore complexes. The suggestion is advanced that structural protection is provided for the fragile nuclear envelope system by the surrounding plasma membrane, which might account for the final nuclear envelope being in the form of relatively intact ghosts with well defined nuclear pore complexes. The nuclear envelope is highly fragmented when the plasma membrane is absent, the nuclear pore complexes showing appreciable breakdown. Thin sectioning supports the results of negative staining and in addition shows the nuclear envelope retained within the plasma membrane to be composed of both inner and outer nuclear membranes, but the nuclear pore complexes are not clearly defined.

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Nuclear envelope organization in Dictyostelium discoideum

The International Journal of Developmental Biology ◽

10.1387/ijdb.190184rg ◽

2019 ◽

Vol 63 (8-9-10) ◽

pp. 509-519 ◽

Cited By ~ 3

Author(s):

Petros Batsios ◽

Ralph Gräf ◽

Michael P. Koonce ◽

Denis A. Larochelle ◽

Irene Meyer

Keyword(s):

Nuclear Envelope ◽

Nuclear Membrane ◽

Nuclear Lamina ◽

Major Constituent ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Linc Complex ◽

Family Protein ◽

Import And Export ◽

Pore Complexes

The nuclear envelope consists of the outer and the inner nuclear membrane, the nuclear lamina and the nuclear pore complexes, which regulate nuclear import and export. The major constituent of the nuclear lamina of Dictyostelium is the lamin NE81. It can form filaments like B-type lamins and it interacts with Sun1, as well as with the LEM/HeH-family protein Src1. Sun1 and Src1 are nuclear envelope transmembrane proteins involved in the centrosome-nucleus connection and nuclear envelope stability at the nucleolar regions, respectively. In conjunction with a KASH-domain protein, Sun1 usually forms a so-called LINC complex. Two proteins with functions reminiscent of KASH-domain proteins at the outer nuclear membrane of Dictyostelium are known; interaptin which serves as an actin connector and the kinesin Kif9 which plays a role in the microtubule-centrosome connector. However, both of these lack the conserved KASH-domain. The link of the centrosome to the nuclear envelope is essential for the insertion of the centrosome into the nuclear envelope and the appropriate spindle formation. Moreover, centrosome insertion is involved in permeabilization of the mitotic nucleus, which ensures access of tubulin dimers and spindle assembly factors. Our recent progress in identifying key molecular players at the nuclear envelope of Dictyostelium promises further insights into the mechanisms of nuclear envelope dynamics.

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Nuclear Envelope and Nuclear Pore Complexes in Neurodegenerative Diseases—New Perspectives for Therapeutic Interventions

Molecular Neurobiology ◽

10.1007/s12035-020-02168-x ◽

2020 ◽

Author(s):

Naomi Hachiya ◽

Marta Sochocka ◽

Anna Brzecka ◽

Takuto Shimizu ◽

Kazimierz Gąsiorowski ◽

...

Keyword(s):

Gene Expression ◽

Neurodegenerative Diseases ◽

Nuclear Envelope ◽

Neurodegenerative Disorders ◽

Nuclear Transport ◽

Therapeutic Interventions ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Pore Complexes ◽

Bidirectional Exchange

Abstract Transport of proteins, transcription factors, and other signaling molecules between the nucleus and cytoplasm is necessary for signal transduction. The study of these transport phenomena is particularly challenging in neurons because of their highly polarized structure. The bidirectional exchange of molecular cargoes across the nuclear envelope (NE) occurs through nuclear pore complexes (NPCs), which are aqueous channels embedded in the nuclear envelope. The NE and NPCs regulate nuclear transport but are also emerging as relevant regulators of chromatin organization and gene expression. The alterations in nuclear transport are regularly identified in affected neurons associated with human neurodegenerative diseases. This review presents insights into the roles played by nuclear transport defects in neurodegenerative disease, focusing primarily on NE proteins and NPCs. The subcellular mislocalization of proteins might be a very desirable means of therapeutic intervention in neurodegenerative disorders.

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Piecing together nuclear pore complex assembly during interphase

The Journal of Cell Biology ◽

10.1083/jcb.200904022 ◽

2009 ◽

Vol 185 (3) ◽

pp. 377-379 ◽

Cited By ~ 9

Author(s):

Michael Rexach

Keyword(s):

Nuclear Envelope ◽

Nuclear Pore Complex ◽

Supramolecular Structures ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Assembly Process ◽

Nuclear Pores ◽

Complex Assembly ◽

Cell Biol ◽

Pore Complexes

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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Expression of TorsinA in a heterologous yeast system reveals interactions with conserved lumenal domains of LINC and nuclear pore complexes

10.1101/421909 ◽

2018 ◽

Author(s):

Madeleine Chalfant ◽

Karl W. Barber ◽

Sapan Borah ◽

David Thaller ◽

C. Patrick Lusk

Keyword(s):

Nuclear Envelope ◽

Genetic Interaction ◽

Spindle Pole ◽

Nuclear Pore ◽

Physical Interaction ◽

Nuclear Pore Complexes ◽

Gene Encoding ◽

Aaa Atpase ◽

Dyt1 Dystonia ◽

Pore Complexes

ABSTRACTDYT1 dystonia is caused by an in-frame deletion of a glutamic acid codon in the gene encoding the AAA+ ATPase TorsinA. TorsinA localizes within the lumen of the nuclear envelope/ER and binds to a membrane-spanning co-factor, LAP1 or LULL1, to form an ATPase; the substrate(s) of TorsinA remain ill defined. Here we use budding yeast, which lack Torsins, to interrogate TorsinA function. We show that TorsinA accumulates at nuclear envelope embedded spindle pole bodies (SPBs) in a way that requires its oligomerization and the conserved SUN-domain protein, Mps3. TorsinA is released from SPBs upon expression of LAP1 and stabilized by LAP1 mutants incapable of stimulating TorsinA ATPase activity, suggesting the recapitulation of a TorsinA-substrate cycle. While the expression of TorsinA or TorsinA-ΔE impacts the fitness of strains expressing mps3 alleles, a genetic interaction with a conserved component of the nuclear pore complex, Pom152, is specific for TorsinA. This specificity is mirrored by a physical interaction between Pom152 and TorsinA, but not TorsinA-ΔE. These data suggest that TorsinA-nucleoporin interactions would be abrogated by TorsinA-ΔE, providing new experimental avenues to interrogate the molecular basis behind nuclear envelope herniations seen in cells lacking TorsinA function.

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