The nucleoporin Nup153 maintains nuclear envelope architecture and is required for cell migration in tumor cells

Nucleoporin 153 (Nup153), a component of the nuclear pore complex (NPC), has been implicated in the interaction of the NPC with the nuclear lamina. Here we show that depletion of Nup153 by RNAi results in alteration of the organization of the nuclear lamina and the nuclear lamin-binding protein Sun1. More striking, Nup153 depletion induces a dramatic cytoskeletal rearrangement that impairs cell migration in human breast carcinoma cells. Our results point to a very prominent role of Nup153 in connection to cell motility that could be exploited in order to develop novel anti-cancer therapy.

Structured summary

MINT-7893777: Lamin-A/C (uniprotkb:P02545) and NUP153 (uniprotkb:P49790) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7893761: sun1 (uniprotkb:Q9D666) and Lamin-A/C (uniprotkb:P02545) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Early embryonic requirement for nucleoporin Nup35/NPP-19 in nuclear assembly

Nuclear pore complexes (NPCs) are gateways for transport between the nucleus and cytoplasm of eukaryotic cells and play crucial roles in regulation of gene expression. NPCs are composed of multiple copies of ∼ 30 different nucleoporins (nups) that display both ubiquitous and cell type specific functions during development. Vertebrate Nup35 (also known as Nup53) was previously described to interact with Nup93, Nup155 and Nup205 and to be required for nuclear envelope (NE) assembly in vitro. Here, we report the first in vivo characterization of a Nup35 mutation, npp-19(tm2886), and its temperature-dependent effects on Caenorhabditis elegans embryogenesis. At restrictive temperature, npp-19(tm2886) embryos exhibit chromosome missegregation, nuclear morphology defects and die around mid-gastrulation. Depletion of Nup35/NPP-19 inhibits NE localization of Nup155/NPP-8, NPC assembly and nuclear lamina formation. Consequently, nuclear envelope function, including nucleo-cytoplasmic transport, is impaired. In contrast, recruitment of Nup107/NPP-5, LEM-2 and nuclear membranes to the chromatin surface is Nup35/NPP-19-independent, suggesting an uncoupling of nuclear membrane targeting and NPC assembly in the absence of Nup35/NPP-19. We propose that Nup35/NPP-19 has an evolutionary conserved role in NE formation and function, and that this role is particularly critical during the rapid cell divisions of early embryogenesis.     

Phosphomimetic mutation of the mitotically phosphorylated serine 1880 compromises the interaction of the transmembrane nucleoporin gp210 with the nuclear pore complex

The nuclear pore complexes (NPCs) reversibly disassemble and reassemble during mitosis. Disassembly of the NPC is accompanied by phosphorylation of many nucleoporins although the function of this is not clear. It was previously shown that in the transmembrane nucleoporin gp210 a single serine residue at position 1880 is specifically phosphorylated during mitosis. Using amino acid substitution combined with live cell imaging, time-lapse microscopy and FRAP, we investigated the role of serine 1880 in binding of gp210 to the NPC in vivo. An alanine substitution mutant (S1880A) was significantly more dynamic at the NPC compared to the wild-type protein, suggesting that serine 1880 is important for binding of gp210 to the NPC. Moreover a glutamate substitution (S1880E) closely mimicking phosphorylated serine specifically interfered with incorporation of gp210 into the NPC and compromised its post-mitotic recruitment to the nuclear envelope of daughter nuclei. Our findings are consistent with the idea that mitotic phosphorylation acts to dissociate gp210 from the structural elements of the NPC.     

Nucleoporins: leaving the nuclear pore complex for a successful mitosis

The nuclear envelope (NE) separates the cytoplasm and the cell nucleus of interphase eukaryotic cells and nuclear pore complexes (NPCs) mediate the macromolecular exchange between these two compartments. The NE and the NPCs of vertebrate cells disassemble during prophase and the nuclear pore proteins (nucleoporins) are distributed within the mitotic cytoplasm. For an increasing number of them active mitotic functions have been assigned over the past few years. Nucleoporins are participating in spindle assembly, kinetochore organisation, and the spindle assembly checkpoint, all processes that control chromosome segregation and are important for maintenance of genome integrity. But nucleoporins are also engaged in early and late mitotic events, such as centrosome positioning and cytokinesis. Here we will highlight recent progress in deciphering the roles for nucleoporins in the distinct steps of mitosis.     

Nucleoporin domain topology is linked to the transport status of the nuclear pore complex

Nuclear pore complexes (NPCs) facilitate macromolecular exchange between the nucleus and cytoplasm of eukaryotic cells. The vertebrate NPC is composed of approximately 30 different proteins (nucleoporins), of which around one third contain phenylalanine-glycine (FG)-repeat domains that are thought to mediate the main interaction between the NPC and soluble transport receptors. We have recently shown that the FG-repeat domain of Nup153 is flexible within the NPC, although this nucleoporin is anchored to the nuclear side of the NPC. By using domain-specific antibodies, we have now mapped the domain topology of Nup214 in Xenopus oocytes and in human somatic cells by immuno-EM. We have found that whereas Nup214 is anchored to the cytoplasmic side of the NPC via its N-terminal and central domain, its FG-repeat domain appears flexible, residing on both sides of the NPC. Moreover, the spatial distribution of the FG-repeat domains of both Nup153 and Nup214 shifts in a transport-dependent manner, suggesting that the location of FG-repeat domains within the NPC correlates with cargo/receptor interactions and that they concomitantly move with cargo through the central pore of the NPC.     

Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.     

Atomic structure of the nuclear pore complex targeting domain of a Nup116 homologue from the yeast, Candida glabrata

The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of ～456 polypeptide chains contributed by ～30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N‐terminal “FG” repeats containing a Gle2p‐binding sequence motif and a NPC targeting domain at its C‐terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882–1034 [CgNup116(882–1034)], at 1.94 Å resolution. The X‐ray structure of CgNup116(882–1034) is consistent with the molecular envelope determined in solution by small‐angle X‐ray scattering. Structural similarities of CgNup116(882–1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Defective DSB repair correlates with abnormal nuclear morphology and is improved with FTI treatment in Hutchinson-Gilford progeria syndrome fibroblasts

Impaired DSB repair has been implicated as a molecular mechanism contributing to the accelerating aging phenotype in Hutchinson-Gilford progeria syndrome (HGPS), but neither the extent nor the cause of the repair deficiency has been fully elucidated. Here we perform a quantitative analysis of the steady-state number of DSBs and the repair kinetics of ionizing radiation (IR)-induced DSBs in HGPS cells. We report an elevated steady-state number of DSBs and impaired repair of IR-induced DSBs, both of which correlated strongly with abnormal nuclear morphology. We recreated the HGPS cellular phenotype in human coronary artery endothelial cells for the first time by lentiviral transduction of GFP-progerin, which also resulted in impaired repair of IR-induced DSBs, and which correlated with abnormal nuclear morphology. Farnesyl transferase inhibitor (FTI) treatment improved the repair of IR-induced DSBs, but only in HGPS cells whose nuclear morphology was also normalized. Interestingly, FTI treatment did not result in a statistically significant reduction in the higher steady-state number of DSBs. We also report a delay in localization of phospho-NBS1 and MRE11, MRN complex repair factors necessary for homologous recombination (HR) repair, to DSBs in HGPS cells. Our results demonstrate a correlation between nuclear structural abnormalities and the DSB repair defect, suggesting a mechanistic link that may involve delayed repair factor localization to DNA damage. Further, our results show that similar to other HGPS phenotypes, FTI treatment has a beneficial effect on DSB repair.     

Binding of the Mex67p/Mtr2p heterodimer to FXFG, GLFG, and FG repeat nucleoporins is essential for nuclear mRNA export

It is not known how Mex67p and Mtr2p, which form a heterodimer essential for mRNA export, transport mRNPs through the nuclear pore. Here, we show that the Mex67p/Mtr2p complex binds to all of the repeat types (GLFG, FXFG, and FG) found in nucleoporins. For this interaction, complex formation between Mex67p and Mtr2p has to occur. MEX67 and MTR2 also genetically interact with different types of repeat nucleoporins, such as Nup116p, Nup159p, Nsp1p, and Rip1p/Nup40p. These data suggest a model in which nuclear mRNA export requires the Mex67p/Mtr2p heterodimeric complex to directly contact several repeat nucleoporins, organized in different nuclear pore complex subcomplexes, as it carries the mRNP cargo through the nuclear pore.     

Dimerization and direct membrane interaction of Nup53 contribute to nuclear pore complex assembly

Nuclear pore complexes (NPCs) fuse the two membranes of the nuclear envelope (NE) to a pore, connecting cytoplasm and nucleoplasm and allowing exchange of macromolecules between these compartments. Most NPC proteins do not contain integral membrane domains and thus it is largely unclear how NPCs are embedded and anchored in the NE. Here, we show that the evolutionary conserved nuclear pore protein Nup53 binds independently of other proteins to membranes, a property that is crucial for NPC assembly and conserved between yeast and vertebrates. The vertebrate protein comprises two membrane binding sites, of which the C‐terminal domain has membrane deforming capabilities, and is specifically required for de novo NPC assembly and insertion into the intact NE during interphase. Dimerization of Nup53 contributes to its membrane interaction and is crucial for its function in NPC assembly.     

Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import

Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin beta cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during import. Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.     

A-type and B-type lamins initiate layer assembly at distinct areas of the nuclear envelope in living cells

To investigate nuclear lamina re-assembly in vivo, Drosophila A-type and B-type lamins were artificially expressed in Drosophila lamin Dm₀null mutant brain cells. Both exogenous lamin C (A-type) and Dm₀ (B-type) formed sub-layers at the nuclear periphery, and efficiently reverted the abnormal clustering of the NPC. Lamin C initially appeared where NPCs were clustered, and subsequently extended along the nuclear periphery accompanied by the recovery of the regular distribution of NPCs. In contrast, lamin Dm₀ did not show association with the clustered NPCs during lamina formation and NPC spacing recovered only after completion of a closed lamin Dm₀ layer. Further, when lamin Dm₀ and C were both expressed, they did not co-polymerize, initiating layer formation in separate regions. Thus, A and B-type lamins reveal differing properties during lamina assembly, with A-type having the primary role in organizing NPC distribution. This previously unknown complexity in the assembly of the nuclear lamina could be the basis for intricate nuclear envelope functions.     

Laminopathy-inducing lamin A mutants can induce redistribution of lamin binding proteins into nuclear aggregates

Lamins, members of the family of intermediate filaments, form a supportive nucleoskeletal structure underlying the nuclear envelope and can also form intranuclear structures. Mutations within the A-type lamin gene cause a variety of degenerative diseases which are collectively referred to as laminopathies. At the molecular level, laminopathies have been shown to be linked to a discontinuous localization pattern of A-type lamins, with some laminopathies containing nuclear lamin A aggregates. Since nuclear aggregate formation could lead to the mislocalization of proteins interacting with A-type lamins, we set out to examine the effects of FLAG-lamin A N195K and R386K protein aggregate formation on the subnuclear distribution of the retinoblastoma protein (pRb) and the sterol responsive element binding protein 1a (SREBP1a) after coexpression as GFP-fusion proteins in HeLa cells. We observed strong recruitment of both proteins into nuclear aggregates. Nuclear aggregate recruitment of the NPC component nucleoporin NUP153 was also observed and found to be dependent on the N-terminus. That these effects were specific was implied by the fact that a number of other coexpressed karyophilic GFP-fusion proteins, such as the nucleoporin NUP98 and kanadaptin, did not coaggregate with FLAG-lamin A N195K or R386K. Immunofluorescence analysis further indicated that the precursor form of lamin A, pre-lamin A, could be found in intranuclear aggregates. Our results imply that redistribution into lamin A-/pre-lamin A-containing aggregates of proteins such as pRb and SREBP1a could represent a key aspect underlying the molecular pathogenesis of certain laminopathies.     

CHMPions of repair: Emerging perspectives on sensing and repairing the nuclear envelope barrier

Understanding how the integrity of the nuclear membranes is protected against internal and external stresses is an emergent challenge. Work reviewed here investigated the mechanisms by which losses of nuclear–cytoplasmic compartmentalization are sensed and ameliorated. Fundamental to these is spatial control over interactions between the endosomal sorting complexes required for transport machinery and LAP2–emerin–MAN1 family inner nuclear membrane proteins, which together promote nuclear envelope sealing in interphase and at the end of mitosis. We suggest that the size of the nuclear envelope hole dictates the mechanism of its repair, with larger holes requiring barrier-to-autointegration factor and the potential triggering of a postmitotic nuclear envelope reassembly pathway in interphase. We also consider why these mechanisms fail at ruptured micronuclei. Together, this work re-emphasizes the need to understand how membrane flow and local lipid metabolism help ensure that the nuclear envelope is refractory to mechanical rupture yet fluid enough to allow its essential dynamics.     

Nup358 integrates nuclear envelope breakdown with kinetochore assembly

Nuclear envelope breakdown (NEBD) and release of condensed chromosomes into the cytoplasm are key events in the early stages of mitosis in metazoans. NEBD involves the disassembly of all major structural elements of the nuclear envelope, including nuclear pore complexes (NPCs), and the dispersal of nuclear membrane components. The breakdown process is facilitated by microtubules of the mitotic spindle. After NEBD, engagement of spindle microtubules with chromosome-associated kinetochores leads to chromatid segregation. Several NPC subunits relocate to kinetochores after NEBD. siRNA-mediated depletion of one of these proteins, Nup358, reveals that it is essential for kinetochore function. In the absence of Nup358, chromosome congression and segregation are severely perturbed. At the same time, the assembly of other kinetochore components is strongly inhibited, leading to aberrant kinetochore structure. The implication is that Nup358 plays an essential role in integrating NEBD with kinetochore maturation and function. Mitotic arrest associated with Nup358 depletion further suggests that mitotic checkpoint complexes may remain active at nonkinetochore sites.     

Understanding the roles of nuclear A- and B-type lamins in brain development

The nuclear lamina is composed mainly of lamins A and C (A-type lamins) and lamins B1 and B2 (B-type lamins). Dogma has held that lamins B1 and B2 play unique and essential roles in the nucleus of every eukaryotic cell. Recent studies have raised doubts about that view but have uncovered crucial roles for lamins B1 and B2 in neuronal migration during the development of the brain. The relevance of lamins A and C in the brain remains unclear, but it is intriguing that prelamin A expression in the brain is low and is regulated by miR-9, a brain-specific microRNA.