Nuclear envelope dysfunction and its contribution to the aging process

The nuclear envelope (NE) is the central organizing unit of the eukaryotic cell serving as a genome protective barrier and mechanotransduction interface between the cytoplasm and the nucleus. The NE is mainly composed of a nuclear lamina and a double membrane connected at specific points where the nuclear pore complexes (NPCs) form. Physiological aging might be generically defined as a functional decline across lifespan observed from the cellular to organismal level. Therefore, during aging and premature aging, several cellular alterations occur, including nuclear‐specific changes, particularly, altered nuclear transport, increased genomic instability induced by DNA damage, and telomere attrition. Here, we highlight and discuss proteins associated with nuclear transport dysfunction induced by aging, particularly nucleoporins, nuclear transport factors, and lamins. Moreover, changes in the structure of chromatin and consequent heterochromatin rearrangement upon aging are discussed. These alterations correlate with NE dysfunction, particularly lamins’ alterations. Finally, telomere attrition is addressed and correlated with altered levels of nuclear lamins and nuclear lamina‐associated proteins. Overall, the identification of molecular mechanisms underlying NE dysfunction, including upstream and downstream events, which have yet to be unraveled, will be determinant not only to our understanding of several pathologies, but as here discussed, in the aging process.     

Tpr is localized within the nuclear basket of the pore complex and has a role in nuclear protein export

Tpr is a coiled-coil protein found near the nucleoplasmic side of the pore complex. Since neither the precise localization of Tpr nor its functions are well defined, we generated antibodies to three regions of Tpr to clarify these issues. Using light and EM immunolocalization, we determined that mammalian Tpr is concentrated within the nuclear basket of the pore complex in a distribution similar to Nup153 and Nup98. Antibody localization together with imaging of GFP-Tpr in living cells revealed that Tpr is in discrete foci inside the nucleus similar to several other nucleoporins but is not present in intranuclear filamentous networks (Zimowska et al., 1997) or in long filaments extending from the pore complex (Cordes et al., 1997) as proposed. Injection of anti-Tpr antibodies into mitotic cells resulted in depletion of Tpr from the nuclear envelope without loss of other pore complex basket proteins. Whereas nuclear import mediated by a basic amino acid signal was unaffected, nuclear export mediated by a leucine-rich signal was retarded significantly. Nuclear injection of anti-Tpr antibodies in interphase cells similarly yielded inhibition of protein export but not import. These results indicate that Tpr is a nucleoporin of the nuclear basket with a role in nuclear protein export.     

Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin

The nucleus of eukaryotes is organized into functional compartments, the two most prominent being heterochromatin and nucleoli. These structures are highly enriched in DNA, proteins or RNA, and thus thought to be crowded. In vitro, molecular crowding induces volume exclusion, hinders diffusion and enhances association, but whether these effects are relevant in vivo remains unclear. Here, we establish that volume exclusion and diffusive hindrance occur in dense nuclear compartments by probing the diffusive behaviour of inert fluorescent tracers in living cells. We also demonstrate that chromatin‐interacting proteins remain transiently trapped in heterochromatin due to crowding induced enhanced affinity. The kinetic signatures of these crowding consequences allow us to derive a fractal model of chromatin organization, which explains why the dynamics of soluble nuclear proteins are affected independently of their size. This model further shows that the fractal architecture differs between heterochromatin and euchromatin, and predicts that chromatin proteins use different target‐search strategies in the two compartments. We propose that fractal crowding is a fundamental principle of nuclear organization, particularly of heterochromatin maintenance.     

The role of nuclear pores in gene regulation,development and disease

Nuclear‐pore complexes (NPCs) are large protein channels that span the nuclear envelope (NE), which is a double membrane that encloses the nuclear genome of eukaryotes. Each of the typically 2,000–4,000 pores in the NE of vertebrate cells is composed of multiple copies of 30 different proteins known as nucleoporins. The evolutionarily conserved NPC proteins have the well‐characterized function of mediating the transport of molecules between the nucleoplasm and the cytoplasm. Mutations in nucleoporins are often linked to specific developmental defects and disease, and the resulting phenotypes are usually interpreted as the consequences of perturbed nuclear transport activity. However, recent evidence suggests that NPCs have additional functions in chromatin organization and gene regulation, some of which might be independent of nuclear transport. Here, we review the transport‐dependent and transport‐independent roles of NPCs in the regulation of nuclear function and gene expression.     

Proteins connecting the nuclear pore complex with the nuclear interior

While much has been learned in recent years about the movement of soluble transport factors across the nuclear pore complex (NPC), comparatively little is known about intranuclear trafficking. We isolated the previously identified Saccharomyces protein Mlp1p (myosin-like protein) by an assay designed to find nuclear envelope (NE) associated proteins that are not nucleoporins. We localized both Mlp1p and a closely related protein that we termed Mlp2p to filamentous structures stretching from the nucleoplasmic face of the NE into the nucleoplasm, similar to the homologous vertebrate and Drosophila Tpr proteins. Mlp1p can be imported into the nucleus by virtue of a nuclear localization sequence (NLS) within its COOH-terminal domain. Overexpression experiments indicate that Mlp1p can form large structures within the nucleus which exclude chromatin but appear highly permeable to proteins. Remarkably, cells harboring a double deletion of MLP1 and MLP2 were viable, although they showed a slower net rate of active nuclear import and faster passive efflux of a reporter protein. Our data indicate that the Tpr homologues are not merely NPC-associated proteins but that they can be part of NPC-independent, peripheral intranuclear structures. In addition, we suggest that the Tpr filaments could provide chromatin-free conduits or tracks to guide the efficient translocation of macromolecules between the nucleoplasm and the NPC.     

Functional Analysis of Tpr: Identification of Nuclear Pore Complex Association and Nuclear Localization Domains and a Role in mRNA Export

Tpr is a 270-kD coiled-coil protein localized to intranuclear filaments of the nuclear pore complex (NPC). The mechanism by which Tpr contributes to the structure and function of the nuclear pore is currently unknown. To gain insight into Tpr function, we expressed the full-length protein and several subdomains in mammalian cell lines and examined their effects on nuclear pore function. Through this analysis, we identified an NH₂-terminal domain that was sufficient for association with the nucleoplasmic aspect of the NPC. In addition, we unexpectedly found that the acidic COOH terminus was efficiently transported into the nuclear interior, an event that was apparently mediated by a putative nuclear localization sequence. Ectopic expression of the full-length Tpr caused a dramatic accumulation of poly(A)⁺ RNA within the nucleus. Similar results were observed with domains that localized to the NPC and the nuclear interior. In contrast, expression of these proteins did not appear to affect nuclear import. These data are consistent with a model in which Tpr is tethered to intranuclear filaments of the NPC by its coiled coil domain leaving the acidic COOH terminus free to interact with soluble transport factors and mediate export of macromolecules from the nucleus.     

Identification of Protein p270/Tpr as a Constitutive Component of the Nuclear Pore Complex–attached Intranuclear Filaments

Using a monoclonal antibody, mAb 203-37, we have identified a polypeptide of M_r ~270 kD (p270) as a general constituent of the intranuclear filaments attached to the nucleoplasmic annulus of the nuclear pore complex (NPC) in diverse kinds of vertebrate cells. Using cDNA cloning and immunobiochemistry, we show that human protein p270 has a predicted molecular mass of 267 kD and is essentially identical to the coiled-coil dominated protein Tpr reported by others to be located on the outer, i.e., cytoplasmic surface of NPCs (Byrd, D.A., D.J. Sweet, N. Pante, K.N. Konstantinov, T. Guan, A.C.S. Saphire, P.J. Mitchell, C.S. Cooper, U. Aebi, and L. Gerace. 1994. J. Cell Biol. 127: 1515–1526). To clarify this controversial localization, we have performed immunoelectron microscopy in diverse kinds of mammalian and amphibian cells with a series of antibodies raised against different epitopes of human and Xenopus laevis p270/Tpr. In these experiments, the protein has been consistently and exclusively detected in the NPC-attached intranuclear filaments, and p270/Tpr-containing filament bundles have been traced into the nuclear interior for up to 350 nm. No reaction has been noted at the cytoplasmic side of NPCs with any of the p270/Tpr antibodies, whereas control antibodies such as those against protein RanBP2/ Nup358 specifically decorate the cytoplasmic annulus of NPCs. Pore complexes of cytoplasmic annulate lamellae in various mammalian and amphibian cells are also devoid of immunodetectable protein p270/Tpr. We conclude that this coiled-coil protein is a general and ubiquitous component of the intranuclear NPC- attached filaments and discuss its possible functions.     

M33 condenses chromatin through nuclear body formation and methylation of both histone H3 lysine 9 and lysine 27

Heterochromatin, a type of condensed DNA in eukaryotic cells, has two main categories: Constitutive heterochromatin, which contains H3K9 methylation, and facultative heterochromatin, which contains H3K27 methylation. Methylated H3K9 and H3K27 serve as docking sites for chromodomain-containing proteins that compact chromatin. M33 (also known as CBX2) is a chromodomain-containing protein that binds H3K27me3 and compacts chromatin in vitro. However, whether M33 mediates chromatin compaction in cellulo remains unknown. Here we show that M33 compacts chromatin into DAPI-intense heterochromatin domains in cells. The formation of these heterochromatin domains requires H3K27me3, which recruits M33 to form nuclear bodies. G9a and SUV39H1 are sequentially recruited into M33 nuclear bodies to create H3K9 methylated chromatin in a process that is independent of HP1α. Finally, M33 decreases progerin-induced nuclear envelope disruption caused by loss of heterochromatin. Our findings demonstrate that M33 mediates the formation of condensed chromatin by forming nuclear bodies containing both H3K27me3 and H3K9me3. Our model of M33-dependent chromatin condensation suggests H3K27 methylation corroborates with H3K9 methylation during the formation of facultative heterochromatin and provides the theoretical basis for developing novel therapies to treat heterochromatin-related diseases.     

Ultrastructural studies of H-1 parvovirus replication : III. Intracellular localization of viral antigens with immunocytochrome c

Immunoelectron microscopy with cytochrome c conjugated anti-H-1 IgG was used to localize antigens of the parvovirus H-1 within synchronized human NB cells. Since glutaraldehyde destroyed H-1 antigenicity, a fixative containing formaldehyde was developed which preserved both cellular ultrastructure and antigenic function. The earliest H-1 specific staining occurred on the heterochromatin bordering the nuclear envelope at 8 h post infection (p.i.). At 10 h p.i., labeling was found on the chromatin associated with the nucleolar surface and tufts of heterochromatin distributed throughout the nucleoplasm. Except for this H-1 labeling, the chromatin appeared indistinguishable from that of uninfected cells. By 12 h p.i., however, coinciding with the abrupt rise in synthesis of H-1 hemagglutinin and infectious virus, H-1 labeled intranuclear chromatin had condensed and migrated toward the nuclear membrane. Also, trabeculae of intranuclear chromatin were tagged with anti-H-1 conjugate as contraction of nucleolar chromatin and disintegration of nucleolar ultrastructure began. Condensation of the nucleolar associated chromatin and nuclear heterochromatin appeared complete by 18–36 h p.i. when thick zones of this H-1 labeled material were observed at the nucleolar and nuclear periphery. Our results indicate that the binding of unassembled H-1 proteins to specific regions of chromatin is associated with their condensation and margination, resulting in early nucleolar destruction and subsequent nuclear damage during H-1 infection.     

Product of the oncogene-activating gene Tpr is a phosphorylated protein of the nuclear pore complex

We have identified a component of the human nuclear pore complex and have shown that it is the product of a gene involved in oncogenic activation. A monoclonal antibody raised against purified nuclear matrix proteins recognizes a single protein with an electrophoretic mobility of approximately 300 kDa and stains the nuclear envelope in a punctate pattern typical of nuclear pores. The antibody was used to screen λgt11 human cDNA libraries, and the resulting clones were sequenced and compared to sequences in the Genbank database. An exact match was found with the human tpr (for translocated promoter region) gene, a gene shown previously to be involved in the oncogenic activation of several protein kinases. Double-label immunofluorescent microscopy with the anti-Tpr antibody and an antibody to the previously characterized nuclear pore complex protein nup153 confirms that Tpr is localized to the nuclear pore complex. Tpr is located on the cytoplasmic face of the nucleus, as demonstrated by immunofluorescent staining of cells permeabilized with digitonin. Tpr is a 2,349-amino acid protein with extensive coiled-coil domains and an acidic globular C-terminus. The protein contains 10 leucine zipper motifs and numerous sites for phosphorylation by a variety of protein kinases. Immunoprecipitation of Tpr from ³²P-orthophosphate-labeled cells shows that it is a phosphoprotein. Potential functions for Tpr and possible mechanisms for the transforming activity of Tpr fusion proteins are discussed. © 1996 Wiley-Liss, Inc.     

Transmission electron microscope studies of the nuclear envelope in Caenorhabditis elegans embryos

Nuclear membranes and nuclear pore complexes (NPCs) are conserved in both animals and plants. However, the lamina composition and the dimensions of NPCs vary between plants, yeast, and vertebrates. In this study, we established a protocol that preserves the structure of Caenorhabditis elegans embryonic cells for high-resolution studies with thin-section transmission electron microscopy (TEM). We show that the NPCs are bigger in C. elegans embryos than in yeast, with dimensions similar to those in higher eukaryotes. We also localized the C. elegans nuclear envelope proteins Ce-lamin and Ce-emerin by pre-embedding gold labeling immunoelectron microscopy. Both proteins are present at or near the inner nuclear membrane. A fraction of Ce-lamin, but not Ce-emerin, is present in the nuclear interior. Removing the nuclear membranes leaves both Ce-lamin and Ce-emerin associated with the chromatin. Eliminating the single lamin protein caused cell death as visualized by characteristic changes in nuclear architecture including condensation of chromatin, clustering of NPCs, membrane blebbing, and the presence of vesicles inside the nucleus. Taken together, these results show evolutionarily conserved protein localization, interactions, and functions of the C. elegans nuclear envelope.     

A nuclear lamina‐chromatin‐Ran GTPase axis modulates nuclear import and DNA damage signaling

The Ran GTPase regulates nuclear import and export by controlling the assembly state of transport complexes. This involves the direct action of RanGTP, which is generated in the nucleus by the chromatin‐associated nucleotide exchange factor, RCC1. Ran interactions with RCC1 contribute to formation of a nuclear:cytoplasmic (N:C) Ran protein gradient in interphase cells. In previous work, we showed that the Ran protein gradient is disrupted in fibroblasts from Hutchinson–Gilford progeria syndrome (HGPS) patients. The Ran gradient disruption in these cells is caused by nuclear membrane association of a mutant form of Lamin A, which induces a global reduction in heterochromatin marked with Histone H3K9me3 and Histone H3K27me3. Here, we have tested the hypothesis that heterochromatin controls the Ran gradient. Chemical inhibition and depletion of the histone methyltransferases (HMTs) G9a and GLP in normal human fibroblasts reduced heterochromatin levels and caused disruption of the Ran gradient, comparable to that observed previously in HGPS fibroblasts. HMT inhibition caused a defect in nuclear localization of TPR, a high molecular weight protein that, owing to its large size, displays a Ran‐dependent import defect in HGPS. We reasoned that pathways dependent on nuclear import of large proteins might be compromised in HGPS. We found that nuclear import of ATM requires the Ran gradient, and disruption of the Ran gradient in HGPS causes a defect in generating nuclear γ‐H2AX in response to ionizing radiation. Our data suggest a lamina–chromatin–Ran axis is important for nuclear transport regulation and contributes to the DNA damage response.     

Defects in nuclear pore assembly lead to activation of an Aurora B-mediated abscission checkpoint

Correct assembly of nuclear pore complexes (NPCs), which directly and indirectly control nuclear environment and architecture, is vital to genomic regulation. We previously found that nucleoporin 153 (Nup 153) is required for timely progression through late mitosis. In this study, we report that disruption of Nup 153 function by either small interfering RNA-mediated depletion or expression of a dominant-interfering Nup 153 fragment results in dramatic mistargeting of the pore basket components Tpr and Nup 50 in midbody-stage cells. We find a concomitant appearance of aberrantly localized active Aurora B and an Aurora B-dependent delay in abscission. Depletion of Nup 50 is also sufficient to increase the number of midbody-stage cells and, likewise, triggers distinctive mislocalization of Aurora B. Together, our results suggest that defects in nuclear pore assembly, and specifically the basket structure, at this time of the cell cycle activate an Aurora B-mediated abscission checkpoint, thereby ensuring that daughter cells are generated only when fully formed NPCs are present.     

Chm7 and Heh1 collaborate to link nuclear pore complex quality control with nuclear envelope sealing

The integrity of the nuclear envelope barrier relies on membrane remodeling by the ESCRTs, which seal nuclear envelope holes and contribute to the quality control of nuclear pore complexes (NPCs); whether these processes are mechanistically related remains poorly defined. Here, we show that the ESCRT‐II/III chimera, Chm7, is recruited to a nuclear envelope subdomain that expands upon inhibition of NPC assembly and is required for the formation of the storage of improperly assembled NPCs (SINC) compartment. Recruitment to sites of NPC assembly is mediated by its ESCRT‐II domain and the LAP2‐emerin‐MAN1 (LEM) family of integral inner nuclear membrane proteins, Heh1 and Heh2. We establish direct binding between Heh2 and the “open” forms of both Chm7 and the ESCRT‐III, Snf7, and between Chm7 and Snf7. Interestingly, Chm7 is required for the viability of yeast strains where double membrane seals have been observed over defective NPCs; deletion of CHM7 in these strains leads to a loss of nuclear compartmentalization suggesting that the sealing of defective NPCs and nuclear envelope ruptures could proceed through similar mechanisms.     

Changes at the nuclear lamina alter binding of pioneer factor Foxa2 in aged liver

Increasing evidence suggests that regulation of heterochromatin at the nuclear envelope underlies metabolic disease susceptibility and age‐dependent metabolic changes, but the mechanism is unknown. Here, we profile lamina‐associated domains (LADs) using lamin B1 ChIP‐Seq in young and old hepatocytes and find that, although lamin B1 resides at a large fraction of domains at both ages, a third of lamin B1‐associated regions are bound exclusively at each age in vivo. Regions occupied by lamin B1 solely in young livers are enriched for the forkhead motif, bound by Foxa pioneer factors. We also show that Foxa2 binds more sites in Zmpste24 mutant mice, a progeroid laminopathy model, similar to increased Foxa2 occupancy in old livers. Aged and Zmpste24‐deficient livers share several features, including nuclear lamina abnormalities, increased Foxa2 binding, de‐repression of PPAR‐ and LXR‐dependent gene expression, and fatty liver. In old livers, additional Foxa2 binding is correlated to loss of lamin B1 and heterochromatin (H3K9me3 occupancy) at these loci. Our observations suggest that changes at the nuclear lamina are linked to altered Foxa2 binding, enabling opening of chromatin and de‐repression of genes encoding lipid synthesis and storage targets that contribute to etiology of hepatic steatosis.     

The deSUMOylase SENP7 promotes chromatin relaxation for homologous recombination DNA repair

SUMO conjugation is known to occur in response to double‐stranded DNA breaks in mammalian cells, but whether SUMO deconjugation has a role remains unclear. Here, we show that the SUMO/Sentrin/Smt3‐specific peptidase, SENP7, interacts with the chromatin repressive KRAB‐associated protein 1 (KAP1) through heterochromatin protein 1 alpha (HP1α). SENP7 promotes the removal of SUMO2/3 from KAP1 and regulates the interaction of the chromatin remodeler CHD3 with chromatin. Consequently, in the presence of CHD3, SENP7 is required for chromatin relaxation in response to DNA damage, for homologous recombination repair and for cellular resistance to DNA‐damaging agents. Thus, deSUMOylation by SENP7 is required to promote a permissive chromatin environment for DNA repair.