Nuclear Pore Components Research Articles

You are who your friends are-nuclear pore proteins as components of chromatin-binding complexes.

Nuclear pore complexes are large multicomponent protein complexes that are embedded in the nuclear envelope, where they mediate nucleocytoplasmic transport. In addition to supporting transport, nuclear pore components, termed nucleoporins (Nups), can interact with chromatin and influence genome function. A subset of Nups can also localize to the nuclear interior and bind chromatin intranuclearly, providing an opportunity to investigate chromatin-associated functions of Nups outside of the transport context. This review focuses on the gene regulatory functions of such intranuclear Nups, with a particular emphasis on their identity as components of several chromatin regulatory complexes. Recent proteomic screens have identified Nups as interacting partners of active and repressive epigenetic machinery, architectural proteins, and DNA replication complexes, providing insight into molecular mechanisms via which Nups regulate gene expression programs. This review summarizes these interactions and discusses their potential functions in the broader framework of nuclear genome organization.

Sensitized piRNA reporter identifies multiple RNA processing factors involved in piRNA-mediated gene silencing.

Metazoans guard their germlines against transposons and other foreign transcripts with PIWI-interacting RNAs (piRNAs). Due to the robust heritability of the silencing initiated by piRNAs in Caenorhabditis elegans (C. elegans), previous screens using C. elegans were strongly biased to uncover members of this pathway in the maintenance process but not in the initiation process. To identify novel piRNA pathway members, we have utilized a sensitized reporter strain which detects defects in initiation, amplification, or regulation of piRNA silencing. Using our reporter, we have identified Integrator complex subunits, nuclear pore components, protein import components, and pre-mRNA splicing factors as essential for piRNA-mediated gene silencing. We found the small nuclear processing cellular machine termed the Integrator complex is required for both type I and type II piRNA production. Notably, we identified a role for nuclear pore and nucleolar components NPP-1/Nup54, NPP-6/Nup160, NPP-7/Nup153, and FIB-1 in promoting the perinuclear localization of anti-silencing CSR-1 Argonaute, as well as a role for Importin factor IMA-3 in nuclear localization of silencing Argonaute HRDE-1. Together, we have shown that piRNA silencing in C. elegans is dependent on evolutionarily ancient RNA processing machinery that has been co-opted to function in the piRNA-mediated genome surveillance pathway.

Genetic marker identification of SEC13 gene for milk production traits in Chinese holstein.

SEC13 homolog, nuclear pore and COPII coat complex component (SEC13) is the core component of the cytoplasmic COPII complex, which mediates material transport from the endoplasmic reticulum to the Golgi complex. Our preliminary work found that SEC13 gene was differentially expressed in dairy cows during different stages of lactation, and involved in metabolic pathways of milk synthesis such as citric acid cycle, fatty acid, starch and sucrose metabolisms, so we considered that the SEC13 might be a candidate gene affecting milk production traits. In this study, we detected the polymorphisms of SEC13 gene and verified their genetic effects on milk yield and composition traits in a Chinese Holstein cow population. By sequencing the whole coding and partial flanking regions of SEC13, we found four single nucleotide polymorphisms (SNPs). Subsequent association analysis showed that these four SNPs were significantly associated with milk yield, fat yield, protein yield or protein percentage in the first and second lactations (p ≤.0351). We also found that two SNPs in SEC13 formed one haplotype block by Haploview4.2, and the block was significantly associated with milk yield, fat yield, fat percentage, protein yield or protein percentage (p ≤ .0373). In addition, we predicted the effect of SNP on 5'region on transcription factor binding sites (TFBSs), and found that the allele A of 22:g.54362761A>G could bind transcription factors (TFs) GATA5, GATA3, HOXD9, HOXA10, CDX1 and Hoxd13; and further dual-luciferase reporter assay verified that the allele A of this SNP inhibited the fluorescence activity. We speculate that the A allele of 22:g.54362761A>G might inhibit the transcriptional activity of SEC13 gene by binding the TFs, which may be a cause mutation affecting the formation of milk production traits in dairy cows. In summary, we proved that SEC13 has a significant genetic effect on milk production traits and the identified significant SNPs could be used as candidate genetic markers for GS SNP chips development; on the other hand, we verified the transcriptional regulation of 22:g.54362761A>G on SEC13 gene, providing research direction for further function validation tests.

Maximum-likelihood model fitting for quantitative analysis of SMLM data

Quantitative data analysis is important for any single-molecule localization microscopy (SMLM) workflow to extract biological insights from the coordinates of the single fluorophores. However, current approaches are restricted to simple geometries or require identical structures. Here, we present LocMoFit (Localization Model Fit), an open-source framework to fit an arbitrary model to localization coordinates. It extracts meaningful parameters from individual structures and can select the most suitable model. In addition to analyzing complex, heterogeneous and dynamic structures for in situ structural biology, we demonstrate how LocMoFit can assemble multi-protein distribution maps of six nuclear pore components, calculate single-particle averages without any assumption about geometry or symmetry, and perform a time-resolved reconstruction of the highly dynamic endocytic process from static snapshots. We provide extensive simulation and visualization routines to validate the robustness of LocMoFit and tutorials to enable any user to increase the information content they can extract from their SMLM data.

Decoupling SARS-CoV-2 ORF6 localization and interferon antagonism.

Like many pathogenic viruses, SARS-CoV-2 must overcome interferon (IFN)-mediated host defenses for infection establishment. To achieve this, SARS-CoV-2 deploys overlapping mechanisms to antagonize IFN production and signaling. The strongest IFN antagonist is the accessory protein ORF6, which localizes to multiple membranous compartments, including the nuclear envelope, where it directly binds nuclear pore component Nup98-Rae1 to inhibit nuclear translocation of activated STAT1 and IRF3 transcription factors. However, this direct cause-and-effect relationship between ORF6 localization and IFN antagonism has yet to be explored experimentally. Here, we use extensive mutagenesis studies to define the structural determinants required for steady-state localization and demonstrate that mis-localized ORF6 variants still potently inhibit nuclear trafficking and IFN signaling. Additionally, expression of a peptide that mimics the ORF6-Nup98 interaction domain robustly blocked nuclear trafficking. Furthermore, pharmacologic and mutational approaches combined to suggest that ORF6 is likely a peripheral membrane protein, as opposed to being a transmembrane protein as previously speculated. Thus, ORF6 localization and IFN antagonism are independent activities, which raises the possibility that ORF6 may have additional functions within membrane networks to enhance virus replication. This article has an associated First Person interview with the first author of the paper.

Nup98-dependent transcriptional memory is established independently of transcription.

Cellular ability to mount an enhanced transcriptional response upon repeated exposure to external cues is termed transcriptional memory, which can be maintained epigenetically through cell divisions and can depend on a nuclear pore component Nup98. The majority of mechanistic knowledge on transcriptional memory has been derived from bulk molecular assays. To gain additional perspective on the mechanism and contribution of Nup98 to memory, we used single-molecule RNA FISH (smFISH) to examine the dynamics of transcription in Drosophila cells upon repeated exposure to the steroid hormone ecdysone. We combined smFISH with mathematical modeling and found that upon hormone exposure, cells rapidly activate a low-level transcriptional response, but simultaneously begin a slow transition into a specialized memory state characterized by a high rate of expression. Strikingly, our modeling predicted that this transition between non-memory and memory states is independent of the transcription stemming from initial activation. We confirmed this prediction experimentally by showing that inhibiting transcription during initial ecdysone exposure did not interfere with memory establishment. Together, our findings reveal that Nup98's role in transcriptional memory is to stabilize the forward rate of conversion from low to high expressing state, and that induced genes engage in two separate behaviors - transcription itself and the establishment of epigenetically propagated transcriptional memory.

Nucleoporin-93 reveals a common feature of aggressive breast cancers: robust nucleocytoplasmic transport of transcription factors.

By establishing multi-omics pipelines, we uncover overexpression and gene copy-number alterations of nucleoporin-93 (NUP93), a nuclear pore component, in aggressive human mammary tumors. NUP93 overexpression enhances transendothelial migration and matrix invasion invitro, along with tumor growth and metastasis in animal models. These findings are supported by analyses of two sets of naturally occurring mutations: rare oncogenic mutations and inactivating familial nephrotic syndrome mutations. Mechanistically, NUP93 binds with importins, boosts nuclear transport of importins' cargoes, such as β-catenin, and activates MYC. Likewise, NUP93 overexpression enhances the ultimate nuclear transport step shared by additional signaling pathways, including TGF-β/SMAD and EGF/ERK. The emerging addiction to nuclear transport exposes vulnerabilities of NUP93-overexpressing tumors. Congruently, myristoylated peptides corresponding to the nuclear translocation signals of SMAD and ERK can inhibit tumor growth and metastasis. Our study sheds light on an emerging hallmark of advanced tumors, which derive benefit from robust nucleocytoplasmic transport.

Correct dosage of X chromosome transcription is controlled by a nuclear pore component.

SUMMARYDosage compensation in Drosophila melanogaster involves a 2-fold transcriptional upregulation of the male X chromosome, which relies on the X-chromosome-binding males-specific lethal (MSL) complex. However, how such 2-fold precision is accomplished remains unclear. Here, we show that a nuclear pore component, Mtor, is involved in setting the correct levels of transcription from the male X chromosome. Using larval tissues, we demonstrate that the depletion of Mtor results in selective upregulation at MSL targets of the male X, beyond the required 2-fold. Mtor and MSL components interact genetically, and depletion of Mtor can rescue the male lethality phenotype of MSL components. Using RNA fluorescence in situ hybridization (FISH) analysis and nascent transcript sequencing, we find that the effect of Mtor is not due to defects in mRNA export but occurs at the level of nascent transcription. These findings demonstrate a physiological role for Mtor in the process of dosage compensation, as a transcriptional attenuator of X chromosome gene expression.

Nucleoporin-93 Overcomes Multiple Nucleocytoplasmic Trafficking Bottlenecks and Permits Robust Metastasis

To identify genetic events driving breast cancer progression, we performed multi-omics integrated analyses that identified overexpression of nucleoporin-93, a nuclear pore component. We show that NUP93 overexpression enhances trans-endothelial migration and matrix invasion, along with metastasis in animal models. These findings were supported by analyses of two sets of naturally occurring mutations: oncogenic mutations and inactivating nephrotic syndrome mutations. Mechanistically, NUP93 activation enhanced the ultimate nuclear transport step shared by multiple signaling pathways, including TGF-beta/SMAD, EGF/ERK and TNF/NF-kB. Likewise, NUP93 boosts nuclear transport of beta-catenin, as well as elevates expression and inhibit degradation of this co-activator. The emerging addiction to nuclear transport exposes vulnerabilities of NUP93-overexpressing tumors. Congruently, myristoylated peptides corresponding to the nuclear translocation signals of SMAD and ERK inhibited growth and metastasis. Our study sheds light on a previously unappreciated hallmark of advanced tumors, which derive benefit from unblocking a set of nucleocytoplasmic bottlenecks.

XPO1 Relieves MYC-Induced Replication Stress Limiting the Immunogenicity of DLBCL Cells

The soluble nuclear pore component XPO1 is a therapeutic target in DLBCL and its selective inhibitor selinexor received FDA approval in relapsed/refractory DLBCL. XPO1 inhibitors block the nuclear export of a subset of cargo proteins. Since the spectrum of XPO1 cargos varies among cell types, the mechanism underlying the anti-lymphoma effect of selinexor has not been fully elucidated. Lack of this knowledge hampers the clinical use of XPO1 inhibitors in terms of patient selection and development of rational therapeutic combinations. We reported that XPO1 is amplified and overexpressed in DLBCL. At the molecular level, we described that XPO1 overexpression functionally remodels the nuclear pore to sustain the expression of DNA damage repair proteins increasing lymphoma cells' tolerance to genotoxic stress. Here, we investigated whether this XPO1 role is relevant for the anti-lymphoma effect of selinexor. Aberrant MYC expression induces DNA damage resulting from replication stress; thus, we speculated that selinexor kills MYC expressing (MYC+) DLBCL cells by limiting their ability to tolerate replication stress. Using B-cells expressing MYC in an inducible manner, we demonstrated that exposure to selinexor (6 h) caused G2-M cell cycle arrest upon MYC expression (p<0.0001, vs. vehicle-treated cells). Cell cycle arrest was associated with increased levels of DNA damage in S-phase as measured by yH2AX (p<0.0001). Furthermore, although MYC increased the expression of key replication stress repair proteins (i.e. RAD51, WEE and BRCA1), this effect was blunted by selinexor. To determine the clinical relevance of this mechanism, we interrogated publicly available datasets of DLBCLs and found that XPO1 amplification and overexpression was associated with MYC translocation (p<0.005), double-hit lymphoma signature (p<0.005), and elevated levels of chromosomal instability (p<0.005). Overall, these observations suggest that MYC-driven lymphomas sustaining high levels of genomic instability may critically depend on XPO1. To therapeutically capitalized this mechanism, we evaluated the effect of selinexor on MYC+ DLBCLs. Clinically achievable doses of selinexor induced cell cycle arrest and apoptosis in 83% (5/6) of cell lines investigated in vitro. In a patient-derived xenograft (PDX) MYC+ DLBCL model, a human-equivalent dose of selinexor impaired tumor growth (p< 0.05, vs. vehicle) without significant toxicities. Reduced tumor growth was secondary to apoptosis as determined by TUNEL (p<0.05). To characterize the biological effects of XPO1 inhibition, we compared the transcriptome of PDX from the previous experiment. Pathway analysis of differentially expressed genes revealed that selinexor induced pathways regulating innate immune signaling. The upregulation of key genes from this pathway (i.e. DDX58, DDX60, IRF7 and STAT1) was validated by qRT-PCR. Since unrepaired DNA damage elicits an innate immune response, we hypothesized that these effects could be mechanistically linked. To test this notion, we exposed MYC+ DLBCL cells to sub-lethal doses of the DNA damaging agent etoposide, selinexor, or their combination. Expression of innate immune genes was significantly higher in cells exposed to the combination compared to either drug alone (p<0.001, for both comparisons), indicating that selinexor increases the immunogenicity of MYC+ DLBCL cells and this effect is enhanced by concomitant replication stress. Furthermore, by analyzing primary DLBCLs we found that poorly immunogenic lymphomas, characterized by an immune deserted microenvironment, expressed higher levels of XPO1 and MYC compared to DLBCL with an immune rich microenvironment (P<0.001, for both genes). To determine a potential effect of selinexor in the function of immune effector cells, we performed single cells secretomic analysis (Isolight) of circulating CD8+ T cells obtained from two DLBCL patients before and after selinexor treatment and found that selinexor increases the secretory activity of CD8+ T cells in both patients. Overall, our study reveals a key oncogenic role for XPO1 in enabling DLBCL cells to tolerate MYC-induced replication stress while limiting their immunogenicity. This suggest that patients with MYC+ DLBCL and elevated genomic instability may benefit from therapeutic approaches based on XPO1 inhibitors in combination with immunomodulatory drugs, an approach we will test prospectively. Disclosures Rutherford: AstraZeneca: Consultancy; Celgene: Consultancy; Dova: Consultancy; Genentech/Roche: Research Funding; Heron: Consultancy; Juno: Consultancy; Karyopharm: Consultancy, Research Funding; Kite: Consultancy; LAM Therapeutics: Research Funding; Regeneron: Research Funding; Seattle Genetics: Consultancy. Martin:Regeneron: Consultancy; Cellectar: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy, Research Funding; Teneobio: Consultancy; Celgene: Consultancy; Bayer: Consultancy; Beigene: Consultancy; Sandoz: Consultancy; I-MAB: Consultancy; Morphosys: Consultancy; Kite: Consultancy; Incyte: Consultancy. Leonard:Miltenyi: Consultancy; Sutro: Consultancy; Roche/Genentech: Consultancy; BMS/Celgene: Consultancy; Regeneron: Consultancy; ADC Therapeutics: Consultancy; MEI Pharma: Consultancy; Bayer: Consultancy; Gilead/Kite: Consultancy; Karyopharm: Consultancy; GenMab: Consultancy; Epizyme: Consultancy; AstraZeneca: Consultancy. Cerchietti:Cellgene: Research Funding; Bristol Myers Squibb: Research Funding.

Recruitment of Host Nuclear Pore Components to the Vicinity of Theileria Schizonts.

Parasitic protozoans of the genus Theileria are intracellular pathogens that induce the cellular transformation of leukocytes, causing uncontrolled proliferation of the infected host cell. The transforming stage of the parasite has a strictly intracellular lifestyle and ensures its distribution to both daughter cells during host cell cytokinesis by aligning itself across the metaphase plate and by binding tightly to central spindle and astral microtubules. Given the importance of the parasite surface in maintaining interactions with host microtubules, we analyzed the ultrastructure of the host-parasite interface using transmission electron microscopy combined with high-resolution fluorescence microscopy and live-cell imaging. We show that porous membranes, termed annulate lamellae (AL), closely associate with the Theileria surface in infected T cells, B cells, and macrophages and are not detectable in noninfected bovine cell lines such as BL20 or BoMACs. AL are membranous structures found in the cytoplasm of fast-proliferating cells such as cancer cells, oocytes, and embryonic cells. Although AL were first observed more than 60 years ago, the function of these organelles is still not known. Indirect immunofluorescence analysis with a pan-nuclear pore complex antibody, combined with overexpression of a panel of nuclear pore proteins, revealed that the parasite recruits nuclear pore complex components close to its surface. Importantly, we show that, in addition to structural components of the nuclear pore complex, nuclear trafficking machinery, including importin beta 1, RanGAP1, and the small GTPase Ran, also accumulated close to the parasite surface.IMPORTANCETheileria schizonts are the only known eukaryotic organisms capable of transforming another eukaryotic cell; as such, probing of the interactions that occur at the host-parasite interface is likely to lead to novel insights into the cell biology underlying leukocyte proliferation and transformation. Little is known about how the parasite communicates with its host or by what route secreted parasite proteins are translocated into the host, and we propose that nuclear trafficking machinery at the parasite surface might play a role in this. The function of AL remains completely unknown, and our work provides a basis for further investigation into the contribution that these porous, cytomembranous structures might make to the survival of fast-growing transformed cells.

Topological data analysis quantifies biological nano-structure from single molecule localization microscopy.

MotivationLocalization microscopy data is represented by a set of spatial coordinates, each corresponding to a single detection, that form a point cloud. This can be analyzed either by rendering an image from these coordinates, or by analyzing the point cloud directly. Analysis of this type has focused on clustering detections into distinct groups which produces measurements such as cluster area, but has limited capacity to quantify complex molecular organization and nano-structure.ResultsWe present a segmentation protocol which, through the application of persistence-based clustering, is capable of probing densely packed structures which vary in scale. An increase in segmentation performance over state-of-the-art methods is demonstrated. Moreover we employ persistent homology to move beyond clustering, and quantify the topological structure within data. This provides new information about the preserved shapes formed by molecular architecture. Our methods are flexible and we demonstrate this by applying them to receptor clustering in platelets, nuclear pore components, endocytic proteins and microtubule networks. Both 2D and 3D implementations are provided within RSMLM, an R package for pointillist-based analysis and batch processing of localization microscopy data.Availability and implementationRSMLM has been released under the GNU General Public License v3.0 and is available at https://github.com/JeremyPike/RSMLM. Tutorials for this library implemented as Binder ready Jupyter notebooks are available at https://github.com/JeremyPike/RSMLM-tutorials.Supplementary information Supplementary data are available at Bioinformatics online.

The nuclear pore proteins Nup88/214 and T-cell acute lymphatic leukemia–associated NUP214 fusion proteins regulate Notch signaling

The Notch receptor is a key mediator of developmental programs and cell-fate decisions. Imbalanced Notch signaling leads to developmental disorders and cancer. To fully characterize the Notch signaling pathway and exploit it in novel therapeutic interventions, a comprehensive view on the regulation and requirements of Notch signaling is needed. Notch is regulated at different levels, ranging from ligand binding, stability to endocytosis. Using an array of different techniques, including reporter gene assays, immunocytochemistry, and ChIP-qPCR we show here, to the best of our knowledge for the first time, regulation of Notch signaling at the level of the nuclear pore. We found that the nuclear pore protein Nup214 (nucleoporin 214) and its interaction partner Nup88 negatively regulate Notch signaling in vitro and in vivo in zebrafish. In mammalian cells, loss of Nup88/214 inhibited nuclear export of recombination signal-binding protein for immunoglobulin κJ region (RBP-J), the DNA-binding component of the Notch pathway. This inhibition increased binding of RBP-J to its cognate promoter regions, resulting in increased downstream Notch signaling. Interestingly, we also found that NUP214 fusion proteins, causative for certain cases of T-cell acute lymphatic leukemia, potentially contribute to tumorigenesis via a Notch-dependent mechanism. In summary, the nuclear pore components Nup88/214 suppress Notch signaling in vitro, and in zebrafish, nuclear RBP-J levels are rate-limiting factors for Notch signaling in mammalian cells, and regulation of nucleocytoplasmic transport of RBP-J may contribute to fine-tuning Notch activity in cells.

Chromatin targeting of nuclear pore proteins induces chromatin decondensation.

Nuclear pore complexes have emerged in recent years as chromatin-binding nuclear scaffolds, able to influence target gene expression. However, how nucleoporins (Nups) exert this control remains poorly understood. Here we show that ectopically tethering Drosophila Nups, especially Sec13, to chromatin is sufficient to induce chromatin decondensation. This decondensation is mediated through chromatin-remodeling complex PBAP, as PBAP is both robustly recruited by Sec13 and required for Sec13-induced decondensation. This phenomenon is not correlated with localization of the target locus to the nuclear periphery, but is correlated with robust recruitment of Nup Elys. Furthermore, we identified a biochemical interaction between endogenous Sec13 and Elys with PBAP, and a role for endogenous Elys in global as well as gene-specific chromatin decompaction. Together, these findings reveal a functional role and mechanism for specific nuclear pore components in promoting an open chromatin state.

The Nup84 complex coordinates the DNA damage response to warrant genome integrity.

DNA lesions interfere with cellular processes such as transcription and replication and need to be adequately resolved to warrant genome integrity. Beyond their primary role in molecule transport, nuclear pore complexes (NPCs) function in other processes such as transcription, nuclear organization and DNA double strand break (DSB) repair. Here we found that the removal of UV-induced DNA lesions by nucleotide excision repair (NER) is compromised in the absence of the Nup84 nuclear pore component. Importantly, nup84Δ cells show an exacerbated sensitivity to UV in early S phase and delayed replication fork progression, suggesting that unrepaired spontaneous DNA lesions persist during S phase. In addition, nup84Δ cells are defective in the repair of replication-born DSBs by sister chromatid recombination (SCR) and rely on post-replicative repair functions for normal proliferation, indicating dysfunctions in the cellular pathways that enable replication on damaged DNA templates. Altogether, our data reveal a central role of the NPC in the DNA damage response to facilitate replication progression through damaged DNA templates by promoting efficient NER and SCR and preventing chromosomal rearrangements.

Organelle Segregation and Quality Control during Meiotic Differentiation

Meiotic differentiation is a complex developmental program that involves extensive cellular remodeling. In budding yeast, under nutrient deprived conditions, a diploid mother cell undergoes two successive meiotic divisions to generate four nuclei. After meiosis, the four nuclei are packaged with a subset of the organelles and cytoplasm originating from the mother to form four haploid gametes. Our lab has found that during the latter process, certain components of the mother cell are not inherited by the gametes and are subsequently produced de novo. For example, the nuclear pore complexes and components of the nucleoli do not segregate with any of the four nuclei, whereas only a subset of the mitochondria is inherited by the gametes. A programmed pathway that entails vacuole permeabilization subsequently degrades the excluded cellular materials. We posit that the restricted inheritance of organelles and other cellular sub‐compartments is required for cellular rejuvenation that occurs as part of gametogenesis.Support or Funding InformationPew Charitable Trusts (00027344), Damon Runyon Cancer Research Foundation (35‐15), March of Dimes (5‐FY15‐99), National Institute of Health (DP2 AG055946‐01) and Glenn Foundation for Medical ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Colorectal cancer cells require glycogen synthase kinase-3β for sustaining mitosis via translocated promoter region (TPR)-dynein interaction.

Glycogen synthase kinase (GSK) 3β, which mediates fundamental cellular signaling pathways, has emerged as a potential therapeutic target for many types of cancer including colorectal cancer (CRC). During mitosis, GSK3β localizes in mitotic spindles and centrosomes, however its function is largely unknown. We previously demonstrated that translocated promoter region (TPR, a nuclear pore component) and dynein (a molecular motor) cooperatively contribute to mitotic spindle formation. Such knowledge encouraged us to investigate putative functional interactions among GSK3β, TPR, and dynein in the mitotic machinery of CRC cells. Here, we show that inhibition of GSK3β attenuated proliferation, induced cell cycle arrest at G2/M phase, and increased apoptosis of CRC cells. Morphologically, GSK3β inhibition disrupted chromosome segregation, mitotic spindle assembly, and centrosome maturation during mitosis, ultimately resulting in mitotic cell death. These changes in CRC cells were associated with decreased expression of TPR and dynein, as well as disruption of their functional colocalization with GSK3β in mitotic spindles and centrosomes. Clinically, we showed that TPR expression was increased in CRC databases and primary tumors of CRC patients. Furthermore, TPR expression in SW480 cells xenografted into mice was reduced following treatment with GSK3β inhibitors. Together, these results indicate that GSK3β sustains steady mitotic processes for proliferation of CRC cells via interaction with TPR and dynein, thereby suggesting that the therapeutic effect of GSK3β inhibition depends on induction of mitotic catastrophe in CRC cells.

Tau Protein Disrupts Nucleocytoplasmic Transport in Alzheimerrs Disease

Tau protein, which normally functions to stabilize microtubules, is the major constituent of neurofibrillary tangles in Alzheimer’s disease (AD). The mechanism underlying tau-associated neural damage remains unclear. We now show that tau can interact with nuclear pore complex (NPC) constituents and affect their structural and functional integrity. Pathological tau leads to dissociation of nuclear pore complex proteins, and impairs nuclear export and import in vitro, in tau overexpressing transgenic mouse models, and in human AD tissue. Moreover, a nuclear pore component, Nup98, surprisingly colocalizes with neurofibrillary tangles in neuronal soma, and both in vivo and in vitro directly interacts with tau to greatly facilitate its aggregation. These data support the hypothesis that tau directly interacts with nuclear pore complex constituents, leading to their mislocalization and to disruption of nuclear pore function, raising the possibility that nuclear pore dysfunction contributes to tau-induced neurotoxicity in Alzheimer’s disease.

The function of the inner nuclear envelope protein SUN1 in mRNA export is regulated by phosphorylation

SUN1, a component of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, functions in mammalian mRNA export through the NXF1-dependent pathway. It associates with mRNP complexes by direct interaction with NXF1. It also binds to the NPC through association with the nuclear pore component Nup153, which is involved in mRNA export. The SUN1-NXF1 association is at least partly regulated by a protein kinase C (PKC) which phosphorylates serine 113 (S113) in the N-terminal domain leading to reduced interaction. The phosphorylation appears to be important for the SUN1 function in nuclear mRNA export since GFP-SUN1 carrying a S113A mutation was less efficient in restoring mRNA export after SUN1 knockdown as compared to the wild type protein. By contrast, GFP-SUN1-S113D resembling the phosphorylated state allowed very efficient export of poly(A)+RNA. Furthermore, probing a possible role of the LINC complex component Nesprin-2 in this process we observed impaired mRNA export in Nesprin-2 knockdown cells. This effect might be independent of SUN1 as expression of a GFP tagged SUN-domain deficient SUN1, which no longer can interact with Nesprin-2, did not affect mRNA export.

Abstract A46: Nup153 and Nup50 promote recruitment of 53BP1 to DNA repair foci by antagonizing BRCA1-dependent events

Abstract This study focuses on an unexpected role for particular nuclear pore proteins in the antagonism between the DNA repair factors BRCA1 and 53BP1, and the impact this has on the response to poly(A) ribose polymerase (PARP) inhibition. Repair of DNA double strand breaks, a cornerstone of genomic integrity, typically follows one of two distinct pathways: the high fidelity process of homologous recombination (HR) repair, in which BRCA1 plays a key role, and the more error-prone process of non-homologous end joining (NHEJ), which relies on 53BP1. The balance between NHEJ and HR depends, in part, on whether 53BP1 predominates in binding to damage sites, where it protects the DNA ends from resection and prevents HR from occurring. Conversely, BRCA1 binding counteracts 53BP1-mediated events to promote HR repair. While mutual antagonism between BRCA1 and 53BP1 has been appreciated for some time, factors that modulate this relationship are only beginning to be elucidated. The nuclear pore component Nup153 has previously been implicated in the DNA damage response, specifically in promoting nuclear import of 53BP1. To further explore the role of Nup153 in this context, we engineered an additional strong nuclear localization sequence (NLS) on 53BP1 and found that this overrides the requirement for Nup153 in nuclear import of 53BP1. While 53BP1-NLS is recruited normally to DNA damage foci in control-treated cells, its accumulation at these sites is still severely impaired in Nup153-depleted cells, indicating that Nup153 has an additional and distinct role in 53BP1 intranuclear targeting. These observations are further underscored by the additional finding that a second nuclear pore protein, Nup50, is also required for intranuclear targeting of 53BP1, yet makes no discernable contribution to 53BP1 nuclear import. Of note, depletion of a third nucleoporin (Tpr), which is part of the same structural element of the nuclear pore, did not alter this DNA damage response pathway. To gain insight into the pathway affected by Nup153 and Nup50 and to understand how their role fits into a clinically relevant context, we probed the consequences of reducing Nup153 and Nup50 levels in cells deficient in BRCA1 activity. We found that the requirement for Nup153 and Nup50 in promoting 53BP1 recruitment to damage foci is abrogated under these circumstances. In cells where HR repair is otherwise defective, however, robust recruitment of 53BP1 to DNA damage foci requires Nup153 and Nup50. Together, our results provide novel insight into 53BP1 regulation during the DNA damage response and are consistent with a model where Nup153 and Nup50 normally promote 53BP1 targeting by counteracting BRCA1-dependent events. These findings have important implications with regard to PARP inhibition, a therapeutic strategy thought to capitalize on defective HR repair by forcing inappropriate use of NHEJ, culminating in cellular toxicity. Accordingly, we found that decreased levels of Nup153 and Nup50 do not affect the 53BP1-dependent pathway in BRCA1-deficient cells exposed to PARP inhibitor treatment, but still impair this response in cells deficient for BRCA2 or when the ATM and ATR signaling pathways are inhibited. Our results indicate that Nup153 and Nup50, as factors that modulate 53BP1 activity, belong to a novel category of biomarkers that could be screened to make better predictions of response to PARP inhibitor treatment. Citation Format: Douglas R. Mackay, Katharine S. Ullman. Nup153 and Nup50 promote recruitment of 53BP1 to DNA repair foci by antagonizing BRCA1-dependent events [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A46.