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Viruses frequently exploit the host's nucleocytoplasmic trafficking machinery to facilitate their replication and evade immune defenses. By encoding specialized proteins and other components, they strategically target host nuclear transport receptors (NTRs) and nucleoporins within the spiderweb-like inner channel of the nuclear pore complex (NPC), enabling efficient access to the host nucleus. This review explores the intricate mechanisms governing the nuclear import and export of viral components, with a focus on the interplay between viral factors and host determinants that are essential for these processes. Given the pivotal role of nucleocytoplasmic shuttling in the viral life cycle, we also examine therapeutic strategies aimed at disrupting the host's nuclear transport pathways. This includes evaluating the efficacy of pharmacological inhibitors in impairing viral replication and assessing their potential as antiviral treatments. Furthermore, we emphasize the need for continued research to develop targeted therapies that leverage vulnerabilities in nucleocytoplasmic trafficking. Emerging high-resolution techniques, such as advanced imaging and computational modeling, are transforming our understanding of the dynamic interactions between viruses and the NPC. These cutting-edge tools are driving progress in identifying novel therapeutic opportunities and uncovering deeper insights into viral pathogenesis. This review highlights the importance of these advancements in paving the way for innovative antiviral strategies. [ABSTRACT FROM AUTHOR]

Adeno-associated viruses (AAVs) are the most extensively researched viral vectors for gene therapy globally. The AAV viral protein 1 (VP1) N-terminus controls the capsid's ability to translocate into the cell nucleus; however, the exact mechanism of this process is largely unknown. In this study, we sought to elucidate the precise interactions between AAV serotype 6 (AAV6), a promising vector for immune disorders, and host transport receptors responsible for vector nuclear localization. Focusing on the positively charged basic areas within the N-terminus of AAV6 VP1, we identified a 53-amino acid region that interacts with nuclear import receptors. We measured the binding affinities between this region and various nuclear import receptors, discovering a notably strong interaction with IMPa5 and IMPa7 in the low nanomolar range. We also elucidated the X-ray crystal structure of this region in complex with an importin alpha (IMPa) isoform, uncovering its binding as a bipartite nuclear localization signal (NLS). Furthermore, we show that using this bipartite NLS, AAV6 VP1 capsid protein can localize to the nucleus of mammalian cells in a manner dependent on the IMPa/IMPß nuclear import pathway. This study provides detailed insights into the interaction between the AAV6 VP1 capsid protein and nuclear import receptors, deepening our knowledge of AAV nuclear import mechanisms and establishing a basis for the improvement of AAV6-based gene therapy vectors. IMPORTANCE AAVs, recognized as the most extensively researched viral vectors for gene therapy globally, offer significant advantages over alternatives due to their small size, non-pathogenic nature, and innate ability for tissue-specific targeting. AAVs are required to localize to the nucleus to perform their role as a gene therapy vector; however, the precise mechanisms that facilitate this process remain unknown. Despite sharing overt genomic similarities with AAV1 and AAV2, AAV6 is a unique serotype. It is currently recognized for its ability to effectively transduce hematopoietic cell lineages and, consequently, is considered promising for the treatment of immune disorders. Identifying the exact mechanisms that permit AAV6 to access the nucleus can open up new avenues for gene therapy vector engineering, which can ultimately lead to increased therapeutic benefits. [ABSTRACT FROM AUTHOR]

The USH1G protein SANS is a small multifunctional scaffold protein. It is involved in several different cellular processes, such as intracellular transport, in the cytoplasm, or splicing of pre-mRNA, in the cell nucleus. Here, we aimed to gain insight into the regulation of the subcellular localization and the nuclear–cytoplasmic shuttling of SANS and its paralog ANKS4B, not yet reported in the nucleus. We identified karyopherins mediating the nuclear import and export by screening the nuclear interactome of SANS. Sequence analyses predicted in silico evolutionarily conserved nuclear localization sequences (NLSs) and nuclear export sequences (NESs) in SANS, but only NESs in ANKS4B, which are suitable for karyopherin binding. Quantifying the nuclear–cytoplasmic localization of wild-type SANS and NLS/NES mutants, we experimentally confirmed in silico predicted NLS and NES functioning in the nuclear–cytoplasmic shuttling in situ in cells. The comparison of SANS and its paralog ANKS4B revealed substantial differences in the interaction with the nuclear splicing protein PRPF31 and in their nuclear localization. Finally, our results on pathogenic USH1G/SANS mutants suggest that the loss of NLSs and NESs and thereby the ability to control nuclear–cytoplasmic shuttling is disease-relevant. [ABSTRACT FROM AUTHOR]

The nuclear transport of proteins plays an important role in mediating the transition from egg to embryo and distinct karyopherins have been implicated in this process. Here, we studied the impact of KPNA2 deficiency on preimplantation embryo development in mice. Loss of KPNA2 results in complete arrest at the 2cell stage and embryos exhibit the inability to activate their embryonic genome as well as a severely disturbed nuclear translocation of Nucleoplasmin 2. Our findings define KPNA2 as a new maternal effect gene. [ABSTRACT FROM AUTHOR]

Coordination and information exchange among the various organelles ensure the precise and orderly functioning of eukaryotic cells. Interaction between the cytoplasm and nucleoplasm is crucial for many physiological processes. Macromolecular protein transport into the nucleus requires assistance from the nuclear transport system. These proteins typically contain a nuclear localisation sequence that guides them to enter the nucleus. Understanding the mechanism of nuclear import of macromolecular proteins is important for comprehending cellular processes. Investigation of disease-related alterations can facilitate the development of novel therapeutic strategies and provide additional evidence for clinical trials. This review provides an overview of the proteins involved in nuclear transport and the mechanisms underlying macromolecular protein transport. [ABSTRACT FROM AUTHOR]

Selective transport through the nuclear pore complex (NPC) depends on the dynamic binding of FG-repeat containing nucleoporins, the FG-nups, with each other and with Karyopherins (Kaps). Here, we assessed the specificity and mechanism by which the aliphatic alcohol 1,6-hexanediol (1,6HD) disrupts the permeability barrier of NPCs in live baker's yeast cells. After a 10-minute exposure to 5% 1,6HD, no notable changes were observed in cell growth, cytosolic pH and ATP levels, or the appearance of organelles. However, effects on the cytoskeleton and Hsp104 were noted. 1,6HD clearly affected the NPC permeability barrier, allowing passive nuclear entry of a 177kDa reporter protein that is normally confined to the cytosol. Moreover, multiple Kaps were displaced from NPCs, and the displacement of Kap122-GFP correlated with the observed passive permeability changes. 1,6HD thus temporarily permeates NPCs, and in line with Kap-centric models, the mechanism includes the release of numerous Kaps from the NPCs. [ABSTRACT FROM AUTHOR]

Epidermal growth factor receptor (EGFR) is transported into the nucleus during irradiation therapy and can cause the radiation resistance. It has been shown that irradiation activates EGFR transport to the nuclear by karyopherin alpha (KPNA) in the classical pathway, whereas ligand‐activated EGFR binds with karyopherin subunit beta 1 (KPNB1). Phosphorylation of T654 located within the nuclear localization signal (NLS) of EGFR is known to activate radiation‐induced nuclear EGFR transport. Therefore, we investigated the binding affinities between the NLS peptides of EGFR (645RRRHIVRKRXLRR657) and KPNA using a single‐molecule pull‐down assay in this study. We also showed the possible assay system for developing small molecules to modulate nuclear EGFR transport by high throughput screening, which potentially reduces the resistance during radiation therapy. [ABSTRACT FROM AUTHOR]

One of the key features of eukaryotic cells is the separation of nuclear and cytoplasmic compartments by a double-layer nuclear envelope. This separation is crucial for timely regulation of gene expression, mRNA biogenesis, cell cycle, and differentiation. Since transcription takes place in the nucleus and the major part of translation in the cytoplasm, proper distribution of biomolecules between these two compartments is ensured by nucleocytoplasmic shuttling proteins – karyopherins. Karyopherins transport biomolecules through nuclear pores bidirectionally in collaboration with Ran GTPases and utilize GTP as the source of energy. Different karyopherins transport different cargo molecules that play important roles in the regulation of cell physiology. In cancer cells, this nucleocytoplasmic transport is significantly dysregulated to support increased demands for the import of cell cycle-promoting biomolecules and export of cell cycle inhibitors and mRNAs. Here, we analysed genomic, transcriptomic and proteomic data from published datasets to comprehensively profile karyopherin genes in hepatocellular carcinoma. We have found out that expression of multiple karyopherin genes is increased in hepatocellular carcinoma in comparison to the normal liver, with importin subunit α-1, exportin 2, importin subunit β-1 and importin 9 being the most over-expressed. Moreover, we have found that increased expression of these genes is associated with higher neoplasm grade as well as significantly worse overall survival of liver cancer patients. Taken together, our bioinformatic data-mining analysis provides a comprehensive genomic and transcriptomic landscape of karyopherins in hepatocellular carcinoma and identifies potential members that could be targeted in order to develop new treatment regimens. [ABSTRACT FROM AUTHOR]

We report the crystal structure of nuclear import receptor Importin-9 bound to its cargo, the histones H2A-H2B. Importin-9 wraps around the core, globular region of H2A-H2B to form an extensive interface. The nature of this interface coupled with quantitative analysis of deletion mutants of H2A-H2B suggests that the NLS-like sequences in the H2A-H2B tails play a minor role in import. Importin-9•H2A-H2B is reminiscent of interactions between histones and histone chaperones in that it precludes H2A-H2B interactions with DNA and H3-H4 as seen in the nucleosome. Like many histone chaperones, which prevent inappropriate non-nucleosomal interactions, Importin-9 also sequesters H2A-H2B from DNA. Importin-9 appears to act as a storage chaperone for H2A-H2B while escorting it to the nucleus. Surprisingly, RanGTP does not dissociate Importin-9•H2A-H2B but assembles into a RanGTP•Importin-9•H2A-H2B complex. The presence of Ran in the complex, however, modulates Imp9-H2A-H2B interactions to facilitate its dissociation by DNA and assembly into a nucleosome.

In budding yeast, a collar of septin filaments at the neck between a mother cell and its bud marks the incipient site for cell division and serves as a scaffold that recruits proteins required for proper spatial and temporal execution of cytokinesis. A set of interacting proteins that localize at or near the bud neck, including Aim44/Gps1, Nba1 and Nis1, also has been implicated in preventing Cdc42-dependent bud site re-establishment at the division site. We found that, at their endogenous level, Aim44 and Nis1 robustly localize sequentially at the septin collar. Strikingly, however, when overproduced, both proteins shift their subcellular distribution predominantly to the nucleus. Aim44 localizes with the inner nuclear envelope, as well as at the plasma membrane, whereas Nis1 accumulates within the nucleus, indicating that these proteins normally undergo nucleocytoplasmic shuttling. Of the 14 yeast karyopherins, Kap123/Yrb4 is the primary importin for Aim44, whereas several importins mediate Nis1 nuclear entry. Conversely, Kap124/Xpo1/Crm1 is the primary exportin for Nis1, whereas both Xpo1 and Cse1/Kap109 likely contribute to Aim44 nuclear export. Even when endogenously expressed, Nis1 accumulates in the nucleus when Nba1 is absent. When either Aim44 or Nis1 are overexpressed, Nba1 is displaced from the bud neck, further consistent with the mutual interactions of these proteins. Collectively, our results indicate that a previously unappreciated level at which localization of septin-associated proteins is controlled is via regulation of their nucleocytoplasmic shuttling, which places constraints on their availability for complex formation with other partners at the bud neck.

Human exposure to arsenicals is associated with devastating diseases such as cancer and neurodegeneration. At the same time, arsenic-based drugs are used as therapeutic agents. The ability of arsenic to directly bind to proteins is correlated with its toxic and therapeutic effects highlighting the importance of elucidating arsenic-protein interactions. In this study, we took a proteomic approach and identified 174 proteins that bind to arsenic in Saccharomyces cerevisiae . Proteins involved in nucleocytoplasmic transport were markedly enriched among the arsenic-binding proteins, and we demonstrate that arsenic-binding to nuclear import factors results in their relocation from the nuclear envelope and subsequent aggregation in the cytosol. Similarly, nuclear pore proteins that make up the nuclear pore complex mislocalized and aggregated in arsenic-exposed cells. Consequently, arsenic was shown to inhibit nuclear protein import and export. We propose a model in which arsenic-binding to nuclear transport factors leads to their mislocalization and aggregation, which disrupts nucleocytoplasmic transport and causes arsenic sensitivity., Competing Interests: Conflict of interest The authors declare no conflicts of interest.

Abstract Background Swi6 acts as a transcription factor in budding yeast, functioning in two different heterodimeric complexes, SBF and MBF, that activate the expression of distinct but overlapping sets of genes. Swi6 undergoes regulated changes in nucleocytoplasmic localization throughout the cell cycle that correlate with changes in gene expression. This study investigates how nucleocytoplasmic transport by multiple transport factors may influence specific Swi6 activities. Results Here we show that the exportin Crm1 is important for Swi6 nuclear export and activity. Loss of a putative Crm1 NES or inhibition of Crm1 activity results in changes in nucleocytoplasmic Swi6 localization. Alteration of the Crm1 NES in Swi6 results in decreased MBF-mediated gene expression, but does not affect SBF reporter expression, suggesting that export of Swi6 by Crm1 regulates a subset of Swi6 transcription activation activity. Finally, alteration of the putative Crm1 NES in Swi6 results in cells that are larger than wild type, and this increase in cell size is exacerbated by deletion of Msn5. Conclusions These data provide evidence that Swi6 has at least two different exportins, Crm1 and Msn5, each of which interacts with a distinct nuclear export signal. We identify a putative nuclear export signal for Crm1 within Swi6, and observe that export by Crm1 or Msn5 independently influences Swi6-regulated expression of a different subset of Swi6-controlled genes. These findings provide new insights into the complex regulation of Swi6 transcription activation activity and the role of nucleocytoplasmic shuttling in regulated gene expression.

Appropriate nucleo-cytoplasmic partitioning of proteins is a vital regulatory mechanism in phytohormone signaling and plant development. However, how this is achieved remains incompletely understood. The Karyopherin (KAP) superfamily is critical for separating the biological processes in the nucleus from those in the cytoplasm. The KAP superfamily is divided into Importin α (IMPα) and Importin β (IMPβ) families and includes the core components in mediating nucleocytoplasmic transport. Recent reports suggest the KAPs play crucial regulatory roles in Arabidopsis development and stress response by regulating the nucleo-cytoplasmic transport of members in hormone signaling. However, the KAP members and their associated molecular mechanisms are still poorly understood in maize. Therefore, we first identified seven IMPα and twenty-seven IMPβ genes in the maize genome and described their evolution traits and the recognition rules for substrates with nuclear localization signals (NLSs) or nuclear export signals (NESs) in plants. Next, we searched for the protein interaction partners of the ZmKAPs and selected the ones with Arabidopsis orthologs functioning in auxin biosynthesis, transport, and signaling to predict their potential function. Finally, we found that several ZmKAPs share similar expression patterns with their interacting proteins, implying their function in root development. Overall, this article focuses on the Karyopherin superfamily in maize and starts with this entry point by systematically comprehending the KAP-mediated nucleo-cytoplasmic transport process in plants, and then predicts the function of the ZmKAPs during maize development, with a perspective on a closely associated regulatory mechanism between the nucleo-cytoplasmic transport and the phytohormone network. [ABSTRACT FROM AUTHOR]

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of banana wilt disease, is one of the most devastating pathogens of bananas (Musa spp.). The nucleocytoplasmic trafficking of proteins and RNAs, mediated by karyopherins (Kaps), is essential for eukaryotes. However, little is known about how Kaps regulate fungal growth and pathogenicity. Here, a Kap119 homolog (FocKap119) was identified in the Foc tropical race 4 strain Foc302. Deletion of FocKap119 led to impaired vegetative growth, conidiation, cell wall integrity, and pathogenicity. Compared to the wild type, the Δ FocKap119 mutant showed increased sensitivity to oxidative stress but greater tolerant to osmotic stress. FocKap119 was found to interact with FocCks1 (cell cycle-dependent kinase subunit 1) in a yeast two-hybrid system, and deletion of FocCks1 showed similar phenotypes as Δ FocKap119. Moreover, whole-transcriptome analysis and qRT-PCR results indicated that deletion of FocKap119 resulted in down-regulation of genes related to key virulence factor biosynthesis, including fusaric acid and beauvericin biosynthetic gene clusters. The Δ FocKap119 mutant also triggered stronger plant defense responses during the early infection compared to the wild type. This study provides insights into how FocKap119 regulated growth, abiotic stress response, and pathogenicity in Foc. • Knockout of FocKap119 could significantly reduce the virulence of Fusarium oxysporum f. sp. cubense. • FocKap119 was involved in regulation of cell wall integrity and toxin biosynthesis. • FocKap119 modulated host defense gene expression during colonization. • The FocKap119-FocCks1 interaction seemed to be involved in regulating fungal growth, stress response, and pathogenicity. [ABSTRACT FROM AUTHOR]

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Background: Swi6 acts as a transcription factor in budding yeast, functioning in two different heterodimeric complexes, SBF and MBF, that activate the expression of distinct but overlapping sets of genes. Swi6 undergoes regulated changes in nucleocytoplasmic localization throughout the cell cycle that correlate with changes in gene expression. This study investigates how nucleocytoplasmic transport by multiple transport factors may influence specific Swi6 activities. Results: Here we show that the exportin Crm1 is important for Swi6 nuclear export and activity. Loss of a putative Crm1 NES or inhibition of Crm1 activity results in changes in nucleocytoplasmic Swi6 localization. Alteration of the Crm1 NES in Swi6 results in decreased MBF-mediated gene expression, but does not affect SBF reporter expression, suggesting that export of Swi6 by Crm1 regulates a subset of Swi6 transcription activation activity. Finally, alteration of the putative Crm1 NES in Swi6 results in cells that are larger than wild type, and this increase in cell size is exacerbated by deletion of Msn5. Conclusions: These data provide evidence that Swi6 has at least two different exportins, Crm1 and Msn5, each of which interacts with a distinct nuclear export signal. We identify a putative nuclear export signal for Crm1 within Swi6, and observe that export by Crm1 or Msn5 independently influences Swi6-regulated expression of a different subset of Swi6-controlled genes. These findings provide new insights into the complex regulation of Swi6 transcription activation activity and the role of nucleocytoplasmic shuttling in regulated gene expression. [ABSTRACT FROM AUTHOR]

Viral structural proteins can have multiple activities. Antivirals that target structural proteins have potential to exhibit multiple antiviral mechanisms. Hepatitis B virus (HBV) core protein (Cp) is involved in most stages of the viral life cycle; it assembles into capsids, packages viral RNA, is a metabolic compartment for reverse transcription, interacts with nuclear trafficking machinery, and disassembles to release the viral genome into the nucleus. During nuclear localization, HBV capsids bind to host importins (e.g., Imp b) via Cp's C-terminal domain (CTD); the CTD is localized to the interior of the capsid and is transiently exposed on the exterior. We used HAP12 as a representative Cp allosteric modulator (CpAM), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV life cycle is poorly understood. We investigate how HAP12 influences the interactions between empty or RNA-filled capsids with Imp β and trypsin in vitro. We show that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrate that Imp b synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize the disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resist the destabilizing effects of HAP12 and Imp b. In summary, we show host protein-induced catalysis of capsid disruption, an unexpected additional mechanism of action for CpAMs. Potentially, untimely capsid disassembly can hamper the HBV life cycle and also cause the virus to become vulnerable to host innate immune responses. [ABSTRACT FROM AUTHOR]

The nucleotide-binding and leucine-rich repeat (NLR) proteins comprise a major class of intracellular immune receptors that are capable of detecting pathogen-derived molecules and activating immunity and cell death in plants. The activity of some NLRs, particularly the Toll-like/interleukin-1 receptor (TIR) type, is highly correlated with their nucleocytoplasmic distribution. However, whether and how the nucleocytoplasmic homeostasis of NLRs is coordinated through a bidirectional nuclear shuttling mechanism remains unclear. Here, we identified a nuclear transport receptor, KA120, which is capable of affecting the nucleocytoplasmic distribution of an NLR protein and is essential in preventing its autoactivation. We showed that the ka120 mutant displays an autoimmune phenotype and NLR-induced transcriptome features. Through a targeted genetic screen using an artificial NLR microRNA library, we identified the TIR-NLR gene SNC1 as a genetic interactor of KA120. Loss-of-function snc1 mutations as well as compromising SNC1 protein activities all substantially suppressed ka120 -induced autoimmune activation, and the enhanced SNC1 activity upon loss of KA120 functionappeared to occur at the protein level. Overexpression of KA120 efficiently repressed SNC1 activity and led to a nearly complete suppression of the autoimmune phenotype caused by the gain-of-function snc1-1 mutation or SNC1 overexpression in transgenic plants. Further florescence imaging analysis indicated that SNC1 undergoes altered nucleocytoplasmic distribution with significantly reduced nuclear signal when KA120 is constitutively expressed, supporting a role of KA120 in coordinating SNC1 nuclear abundance and activity. Consistently, compromising the SNC1 nuclear level by disrupting the nuclear pore complex could also partially rescue ka120 -induced autoimmunity. Collectively, our study demonstrates that KA120 is essential to avoid autoimmune activation in the absence of pathogens and is required to constrain the nuclear activity of SNC1, possibly through coordinating SNC1 nucleocytoplasmic homeostasis as a potential mechanism. This study identified a nuclear transport receptor, KA120, which is essential for repressing NLR-mediated immune activation in plants. This function of KA120 occurs at least partially by constraining the nuclear activity of SNC1 protein. [ABSTRACT FROM AUTHOR]

Neurodegenerative proteinopathies are characterised by progressive cell loss that is preceded by the mislocalisation and aberrant accumulation of proteins prone to aggregation. Despite their different physiological functions, disease-related proteins like tau, alpha-synuclein, Tar DNA binding protein-43, Fused in sarcoma and mutant Huntingtin, all share low complexity regions that can mediate their liquid-liquid phase transitions. The proteins' phase transitions can range from native monomers to soluble oligomers, liquid droplets and further to irreversible, often-mislocalised aggregates that characterise the stages and severity of neurodegenerative diseases. Recent advances into the underlying pathogenic mechanisms have associated mislocalisation and aberrant accumulation of disease-related proteins with defective nucleocytoplasmic transport and its mediators called karyopherins. These studies identify karyopherin abnormalities in amyotrophic lateral sclerosis, frontotemporal dementia, Alzheimer's disease, and synucleinopathies including Parkinson's disease and dementia with Lewy bodies, that range from altered expression levels to the subcellular mislocalisation and aggregation of karyopherin alpha and beta proteins. The reported findings reveal that in addition to their classical function in nuclear import and export, karyopherins can also act as chaperones by shielding aggregation-prone proteins against misfolding, accumulation and irreversible phase-transition into insoluble aggregates. Karyopherin abnormalities can, therefore, be both the cause and consequence of protein mislocalisation and aggregate formation in degenerative proteinopathies. The resulting vicious feedback cycle of karyopherin pathology and proteinopathy identifies karyopherin abnormalities as a common denominator of onset and progression of neurodegenerative disease. Pharmacological targeting of karyopherins, already in clinical trials as therapeutic intervention targeting cancers such as glioblastoma and viral infections like COVID-19, may therefore represent a promising new avenue for disease-modifying treatments in neurodegenerative proteinopathies. [ABSTRACT FROM AUTHOR]

Spermatogenesis is driven by an ordered series of events, which rely on trafficking of specific proteins between nucleus and cytoplasm. The karyopherin α family of proteins mediates movement of specific cargo proteins when bound to karyopherin β. Karyopherin α genes have distinct expression patterns in mouse testis, implying they may have unique roles during mammalian spermatogenesis. Here, we use a loss-of-function approach to determine specifically the role of Kpna6 in spermatogenesis and male fertility.We show that ablation of Kpna6 in male mice leads to infertility and has multiple cumulative effects on both germ cells and Sertoli cells. Kpna6-deficient mice exhibit impaired Sertoli cell function, including loss of Sertoli cells and a compromised nuclear localization of the androgen receptor. Furthermore, our data demonstrate devastating defects on spermiogenesis, including incomplete sperm maturation and a massive reduction in sperm number, accompanied by disturbed histone-protamine exchange, differential localization of the transcriptional regulator BRWD1 and altered expression of RFX2 target genes. Our work uncovers an essential role of Kpna6 in spermatogenesis and, hence, in male fertility. [ABSTRACT FROM AUTHOR]

Abstract Background Members of the karyopherin superfamily serve as nuclear transport receptors/adaptor proteins and provide exchange of macromolecules between the nucleo- and cytoplasm. Emerging evidence suggests a subset of karyopherins to be dysregulated in hepatocarcinogenesis including karyopherin-α2 (KPNA2). However, the functional and regulatory role of KPNA2 in liver cancer remains incompletely understood. Methods Quantitative proteomics (LC-MS/MS, ~ 1750 proteins in total) was used to study changes in global protein abundance upon siRNA-mediated KPNA2 knockdown in HCC cells. Functional and mechanistic analyses included colony formation and 2D migration assays, co-immunoprecipitation (CoIP), chromatin immunoprecipitation (ChIP), qRT-PCR, immmunblotting, and subcellular fractionation. In vitro results were correlated with data derived from a murine HCC model and HCC patient samples (3 cohorts, n > 600 in total). Results The proteomic approach revealed the pro-tumorigenic, microtubule (MT) interacting protein stathmin (STMN1) among the most downregulated proteins upon KPNA2 depletion in HCC cells. We further observed that KPNA2 knockdown leads to reduced tumor cell migration and colony formation of HCC cells, which could be phenocopied by direct knockdown of stathmin. As the underlying regulatory mechanism, we uncovered E2F1 and TFDP1 as transport substrates of KPNA2 being retained in the cytoplasm upon KPNA2 ablation, thereby resulting in reduced STMN1 expression. Finally, murine and human HCC data indicate significant correlations of STMN1 expression with E2F1/TFPD1 and with KPNA2 expression and their association with poor prognosis in HCC patients. Conclusion Our data suggest that KPNA2 regulates STMN1 by import of E2F1/TFDP1 and thereby provide a novel link between nuclear transport and MT-interacting proteins in HCC with functional and prognostic significance.

Transportin-1 (Trn1), also known as karyopherin-β2 (Kapβ2), is probably the best-characterized nuclear import receptor of the karyopherin-β family after Importin-β, but certain aspects of its functions in cells are still puzzling or are just recently emerging. Since the initial identification of Trn1 as the nuclear import receptor of hnRNP A1 ∼25 years ago, several molecular and structural studies have unveiled and refined our understanding of Trn1-mediated nuclear import. In particular, the understanding at a molecular level of the NLS recognition by Trn1 made a decisive step forward with the identification of a new class of NLSs called PY-NLSs, which constitute the best-characterized substrates of Trn1. Besides PY-NLSs, many Trn1 cargoes harbour NLSs that do not resemble the archetypical PY-NLS, which complicates the global understanding of cargo recognition by Trn1. Although PY-NLS recognition is well established and supported by several structures, the recognition of non-PY-NLSs by Trn1 is far less understood, but recent reports have started to shed light on the recognition of this type of NLSs. Aside from its principal and long-established activity as a nuclear import receptor, Trn1 was shown more recently to moonlight outside nuclear import. Trn1 has for instance been caught in participating in virus uncoating, ciliary transport and in modulating the phase separation properties of aggregation-prone proteins. Here, we focus on the structural and functional aspects of Trn1-mediated nuclear import, as well as on the moonlighting activities of Trn1.

Entering the nucleus is important for Porcine circovirus type 2 (PCV2) replication. Karyopherins (KPNs) mediate the nuclear import of many cytoplasmic proteins. Our previous study showed that KPNA3 is involved in interferon production during PCV2 infection induced by Poly I:C and ISD (Interferon stimulatory DNA). However, it remains unclear whether PCV2 replication is associated with KPNA3. In the present study, knockdown of KPNA3 promoted the replication of PCV2, whereas overexpression of KPNA3 inhibited PCV2 replication in PK-15 cells. Furthermore, KPNA3 knockdown inhibited IRF3 and reduced the expression of antiviral genes including IFN-β, ISG54, Mx1 and ISG56, while the opposite results were obtained after KPNA3 overexpression. KPNA3 knockdown also promoted p65 nuclear translocation and increased the mRNA expression of IL-10 and IL-1β. These results suggested that KPNA3 facilitates IRF3 entry into the nucleus and the production of an antiviral response, resulting in PCV2 replication inhibition and blockage of NF-κB signal activation. [ABSTRACT FROM AUTHOR]

The progression of multiple myeloma is accompanied by complex cytogenetic and epigenetic alterations that include mutation or functional inactivation of tumor suppressor proteins and overexpression of oncoproteins. Patients whose myeloma is refractory to the three major classes of drugs including immunomodulatory agents, proteasome inhibitors and anti-CD38 monoclonal antibodies have a very poor prognosis. Drugs with novel mechanisms of action that can bypass resistance mechanisms are sorely needed for this group of patients. Selinexor represents a novel, oral agent with an innovative mechanism of action that offers a significant therapeutic advance in this group of heavily treated patients. Moreover, this novel mechanism may provide additional options for patients with less refractory disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The nucleoporins Nup60p, Nup2p, and Nup1p form part of the nuclear basket structure of the Saccharomyces cerevisiae nuclear pore complex (NPC). Here, we show that these necleoporins can be isolated from yeast extracts by affinity chromatography on karyopherin Kap95p-coated beads. To characterize Nup60p further, Nup60p-coated beads were used to capture its interacting proteins from extracts. We find that Nup60p binds to Nup2p and serves as a docking site for Kap95p-Kap60p heterodimers and Kap123p. Nup60p also binds Gsp1p-GTP and its guanine nucleotide exchange factor Prp20p, and functions as a Gsp1p guanine nucleotide dissociation inhibitor by reducing the activity of Prp20p. Yeast lacking Nup60p exhibit minor defects in nuclear export of Kap60p, nuclear import of Kap95p-Kap60p-dependent cargoes, and diffusion of small proteins across the NPC. Yeast lacking Nup60p also fail to anchor Nup2p at the NPC, resulting in the mislocalization of Nup2p to the nucleoplasm and cytoplasm. Purified Nup60p and Nup2p bind each other directly, but the stability of the complex is compromised when Kap60p binds Nup2p. Gsp1p-GTP enhances by 10-fold the affinity between Nup60p and Nup2p, and restores binding of Nup2p-Kap60p complexes to Nup60p. The results suggest a dynamic interaction, controlled by the nucleoplasmic concentration of Gsp1p-GTP, between Nup60p and Nup2p at the NPC.

Proteasomes are key proteases in regulating protein homeostasis. Their holo-enzymes are composed of 40 different subunits which are arranged in a proteolytic core (CP) flanked by one to two regulatory particles (RP). Proteasomal proteolysis is essential for the degradation of proteins which control time-sensitive processes like cell cycle progression and stress response. In dividing yeast and human cells, proteasomes are primarily nuclear suggesting that proteasomal proteolysis is mainly required in the nucleus during cell proliferation. In yeast, which have a closed mitosis, proteasomes are imported into the nucleus as immature precursors via the classical import pathway. During quiescence, the reversible absence of proliferation induced by nutrient depletion or growth factor deprivation, proteasomes move from the nucleus into the cytoplasm. In the cytoplasm of quiescent yeast, proteasomes are dissociated into CP and RP and stored in membrane-less cytoplasmic foci, named proteasome storage granules (PSGs). With the resumption of growth, PSGs clear and mature proteasomes are transported into the nucleus by Blm10, a conserved 240 kDa protein and proteasome-intrinsic import receptor. How proteasomes are exported from the nucleus into the cytoplasm is unknown.

Ran is a small GTPase whose nucleotide-bound forms cycle through nuclear pore complexes (NPCs) to direct nucleocytoplasmic transport (NCT). Generally, Ran guanosine triphosphate (RanGTP) binds cargocarrying karyopherin receptors (Kaps) in the nucleus and releases them into the cytoplasm following hydrolysis to Ran guanosine diphosphate (RanGDP). This generates a remarkably steep Ran gradient across the nuclear envelope that sustains compartment-specific cargo delivery and accumulation. However, because NPCs are permeable to small molecules of comparable size, it is unclear how an uncontrolled mixing of RanGTP and RanGDP is prevented. Here, we find that an NPCenriched pool of karyopherin subunit beta 1 (KPNB1, hereafter referred to as Kapß1) selectively mediates Ran diffusion across the pore but not passive molecules of similar size (e.g. GFP). This is due to RanGTP having a stronger binding interaction with Kapß1 than RanGDP. For this reason, the RanGDP importer, nuclear transport factor 2, facilitates the return of RanGDP into the nucleus following GTP hydrolysis. Accordingly, the enrichment of Kapß1 at NPCs may function as a retention mechanism that preserves the sharp transition of RanGTP and RanGDP in the nucleus and cytoplasm, respectively. [ABSTRACT FROM AUTHOR]

In the work presented here, we investigated how bromodomain-containing protein 7 (BRD7), a subunit associated with switch/sucrose non-fermentable (SWI/SNF) chromatin remodelling complexes, is trafficked between cellular compartments during embryo development. SWI/SNF complexes are multi-subunit complexes that contain a core catalytic subunit (SWI/SNF related, Matrix associated, Actin dependent Regulator of Chromatin, subfamily A, member 4, or member 2; SMARCA4 or SMARCA2) and a collection of additional subunits that guide the complexes to their appropriate loci; BRD7 is one of these additional subunits. We hypothesised that BRD7 is exported from the nuclei of porcine oocytes and embryos in a Chromosome Region Maintenance 1 (CRM1)-dependent manner and imported into the nuclei using the karyopherin α/β1 heterodimer. Porcine oocytes and embryos were treated with inhibitors of CRM1-mediated nuclear export and karyopherin α/β1-mediated nuclear import to test this hypothesis. An RNA interference assay and a dominant negative overexpression assay were also performed to determine if karyopherin α7 serves a specific role in BRD7 trafficking. Our findings indicate that BRD7 shuttles between nuclear and cytoplasmic compartments during cleavage development. The shuttling of BRD7 indicates that it serves a unique role in remodelling chromatin during this developmental window. Alterations in gene expression in early embryos can impact developmental competence. The aim of the work presented here was to determine how a chromatin remodelling protein, BRD7, is trafficked in the cells of cleavage-stage porcine embryos. Our findings provide new insight into chromatin remodelling dynamics and may further our understanding of the factors that lead to aberrant gene expression. [ABSTRACT FROM AUTHOR]

The nuclear pore is the gatekeeper of nucleocytoplasmic transport and signaling through which a vast flux of information is continuously exchanged between the nuclear and cytoplasmic compartments to maintain cellular homeostasis. A unifying and organizing principle has recently emerged that cements the notion that several forms of amyotrophic lateral sclerosis (ALS), and growing number of other neurodegenerative diseases, co-opt the dysregulation of nucleocytoplasmic transport and that this impairment is a pathogenic driver of neurodegeneration. The understanding of shared pathomechanisms that underpin neurodegenerative diseases with impairments in nucleocytoplasmic transport and how these interface with current concepts of nucleocytoplasmic transport is bound to illuminate this fundamental biological process in a yet more physiological context. Here, I summarize unresolved questions and evidence and extend basic and critical concepts and challenges of nucleocytoplasmic transport and its role in the pathogenesis of neurodegenerative diseases, such as ALS. These principles will help to appreciate the roles of nucleocytoplasmic transport in the pathogenesis of ALS and other neurodegenerative diseases, and generate a framework for new ideas of the susceptibility of motoneurons, and possibly other neurons, to degeneration by dysregulation of nucleocytoplasmic transport. [ABSTRACT FROM AUTHOR]

Protein transport between the nucleus and cytoplasm of eukaryotic cells is tightly regulated, providing a mechanism for controlling intracellular localization of proteins, and regulating gene expression. In this study, we have investigated the importance of nucleocytoplasmic transport mediated by the karyopherin Kap108 in regulating cellular responses to oxidative stress in Saccharomyces cerevisiae. We carried out microarray analyses on wild-type and kap108 mutant cells grown under normal conditions, shortly after introduction of oxidative stress, after 1 hr of oxidative stress, and 1 hr after oxidative stress was removed. We observe more than 500 genes that undergo a 40% or greater change in differential expression between wild-type and kap108Δ cells under at least one of these conditions. Genes undergoing changes in expression can be categorized in two general groups: 1) those that are differentially expressed between wild-type and kap108Δ cells, no matter the oxidative stress conditions; and 2) those that have patterns of response dependent upon both the absence of Kap108, and introduction or removal of oxidative stress. Gene ontology analysis reveals that, among the genes whose expression is reduced in the absence of Kap108 are those involved in stress response and intracellular transport, while those overexpressed are largely involved in mating and pheromone response. We also identified 25 clusters of genes that undergo similar patterns of change in gene expression when oxidative stresses are added and subsequently removed, including genes involved in stress response, oxidation–reduction processing, iron homeostasis, ascospore wall assembly, transmembrane transport, and cell fusion during mating. These data suggest that Kap108 is important for regulating expression of genes involved in a variety of specific cell functions.

We report the crystal structure of nuclear import receptor Importin-9 bound to its cargo, the histones H2A-H2B. Importin-9 wraps around the core, globular region of H2A-H2B to form an extensive interface. The nature of this interface coupled with quantitative analysis of deletion mutants of H2A-H2B suggests that the NLS-like sequences in the H2A-H2B tails play a minor role in import. Importin-9•H2A-H2B is reminiscent of interactions between histones and histone chaperones in that it precludes H2A-H2B interactions with DNA and H3-H4 as seen in the nucleosome. Like many histone chaperones, which prevent inappropriate non-nucleosomal interactions, Importin-9 also sequesters H2A-H2B from DNA. Importin-9 appears to act as a storage chaperone for H2A-H2B while escorting it to the nucleus. Surprisingly, RanGTP does not dissociate Importin-9•H2A-H2B but assembles into a RanGTP•Importin-9•H2A-H2B complex. The presence of Ran in the complex, however, modulates Imp9-H2A-H2B interactions to facilitate its dissociation by DNA and assembly into a nucleosome.

The building of the brain is a multistep process that requires the coordinate expression of thousands of genes and an intense nucleocytoplasmic transport of RNA and proteins. This transport is mediated by karyopherins that comprise importins and exportins. Here, we investigated the role of the ß-importin, importin-8 (IPO8) during mouse cerebral corticogenesis as several of its cargoes have been shown to be essential during this process. First, we showed that Ipo8 mRNA is expressed in mouse brain at various embryonic ages with a clear signal in the sub-ventricular/ventricular zone (SVZ/VZ), the cerebral cortical plate (CP) and the ganglionic eminences. We found that acute knockdown of IPO8 in cortical progenitors reduced both their proliferation and cell cycle exit leading to the increase in apical progenitor pool without influencing the number of basal progenitors (BPs). Projection neurons ultimately reached their appropriate cerebral cortical layer, but their dendritogenesis was specifically affected, resulting in neurons with reduced dendrite complexity. IPO8 knockdown also slowed the migration of cortical interneurons. Together, our data demonstrate that IPO8 contribute to the coordination of several critical steps of cerebral cortex development. These results suggest that the impairment of IPO8 function might be associated with some diseases of neuronal migration defects.

The karyopherin (Kap) family of nuclear transport factors facilitates macromolecular transport through nuclear pore complexes (NPCs). The binding of Kaps to their cargos can also regulate, both temporally and spatially, the interactions of the cargo protein with interacting partners. Here, we show that the essential yeast Kap, Kap121, binds Dam1 and Duo1, components of the microtubule (MT)-associated Dam1 complex required for linking dynamic MT ends with kinetochores (KTs). Like mutations in the Dam1 complex, loss of Kap121 function compromises the formation of normal KT-MT attachments during mitosis. We show that the stability of the Dam1 complex in vivo is dependent on its association with Kap121. Furthermore, we show that the Kap121/Duo1 complex is maintained in the presence of RanGTP but Kap121 is released by the cooperative actions of RanGTP and tubulin. We propose that Kap121 stabilizes the Dam1 complex and participates in escorting it to spindle MTs.

Coordination and information exchange among the various organelles ensure the precise and orderly functioning of eukaryotic cells. Interaction between the cytoplasm and nucleoplasm is crucial for many physiological processes. Macromolecular protein transport into the nucleus requires assistance from the nuclear transport system. These proteins typically contain a nuclear localisation sequence that guides them to enter the nucleus. Understanding the mechanism of nuclear import of macromolecular proteins is important for comprehending cellular processes. Investigation of disease-related alterations can facilitate the development of novel therapeutic strategies and provide additional evidence for clinical trials. This review provides an overview of the proteins involved in nuclear transport and the mechanisms underlying macromolecular protein transport.

Adeno-associated viruses (AAV) are one of the world's most promising gene therapy vectors and as a result, are one of the most intensively studied viral vectors. Despite a wealth of research into these vectors, the precise characterisation of AAVs to translocate into the host cell nucleus remains unclear. Recently we identified the nuclear localization signals of an AAV porcine strain and determined its mechanism of binding to host importin proteins. To expand our understanding of diverse AAV import mechanisms we sought to determine the mechanism in which the Cap protein from a bat-infecting AAV can interact with transport receptor importins for translocation into the nucleus. Using a high-resolution crystal structure and quantitative assays, we were able to not only determine the exact region and residues of the N-terminal domain of the Cap protein which constitute the functional NLS for binding with the importin alpha two protein, but also reveal the differences in binding affinity across the importin-alpha isoforms. Collectively our results allow for a detailed molecular view of the way AAV Cap proteins interact with host proteins for localization into the cell nucleus.

Proteasomes are highly conserved protease complexes responsible for the degradation of aberrant and short-lived proteins. In highly proliferating yeast and mammalian cells, proteasomes are predominantly nuclear. During quiescence and cell cycle arrest, proteasomes accumulate in granules in close proximity to the nuclear envelope/ER. With prolonged quiescence in yeast, these proteasome granules pinch off as membraneless organelles, and migrate as stable entities through the cytoplasm. Upon exit from quiescence, the proteasome granules clear and the proteasomes are rapidly transported into the nucleus, a process reflecting the dynamic nature of these multisubunit complexes. Due to the scarcity of studies on the nuclear transport of mammalian proteasomes, we summarised the current knowledge on the nuclear import of yeast proteasomes. This pathway uses canonical nuclear localisation signals within proteasomal subunits and Srp1/Kap95, and the canonical import receptor, named importin/karyopherin αβ. Blm10, a conserved 240 kDa protein, which is structurally related to Kap95, provides an alternative import pathway. Two models exist upon which either inactive precursor complexes or active holo-enzymes serve as the import cargo. Here, we reconcile both models and suggest that the import of inactive precursor complexes predominates in dividing cells, while the import of mature enzymes mainly occurs upon exit from quiescence.

Accumulation and aggregation of TDP-43 is a major pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43 inclusions also characterize patients with GGGGCC (G4C2) hexanucleotide repeat expansion in C9orf72 that causes the most common genetic form of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Functional studies in cell and animal models have identified pathogenic mechanisms including repeat-induced RNA toxicity and accumulation of G4C2-derived dipeptide-repeat proteins. The role of TDP-43 dysfunction in C9ALS/FTD, however, remains elusive. We found G4C2-derived dipeptide-repeat protein but not G4C2-RNA accumulation caused TDP-43 proteinopathy that triggered onset and progression of disease in Drosophila models of C9ALS/FTD. Timing and extent of TDP-43 dysfunction was dependent on levels and identity of dipeptide-repeat proteins produced, with poly-GR causing early and poly-GA/poly-GP causing late onset of disease. Accumulating cytosolic, but not insoluble aggregated TDP-43 caused karyopherin-α2/4 (KPNA2/4) pathology, increased levels of dipeptide-repeat proteins and enhanced G4C2-related toxicity. Comparable KPNA4 pathology was observed in both sporadic frontotemporal dementia and C9ALS/FTD patient brains characterized by its nuclear depletion and cytosolic accumulation, irrespective of TDP-43 or dipeptide-repeat protein aggregates. These findings identify a vicious feedback cycle for dipeptide-repeat protein-mediated TDP-43 and subsequent KPNA pathology, which becomes self-sufficient of the initiating trigger and causes C9-related neurodegeneration. [ABSTRACT FROM AUTHOR]

The building of the brain is a multistep process that requires the coordinate expression of thousands of genes and an intense nucleocytoplasmic transport of RNA and proteins. This transport is mediated by karyopherins that comprise importins and exportins. Here, we investigated the role of the ß-importin, importin-8 (IPO8) during mouse cerebral corticogenesis as several of its cargoes have been shown to be essential during this process. First, we showed that Ipo8 mRNA is expressed in mouse brain at various embryonic ages with a clear signal in the sub-ventricular/ventricular zone (SVZ/VZ), the cerebral cortical plate (CP) and the ganglionic eminences. We found that acute knockdown of IPO8 in cortical progenitors reduced both their proliferation and cell cycle exit leading to the increase in apical progenitor pool without influencing the number of basal progenitors (BPs). Projection neurons ultimately reached their appropriate cerebral cortical layer, but their dendritogenesis was specifically affected, resulting in neurons with reduced dendrite complexity. IPO8 knockdown also slowed the migration of cortical interneurons. Together, our data demonstrate that IPO8 contribute to the coordination of several critical steps of cerebral cortex development. These results suggest that the impairment of IPO8 function might be associated with some diseases of neuronal migration defects. [ABSTRACT FROM AUTHOR]

Interferon (IFN)-mediated antiviral response is an important part of host defense. Previous studies reported that porcine circovirus type 2 (PCV2) inhibits interferon production, but the mechanism is still poorly understood. In this study, PCV2 suppresses IFN-β and IRF3 promoters and mRNA level of IFN-β induced by ISD or Poly(I:C), but has no effect on the activation of AP-1 and NF-κB. Furthermore, PCV2 decreases the mRNA level of IFN-β and IFN-β promoter activity driven by STING, TBK1, IRF3, and IRF3/5D, and causes a reduction in the protein level of nuclear p-IRF3. In addition, PCV2 interrupts the interaction of KPNA3, rather than KPNA4, with p-IRF3. Overexpression of KPNA3 restores IFN-β promoter activity. These results indicate that PCV2 disrupts the interaction of KPNA3 with p-IRF3 and blocks p-IRF3 translocation to the nucleus, thereby inhibiting IFN-β induction in PK-15 cells. [ABSTRACT FROM AUTHOR]

The nucleocytoplasmic exchange is of fundamental importance to eukaryotic life and is mediated by karyopherins, a superfamily of nuclear transport receptors. However, the function and cargo spectrum of plant karyopherins are largely obscure. Here, we report proximity-labeling-based proteomic profiling of in vivo substrates of KA120, a karyopherin-β required for suppressing autoimmune induction in Arabidopsis. We identify multiple components of the MOS4-associated complex (MAC), a conserved splicing regulatory protein complex. Surprisingly, we find that KA120 does not affect the nucleocytoplasmic distribution of MAC proteins but rather prevents their protein condensation in the nucleus. Furthermore, we demonstrate that MAC condensation is robustly induced by pathogen infection, which is sufficient to activate defense gene expression, possibly by sequestrating negative immune regulators via phase transition. Our study reveals a noncanonical chaperoning activity of a plant karyopherin, which modulates the nuclear condensation of an evolutionarily conserved splicing regulatory complex to coordinate plant immune activation. [Display omitted] • Loss of KA120 leads to MAC-dependent autoimmune activation in plants • KA120 interacts with MAC proteins and prevents spontaneous nuclear condensation of MAC • MAC condensation is robustly induced by pathogen-triggered immune activation • The assembly of MAC condensates is sufficient to activate defense response in plants Jia et al. report that KA120, a conserved nuclear transport receptor, processes a noncanonical chaperoning activity to prevent spontaneous nuclear condensation of the MAC protein complex, an essential regulator of pre-mRNA splicing. However, pathogen infection triggers MAC condensate assembly in plants, sequestering negative immune regulators to coordinate host defense response. [ABSTRACT FROM AUTHOR]

Importin proteins were originally characterized for their central role in protein transport through the nuclear pores, the only intracellular entry to the nucleus. This vital function must be tightly regulated to control access by transcription factors and other nuclear proteins to genomic DNA, to achieve appropriate modulation of cellular behaviors affecting cell fate. Importin-mediated nucleocytoplasmic transport relies on their specific recognition of cargoes, with each importin binding to distinct and overlapping protein subsets. Knowledge of importin function has expanded substantially in regard to three key developmental systems: embryonic stem cells, muscle cells and the germ line. In the decade since the potential for regulated nucleocytoplasmic transport to contribute to spermatogenesis was proposed, we and others have shown that the importins that ferry transcription factors into the nucleus perform additional roles, which control cell fate. This review presents key findings from studies of mammalian spermatogenesis that reveal potential new pathways by which male fertility and infertility arise. These studies of germline genesis illuminate new ways in which importin proteins govern cellular differentiation, including via directing proteins to distinct intracellular compartments and by determining cellular stress responses.

Understanding and sustaining biodiversity is a multi-disciplinary science that benefits highly from the creation of organized and accessible collections of biomaterials (Genome Resource Banks). Large cryo-collections are invaluable tools for understanding, cataloging, and protecting the genetic diversity of the world′s unique animals and plants. Specifically, the systematic collection and preservation of semen from rare species has been developed significantly in recent decades with some biobanks now being actively used for endangered species management and propagation (including the introduction of species such as the black-footed ferret and the giant panda). Innovations emerging from the growing field of male fertility preservation for humans, livestock species, and laboratory animals are also becoming relevant to the protection and the propagation of valuable domestic and wild species. These new approaches extend beyond the "classical" methods associated with sperm freezing to include testicular tissue preservation combined with xenografting or in vitro culture, all of which have potential for rescuing vast amounts of unused germplasm. There also are other options under development that are predicted to have a high impact within the next decade (stem cell technologies, bio-stabilization of sperm cells at ambient temperatures, and the use of genomics tools). However, biobanking efforts and new fertility preservation strategies have to expand the way beyond mammalian species, which will offer knowledge and tools to better manage species that serve as valuable biomedical models or require assistance to reverse endangerment.

Evidence is increasing that the integrity of sperm DNA may also be related to implantation failure and recurrent miscarriage (RM). To investigate this, the sperm DNA fragmentation in partners of 35 women with recurrent implantation failure (RIF) following in vitro fertilization, 16 women diagnosed with RM and seven recent fathers (control) were examined. Sperm were examined pre- and post-density centrifugation by the sperm chromatin dispersion (SCD) test and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. There were no significant differences in the age of either partner or sperm concentration, motility or morphology between three groups. Moreover, there were no obvious differences in sperm DNA fragmentation measured by either test. However, whilst on average sperm DNA fragmentation in all groups was statistically lower in prepared sperm when measured by the SCD test, this was not seen with the results from the TUNEL assay. These results do not support the hypothesis that sperm DNA fragmentation is an important cause of RIF or RM, or that sperm DNA integrity testing has value in such patients. It also highlights significant differences between test methodologies and sperm preparation methods in interpreting the data from sperm DNA fragmentation tests.