Transportin-2 plays a critical role in nucleocytoplasmic shuttling of oestrogen receptor-α.

Oestrogen receptor-α (ERα) shuttles continuously between the nucleus and the cytoplasm, and functions as an oestrogen-dependent transcription factor in the nucleus and as an active mediator of signalling pathways, such as phosphatidylinositol 3-kinase (PI3K)/AKT, in the cytoplasm. However, little is known regarding the mechanism of ERα nucleocytoplasmic shuttling. In this study, we found that ERα is transported into the nucleus by importin-α/ß1. Furthermore, we found that Transportin-2 (TNPO2) is involved in 17ß-oestradiol (E2)-dependent cytoplasmic localisation of ERα. Interestingly, it was found that TNPO2 does not mediate nuclear export, but rather is involved in the cytoplasmic retention of ERα via the proline/tyrosine (PY) motifs. Moreover, we found that TNPO2 competitively binds to the basic nuclear localisation signal (NLS) of ERα with importin-α to inhibit importin-α/ß-dependent ERα nuclear import. Finally, we confirmed that TNPO2 knockdown enhances the nuclear localisation of wild-type ERα and reduces PI3K/AKT phosphorylation in the presence of E2. These results reveal that TNPO2 regulates nucleocytoplasmic shuttling and cytoplasmic retention of ERα, so that ERα has precise functions depending on the stimulation.

Nuclear CD44 Mediated by Importin ß Participated in Naïve Genes Transcriptional Regulation in C3A-iCSCs.

CD44 is one of biomarkers of liver cancer stem cells (CSCs). The investigation of mechanism of CD44 translocation helps to uncover new molecular pathways participated in the regulation of various cellular processes in CSCs. In the present study, we observed the translocation of CD44 from cytoplasm to nuclear in the reprogramming process of C3A cells, full-length CD44 presented in the nucleus of liver iCSCs. CD44 was bound with importin ß and transportin 1 in liver iCSCs. Inhibition of importin ß transport leads to reduction of CD44 in the nucleus. Translocation of CD44 is also influenced by importin α. Besides, overexpression of naïve pluripotent genes, KLF2, KLF5, DNMT3L, GBX2, ZFP42, ESRRB and DPPA4 were found in liver iCSCs. Inhibition of CD44 leads to the reduction of these naïve genes. Luciferase and chromatin immunoprecipitation (ChIP) assays further identified nuclear CD44 bound to the promoter regions of naïve genes, KLF2, KLF5, and ESRRB functioned as transcriptional activators in liver iCSCs. Our present work provides new insight into the dynamic states and functions of CD44 in iCSCs.

Mitotic Motor KIFC1 Is an Organizer of Microtubules in the Axon.

KIFC1 (also called HSET or kinesin-14a) is best known as a multifunctional motor protein essential for mitosis. The present studies are the first to explore KIFC1 in terminally postmitotic neurons. Using RNA interference to partially deplete KIFC1 from rat neurons (from animals of either gender) in culture, pharmacologic agents that inhibit KIFC1, and expression of mutant KIFC1 constructs, we demonstrate critical roles for KIFC1 in regulating axonal growth and retraction as well as growth cone morphology. Experimental manipulations of KIFC1 elicit morphological changes in the axon as well as changes in the organization, distribution, and polarity orientation of its microtubules. Together, the results indicate a mechanism by which KIFC1 binds to microtubules in the axon and slides them into alignment in an ATP-dependent fashion and then cross-links them in an ATP-independent fashion to oppose their subsequent sliding by other motors.SIGNIFICANCE STATEMENT Here, we establish that KIFC1, a molecular motor well characterized in mitosis, is robustly expressed in neurons, where it has profound influence on the organization of microtubules in a number of different functional contexts. KIFC1 may help answer long-standing questions in cellular neuroscience such as, mechanistically, how growth cones stall and how axonal microtubules resist forces that would otherwise cause the axon to retract. Knowledge about KIFC1 may help researchers to devise strategies for treating disorders of the nervous system involving axonal retraction given that KIFC1 is expressed in adult neurons as well as developing neurons.

[Spatio-temporal expression and function of KIFC1 in acrosomogenesis].

OBJECTIVE: To investigate the spatio-temporal expression and function of the end-directed KIFC1 (kinesin family member C1) protein during acrosomogenesis. METHODS: The expression and location of KIFC1 were analyzed by flow separation and immunofluorescence, and the small interference RNA (RNAi1) of KIFC1 with high-interference efficiency was screened using in vitro GC2-spd cell lines. The KIFC1 RNAi1 mixed with 0.5% trypan blue solution was microinjected into the testicular seminiferous tubules and negative Con-RNAi1 into the contralateral testis of a 3-week-old Balb/c mouse, followed by morphological analysis of the sperm collected from the testis tail at 3 weeks after injection. RESULTS: The expression of KIFC1 was observed mainly in the cytoplasm of the sperm cells in the early stage of sperm deformation, in the acrosomal vesicle and acrosome in the middle stage, and in the residual body in the late stage, but vanished during sperm maturation. The sperm head deformity rate was significantly higher in the RNAi1 than in the negative control group (ï¼»32.12 ± 0.25ï¼½% vs ï¼»7.06 ± 1.25ï¼½%, P < 0.01). CONCLUSIONS: The KIFC1 protein may play an important role in the formation of spermatozoa, mainly affecting acrosomogenesis.

Importins α and ß signaling mediates endothelial cell inflammation and barrier disruption.

Nucleocytoplasmic shuttling via importins is central to the function of eukaryotic cells and an integral part of the processes that lead to many human diseases. In this study, we addressed the role of α and ß importins in the mechanism of endothelial cell (EC) inflammation and permeability, important pathogenic features of many inflammatory diseases such as acute lung injury and atherosclerosis. RNAi-mediated knockdown of importin α4 or α3 each inhibited NF-κB activation, proinflammatory gene (ICAM-1, VCAM-1, and IL-6) expression, and thereby endothelial adhesivity towards HL-60 cells, upon thrombin challenge. The inhibitory effect of α4 and α3 knockdown was associated with impaired nuclear import and consequently, DNA binding of RelA/p65 subunit of NF-κB and occurred independently of IκBα degradation. Intriguingly, knockdown of importins α4 and α3 also inhibited thrombin-induced RelA/p65 phosphorylation at Ser536, showing a novel role of α importins in regulating transcriptional activity of RelA/p65. Similarly, knockdown of importin ß1, but not ß2, blocked thrombin-induced activation of RelA/p65 and its target genes. In parallel studies, TNFα-mediated inflammatory responses in EC were refractory to knockdown of importins α4, α3 or ß1, indicating a stimulus-specific regulation of RelA/p65 and EC inflammation by these importins. Importantly, α4, α3, or ß1 knockdown also protected against thrombin-induced EC barrier disruption by inhibiting the loss of VE-cadherin at adherens junctions and by regulating actin cytoskeletal rearrangement. These results identify α4, α3 and ß1 as critical mediators of EC inflammation and permeability associated with intravascular coagulation.

The non-classical nuclear import carrier Transportin 1 modulates circadian rhythms through its effect on PER1 nuclear localization.

Circadian clocks are molecular timekeeping mechanisms that allow organisms to anticipate daily changes in their environment. The fundamental cellular basis of these clocks is delayed negative feedback gene regulation with PERIOD and CRYPTOCHROME containing protein complexes as main inhibitory elements. For a correct circadian period, it is essential that such clock protein complexes accumulate in the nucleus in a precisely timed manner, a mechanism that is poorly understood. We performed a systematic RNAi-mediated screen in human cells and identified 15 genes associated with the nucleo-cytoplasmic translocation machinery, whose expression is important for circadian clock dynamics. Among them was Transportin 1 (TNPO1), a non-classical nuclear import carrier, whose knockdown and knockout led to short circadian periods. TNPO1 was found in endogenous clock protein complexes and particularly binds to PER1 regulating its (but not PER2's) nuclear localization. While PER1 is also transported to the nucleus by the classical, Importin ß-mediated pathway, TNPO1 depletion slowed down PER1 nuclear import rate as revealed by fluorescence recovery after photobleaching (FRAP) experiments. In addition, we found that TNPO1-mediated nuclear import may constitute a novel input pathway of how cellular redox state signals to the clock, since redox stress increases binding of TNPO1 to PER1 and decreases its nuclear localization. Together, our RNAi screen knocking down import carriers (but also export carriers) results in short and long circadian periods indicating that the regulatory pathways that control the timing of clock protein subcellular localization are far more complex than previously assumed. TNPO1 is one of the novel players essential for normal circadian periods and potentially for redox regulation of the clock.

Nuclear import inhibitor N-(4-hydroxyphenyl) retinamide targets Zika virus (ZIKV) nonstructural protein 5 to inhibit ZIKV infection.

In the absence of approved therapeutics, Zika virus (ZIKV)'s recent prolific outbreaks in the Americas, together with impacts on unborn fetuses of infected mothers, make it a pressing human health concern worldwide. Although a key player in viral replication in the infected host cell cytoplasm, ZIKV non-structural protein 5 (NS5) appears to contribute integrally to pathogenesis by localising in the host cell nucleus, in similar fashion to NS5 from Dengue virus (DENV). We show here for the first time that ZIKV NS5 is recognized with high nanomolar affinity by the host cell importin α/ß1 heterodimer, and that this interaction can be blocked by the novel DENV NS5 targeting inhibitor N-(4-hydroxyphenyl) retinamide (4-HPR). Importantly, we show that 4-HPR has potent anti-ZIKV activity at low µM concentrations. With an established safety profile for human use, 4-HPR represents an exciting possibility as an anti-ZIKV agent.

Characterization of Prototype Foamy Virus Infectivity in Transportin 3 Knockdown Human 293t Cell Line.

The foamy viruses are currently considered essential for development as vectors for gene delivery. Previous studies demonstrated that prototype foamy virus (PFV) can infect and replicate prevalently in a variety of cell types for its exclusive replication strategy. However, the virus-host interaction, especially PFV-transportin3 (TNPO3), is still poorly understood. In our investigation of the role of TNPO3 in PFV infection, we found lower virus production in TNPO3 knockdown (KD) cells compared with wild-type 293T cells. PCR analysis revealed that viral DNAs were mostly altered to circular forms: both 1-long terminal repeat (1-LTR) and 2-LTR in TNPO3 KD cells. We therefore suggest that TNPO3 is required for successful PFV replication, at least at/after the nuclear entry step of viral DNA. These findings highlight the obscure mysteries of PFV-host interaction and the requirement of TNPO3 for productive infection of PFV in 293T cells.

The presence of heterogeneous nuclear ribonucleoproteins in frontotemporal lobar degeneration with FUS-positive inclusions.

Frontotemporal lobar degeneration with fused in sarcoma-positive inclusions (FTLD-FUS) is a disease with unknown cause. Transportin 1 is abundantly found in FUS-positive inclusions and responsible for the nuclear import of the FET proteins of which FUS is a member. The presence of all FET proteins in pathological inclusions suggests a disturbance of transportin 1-mediated nuclear import. FUS also belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) protein family. We investigated whether hnRNP proteins are associated with FUS pathology implicating dysfunctional nuclear export in the pathogenesis of FTLD-FUS. hnRNP proteins were investigated in affected brain regions in FTLD-FUS using immunohistochemistry, biochemical analysis, and the expression analysis. We demonstrated the presence of several hnRNP proteins in pathological inclusions including neuronal cytoplasmic inclusions and dystrophic neurites. The biochemical analysis revealed a shift in the location of hnRNP A1 from the nucleus to the cytoplasm. The expression analysis revealed an increase in several hnRNP proteins in FTLD-FUS. These results implicate a wider dysregulation of movement between intracellular compartments, than mechanisms only affecting the nuclear import of FUS proteins.

Cellular and viral determinants of retroviral nuclear entry.

Retroviruses must integrate their cDNA into the host genome to generate proviruses. Viral DNA-protein complexes interact with cellular proteins and produce pre-integration complexes, which carry the viral genome and cross the nuclear pore channel to enter the nucleus and integrate viral DNA into host chromosomal DNA. If the reverse transcripts fail to integrate, linear or circular DNA species such as 1- and 2-long terminal repeats are generated. Such complexes encounter numerous cellular proteins in the cytoplasm, which restrict viral infection and protect the nucleus. To overcome host cell defenses, the pathogens have evolved several evasion strategies. Viral proteins often contain nuclear localization signals, allowing entry into the nucleus. Among more than 1000 proteins identified as required for HIV infection by RNA interference screening, karyopherins, cleavage and polyadenylation specific factor 6, and nucleoporins have been predominantly studied. This review discusses current opinions about the synergistic relationship between the viral and cellular factors involved in nuclear import, with focus on the unveiled mysteries of the host-pathogen interaction, and highlights novel approaches to pinpoint therapeutic targets.

Hyper-dependence of breast cancer cell types on the nuclear transporter Importin ß1.

We previously reported that overexpression of members of the Importin (Imp) superfamily of nuclear transporters results in increased nuclear trafficking through conventional transport pathways in tumour cells. Here we show for the first time that the extent of overexpression of Impß1 correlates with disease state in the MCF10 human breast tumour progression system. Excitingly, we find that targeting Impß1 activity through siRNA is >30 times more efficient in decreasing the viability of malignant ductal carcinoma cells compared to isogenic non-transformed counterparts, and is highly potent and tumour selective at subnanomolar concentrations. Tumour cell selectivity of the siRNA effects was unique to Impß1 and not other Imps, with flow cytometric analysis showing >60% increased cell death compared to controls concomitant with reduced nuclear import efficiency as indicated by confocal microscopic analysis. This hypersensitivity of malignant cell types to Impß1 knockdown raises the exciting possibility of anti-cancer therapies targeted at Impß1.

Role of a nuclear localization signal on the minor capsid proteins VP2 and VP3 in BKPyV nuclear entry.

BK Polyomavirus (BKPyV) is a ubiquitous nonenveloped human virus that can cause severe disease in immunocompromised populations. After internalization into renal proximal tubule epithelial cells, BKPyV traffics through the ER and enters the cytosol. However, it is unclear how the virus enters the nucleus. In this study, we elucidate a role for the nuclear localization signal located on the minor capsid proteins VP2 and VP3 during infection. Site-directed mutagenesis of a single lysine in the basic region of the C-terminus of the minor capsid proteins abrogated their nuclear localization, and the analogous genomic mutation reduced infectivity. Additionally, through use of the inhibitor ivermectin and knockdown of importin ß1, we found that the importin α/ß pathway is involved during infection. Overall these data are the first to show the significance of the NLS of the BKPyV minor capsid proteins during infection in a natural host cell.

Osteoblast regulation via ligand-activated nuclear trafficking of the oxytocin receptor.

We report that oxytocin (Oxt) receptors (Oxtrs), on stimulation by the ligand Oxt, translocate into the nucleus of osteoblasts, implicating this process in the action of Oxt on osteoblast maturation. Sequential immunocytochemistry of intact cells or isolated nucleoplasts stripped of the outer nuclear membrane showed progressive nuclear localization of the Oxtr; this nuclear translocation was confirmed by monitoring the movement of Oxtr-EGFP as well as by immunogold labeling. Nuclear Oxtr localization was conclusively shown by Western immunoblotting and MS of nuclear lysate proteins. We found that the passage of Oxtrs into the nucleus was facilitated by successive interactions with ß-arrestins (Arrbs), the small GTPase Rab5, importin-ß (Kpnb1), and transportin-1 (Tnpo1). siRNA-mediated knockdown of Arrb1, Arrb2, or Tnpo1 abrogated Oxt-induced expression of the osteoblast differentiation genes osterix (Sp7), Atf4, bone sialoprotein (Ibsp), and osteocalcin (Bglap) without affecting Erk phosphorylation. Likewise and again, without affecting pErk, inhibiting Arrb recruitment by mutating Ser rich clusters of the nuclear localization signal to Ala abolished nuclear import and Oxtr-induced gene expression. These studies define a previously unidentified mechanism for Oxtr action on bone and open possibilities for direct transcriptional modulation by nuclear G protein-coupled receptors.

The α-importome of mammalian germ cell maturation provides novel insights for importin biology.

Importin α proteins function as adaptors to connect a cargo protein and importin ß1 in the classical nuclear import pathway. Here we measure for the first time the stoichiometry of importins α2, α3, α4, and ß1 in primary cells corresponding to 2 successive stages of rat spermatogenesis: meiotic spermatocytes and haploid round spermatids. Importin α2 levels were more than 2-fold higher in spermatocytes than in spermatids, while importins α4 and ß1 levels did not differ significantly. We performed a comprehensive proteomics analysis to identify binding proteins in spermatocytes and spermatids using recombinant importin α2 and α4 proteins. Among the 100 candidate partners, 42 contained a strong classical nuclear localization signal (cNLS; score of>6 by cNLS Mapper), while 8 nuclear proteins lacked any cNLS. In addition, we developed a new strategy to predict which cargoes bind to importin α through the conserved C-terminal acidic domain (ARM repeats 9-10), and provided functional validation of a predicted importin α C-terminal binding segment in Senataxin and Smarca4. Evaluation of this set of candidate binding partners from spermatogenic cells using several bioinformatics approaches provides new evidence that individual importin αs may serve unique and nonredundant roles in mediating cellular differentiation.

Transportin-1 and Transportin-2: protein nuclear import and beyond.

Nearly 20 years after its identification as a new ß-karyopherin mediating the nuclear import of the RNA-binding protein hnRNP A1, Transportin-1 is still commonly overlooked in comparison with its best known cousin, Importin-ß. Transportin-1 is nonetheless a considerable player in nucleo-cytoplasmic transport. Over the past few years, significant progress has been made in the characterization of the nuclear localization signals (NLSs) that Transportin-1 recognizes, thereby providing the molecular basis of its diversified repertoire of cargoes. The recent discovery that mutations in the Transportin-dependent NLS of FUS cause mislocalization of this protein and result in amyotrophic lateral sclerosis illustrates the importance of Transportin-dependent import for human health. Besides, new functions of Transportin-1 are emerging in processes other than nuclear import. Here, we summarize what is known about Transportin-1 and the related ß-karyopherin Transportin-2.

Microtubule-dependent regulation of mitotic protein degradation.

Accurate cell division depends on tightly regulated ubiquitylation events catalyzed by the anaphase-promoting complex (APC/C). Among its many substrates, the APC/C triggers the degradation of proteins that stabilize the mitotic spindle, and loss or accumulation of such spindle assembly factors can result in aneuploidy and cancer. Although critical for cell division, it has remained poorly understood how the timing of spindle assembly factor degradation is established during mitosis. Here, we report that active spindle assembly factors are protected from APC/C-dependent degradation by microtubules. In contrast, those molecules that are not bound to microtubules are highly susceptible to proteolysis and turned over immediately after APC/C activation. The correct timing of spindle assembly factor degradation, as achieved by this regulatory circuit, is required for accurate spindle structure and function. We propose that the localized stabilization of APC/C substrates provides a mechanism for the selective disposal of cell-cycle regulators that have fulfilled their mitotic roles.

Wolbachia interferes with the intracellular distribution of Argonaute 1 in the dengue vector Aedes aegypti by manipulating the host microRNAs.

Argonaute proteins (AGOs) are vital components of the RNA-induced silencing complex in gene silencing. AGOs are indispensable for microRNA (miRNA) biogenesis as well as function, and are intracellularly localized to both the cytoplasm and the nucleus. Cytoplasmic AGO-miRNA complexes are mainly involved in cleavage or translational repression of target mRNAs while the nuclear ones are engaged in transcriptional gene silencing, methylation, chromatin remodeling, and splicing. In insects, AGO1 and AGO2 are involved in RNA interference and miRNA pathways but the components involved in their trafficking between the nucleus and the cytoplasm are not known. In this study, we found that importin ß-4 facilitates AGO1 distribution to the nucleus, which is regulated by aae-miR-981 miRNA. The results also revealed association of prohibitin with AGO1 that may play an important role in its stability. Importantly, we found that AGO1 distribution to the nucleus is blocked by Wolbachia, an endosymbiotic bacterium introduced into the Dengue vector, Aedes aegypti. Our results provide basic mechanisms on intracellular trafficking of AGO1 in insects and how this may be altered by Wolbachia, which may affect trafficking of miRNAs to the nucleus leading to alteration in epigenetic effects.

Genome-wide investigation of the role of the tRNA nuclear-cytoplasmic trafficking pathway in regulation of the yeast Saccharomyces cerevisiae transcriptome and proteome.

In eukaryotic cells, tRNAs are transcribed and partially processed in the nucleus before they are exported to the cytoplasm, where they have an essential role in protein synthesis. Surprisingly, mature cytoplasmic tRNAs shuttle between nucleus and cytoplasm, and tRNA subcellular distribution is nutrient dependent. At least three members of the ß-importin family, Los1, Mtr10, and Msn5, function in tRNA nuclear-cytoplasmic intracellular movement. To test the hypothesis that the tRNA retrograde pathway regulates the translation of particular transcripts, we compared the expression profiles from nontranslating mRNAs and polyribosome-associated translating mRNAs collected from msn5Δ, mtr10Δ, and wild-type cells under fed or acute amino acid depletion conditions. Our microarray data revealed that the methionine, arginine, and leucine biosynthesis pathways are targets of the tRNA retrograde process. We confirmed the microarray data by Northern and Western blot analyses. The levels of some of the particular target mRNAs were reduced, while others appeared not to be affected. However, the protein levels of all tested targets in these pathways were greatly decreased when tRNA nuclear import or reexport to the cytoplasm was disrupted. This study provides information that tRNA nuclear-cytoplasmic dynamics is connected to the biogenesis of proteins involved in amino acid biosynthesis.