Importin α3 regulates chronic pain pathways in peripheral sensory neurons.

How is neuropathic pain regulated in peripheral sensory neurons? Importins are key regulators of nucleocytoplasmic transport. In this study, we found that importin α3 (also known as karyopherin subunit alpha 4) can control pain responsiveness in peripheral sensory neurons in mice. Importin α3 knockout or sensory neuron-specific knockdown in mice reduced responsiveness to diverse noxious stimuli and increased tolerance to neuropathic pain. Importin α3-bound c-Fos and importin α3-deficient neurons were impaired in c-Fos nuclear import. Knockdown or dominant-negative inhibition of c-Fos or c-Jun in sensory neurons reduced neuropathic pain. In silico screens identified drugs that mimic importin α3 deficiency. These drugs attenuated neuropathic pain and reduced c-Fos nuclear localization. Thus, perturbing c-Fos nuclear import by importin α3 in peripheral neurons can promote analgesia.

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.

Upregulated KPNA2 promotes hepatocellular carcinoma progression and indicates prognostic significance across human cancer types.

Hepatocellular carcinoma (HCC) is one of the most aggressive cancers worldwide. Identification of the molecular mechanisms underlying the development and progression of HCC is particularly important. Here, we demonstrated the expression pattern, clinical significance, and function of Karyopherin α2 (KPNA2) in HCC. The expression of KPNA2 was upregulated in tumor tissue and negatively associated with the survival time, and a significant correlation between KPNA2 expression and aggressive clinical characteristics was established. Both in vitro and in vivo experiments demonstrated that knockdown of KPNA2 reduced migration and proliferation capacities of HCC cells, while over-expression of KPNA2 increased these malignant characteristics. The analysis of the Cancer Genome Atlas cohorts also reveals that high-KPNA2 expression is associated with poor outcome in multiple cancer types. In addition, gene sets enrichment analysis exhibited cell cycle and DNA replication as the top altered pathways in the high-KPNA2 expression group in HCC and other two cancer types. Overall, this study identified KPNA2 as a potential diagnostic and prognostic biomarker in HCC and other neoplasms, probably by regulating cell cycle and DNA replication.

Importin α Partitioning to the Plasma Membrane Regulates Intracellular Scaling.

Early embryogenesis is accompanied by reductive cell divisions requiring that subcellular structures adapt to a range of cell sizes. The interphase nucleus and mitotic spindle scale with cell size through both physical and biochemical mechanisms, but control systems that coordinately scale intracellular structures are unknown. We show that the nuclear transport receptor importin α is modified by palmitoylation, which targets it to the plasma membrane and modulates its binding to nuclear localization signal (NLS)-containing proteins that regulate nuclear and spindle size in Xenopus egg extracts. Reconstitution of importin α targeting to the outer boundary of extract droplets mimicking cell-like compartments recapitulated scaling relationships observed during embryogenesis, which were altered by inhibitors that shift levels of importin α palmitoylation. Modulation of importin α palmitoylation in human cells similarly affected nuclear and spindle size. These experiments identify importin α as a conserved surface area-to-volume sensor that scales intracellular structures to cell size.

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.

Importin α1 is required for nuclear import of herpes simplex virus proteins and capsid assembly in fibroblasts and neurons.

Herpesviruses are large DNA viruses which depend on many nuclear functions, and therefore on host transport factors to ensure specific nuclear import of viral and host components. While some import cargoes bind directly to certain transport factors, most recruit importin ß1 via importin α. We identified importin α1 in a small targeted siRNA screen to be important for herpes simplex virus (HSV-1) gene expression. Production of infectious virions was delayed in the absence of importin α1, but not in cells lacking importin α3 or importin α4. While nuclear targeting of the incoming capsids, of the HSV-1 transcription activator VP16, and of the viral genomes were not affected, the nuclear import of the HSV-1 proteins ICP4 and ICP0, required for efficient viral transcription, and of ICP8 and pUL42, necessary for DNA replication, were reduced. Furthermore, quantitative electron microscopy showed that fibroblasts lacking importin α1 contained overall fewer nuclear capsids, but an increased proportion of mature nuclear capsids indicating that capsid formation and capsid egress into the cytoplasm were impaired. In neurons, importin α1 was also not required for nuclear targeting of incoming capsids, but for nuclear import of ICP4 and for the formation of nuclear capsid assembly compartments. Our data suggest that importin α1 is specifically required for the nuclear localization of several important HSV1 proteins, capsid assembly, and capsid egress into the cytoplasm, and may become rate limiting in situ upon infection at low multiplicity or in terminally differentiated cells such as neurons.

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.

Karyopherin Alpha Proteins Regulate Oligodendrocyte Differentiation.

Proper regulation of the coordinated transcriptional program that drives oligodendrocyte (OL) differentiation is essential for central nervous system myelin formation and repair. Nuclear import, mediated in part by a group of karyopherin alpha (Kpna) proteins, regulates transcription factor access to the genome. Understanding how canonical nuclear import functions to control genomic access in OL differentiation may aid in the creation of novel therapeutics to stimulate myelination and remyelination. Here, we show that members of the Kpna family regulate OL differentiation, and may play distinct roles downstream of different pro-myelinating stimuli. Multiple family members are expressed in OLs, and their pharmacologic inactivation dose-dependently decreases the rate of differentiation. Additionally, upon differentiation, the three major Kpna subtypes (P/α2, Q/α3, S/α1) display differential responses to the pro-myelinating cues T3 and CNTF. Most notably, the Q/α3 karyopherin Kpna4 is strongly upregulated by CNTF treatment both compared with T3 treatment and other Kpna responses. Kpna4 inactivation results in inhibition of CNTF-induced OL differentiation, in the absence of changes in proliferation or viability. Collectively, these findings suggest that canonical nuclear import is an integral component of OL differentiation, and that specific Kpnas may serve vital and distinct functions downstream of different pro-myelinating cues.

Karyopherin α2 induces apoptosis in tongue squamous cell carcinoma CAL-27 cells through the p53 pathway.

Tumor onset and progression are associated with dysfunction of the nuclear transport machinery at the level of import and export receptors. However, the role of Karyopherin α2 (KPNA2) in human tongue squamous cell carcinoma (TSCC) remains unknown. We assessed the proliferation, apoptosis and migration of TSCC CAL-27 cells using wound healing, Transwell and MTT assays, western blotting, electron microscopy and acridine orange/ethidium bromide staining following knockdown of KPNA2. The results revealed the antiproliferative, proapoptotic and anti-migratory effects of KPNA2 silencing on the TSCC CAL-27 cells. Moreover, the knockdown of KPNA2 proved to be accompanied by the upregulation of active caspase-3, cytochrome c, Bax, Bad and decreased expression of Bcl-2, p-Bad and XIAP. KPNA2 activated the caspase-dependent pathway in the CAL-27 cells with upregulation of p53, p21Cip1/Waf1 and p16INK4a. Thus, the present study demonstrated that p53/p21Cip1/Waf1/p16INK4a may be an important pathway involved in the function of KPNA2 in TSCC CAL-27 cells.

Quantitative proteomics reveals a novel role of karyopherin alpha 2 in cell migration through the regulation of vimentin-pErk protein complex levels in lung cancer.

Karyopherin alpha 2 (KPNA2) is overexpressed in various human cancers and is associated with cancer invasiveness and poor prognosis. Herein, to understand the essential role of KPNA2 protein complexes in cancer progression, we applied stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomic strategy combined with immunoprecipitation (IP) to investigate the differential KPNA2 protein complexes in lung adenocarcinoma cell lines with different invasiveness potentials. We found that 64 KPNA2-interaction proteins displayed a 2-fold difference in abundance between CL1-5 (high invasiveness) and CL1-0 (low invasiveness) cells. Pathway map analysis revealed that the formation of complexes containing KPNA2 and cytoskeleton-remodeling-related proteins, including actin, beta tubulin, tubulin heterodimers, vimentin, keratin 8, keratin 18, and plectin, was associated with cancer invasiveness. IP demonstrated that the levels of KPNA2-vimentin-pErk complexes were significantly higher in CL1-5 cells than in CL1-0 cells. The KPNA2-vimentin-pErk complex was also up-regulated in the advanced stage compared with the early-stage lung adenocarcinoma tissues. Importantly, the levels of pErk as well as cell migration ability were significantly reduced in KPNA2-knockdown cells; however, migration was restored by treatment with pErk phosphatase inhibitors. Collectively, our results demonstrate the usefulness of a SILAC-based proteomic strategy for identifying invasiveness-associated KPNA2 protein complexes and provide new insight into the KPNA2-mediated modulation of cell migration.

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.

Importin 8 regulates the transport of mature microRNAs into the cell nucleus.

Mature microRNAs (miRNAs), ∼ 22-nucleotide noncoding RNAs regulating target gene expression at the post-transcriptional level, have been recently shown to be transported into the nucleus where they modulate the biogenesis of other miRNAs or their own expression. However, the mechanism that governs the transport of mature miRNAs from cytoplasm to nucleus remains unknown. Here, we report that importin 8 (IPO8), a member of the karyopherin ß (also named the protein import receptor importin ß) family, plays a critical role in mediating the cytoplasm-to-nucleus transport of mature miRNAs. Specifically knocking down IPO8 but not other karyopherin ß family proteins via siRNA significantly decreases the nuclear transport of various known nucleus-enriched miRNAs without affecting their total cellular levels. IPO8-mediated nuclear transport of mature miRNAs is also dependent on the association of IPO8 with the Argonaute 2 (Ago2) complex. Cross-immunoprecipitation and Western blot analysis show that IPO8 is physically associated with Ago2. Knocking down IPO8 via siRNA markedly decreases the nuclear transport of Ago2 but does not affect the total cellular Ago2 level. Furthermore, dissociating the binding of miRNAs with Ago2 by trypaflavine strongly reduces the IPO8-mediated nuclear transport of miRNAs.

KPNA7, a nuclear transport receptor, promotes malignant properties of pancreatic cancer cells in vitro.

Pancreatic cancer is an aggressive malignancy and one of the leading causes of cancer deaths. The high mortality rate is mostly due to the lack of appropriate tools for early detection of the disease and a shortage of effective therapies. We have previously shown that karyopherin alpha 7 (KPNA7), the newest member of the alpha karyopherin family of nuclear import receptors, is frequently amplified and overexpressed in pancreatic cancer. Here, we report that KPNA7 expression is absent in practically all normal human adult tissues but elevated in several pancreatic cancer cell lines. Inhibition of KPNA7 expression in AsPC-1 and Hs700T pancreatic cancer cells led to a reduction in cell growth and decreased anchorage independent growth, as well as increased autophagy. The cell growth effects were accompanied by an induction of the cell cycle regulator p21 and a G1 arrest of the cell cycle. Interestingly, the p21 induction was caused by increased mRNA synthesis and not defective nuclear transport. These data strongly demonstrate that KPNA7 silencing inhibits the malignant properties of pancreatic cancer cells in vitro and thereby provide the first evidence on the functional role for KPNA7 in human cancer.

MiR-223 downregulation promotes glomerular endothelial cell activation by upregulating importin α4 and α5 in IgA nephropathy.

Glomerular endothelial cells (GEnCs) contribute to renal injuries in IgA nephropathy (IgAN). Here we profiled microRNAs (miRNAs) in GEnCs treated with conditioned medium from human mesangial cells in vitro. Levels of miR-223 in GEnCs decreased after incubation with the medium prepared with pIgA from patients with glomerular endothelial proliferation and were also decreased in the glomerular tissues of patients with glomerular endothelial proliferation. Mesangial-derived IL-6 caused miR-223 levels to decrease. The addition of exogenous miR-223 inhibited cell proliferation, ICAM-1 expression, and monocyte adhesion. The NF-κB and STAT3 signaling pathways collaborate during the activation process. MiR-223 mimics inhibited the nuclear localization and DNA binding of p65 and STAT3 but had no effect on the expression of upstream molecules. Instead, importin α4 and α5 (multipurpose nuclear transport receptors), validated as targets of miR-223, were responsible for the nuclear transport of p65 and STAT3. Importin α4 and α5 siRNA inhibited the nuclear localization of p65 and STAT3 and prevented cell proliferation and monocyte adhesion. The level of miR-223 in circulating endothelial cells was decreased and related to the clinical and pathological parameters. Thus, miR-223 downregulation promotes glomerular endothelial cell activation by upregulating importin α4 and α5 in IgAN. Monitoring the level of miR-223 in circulating endothelial cells may provide a noninvasive method for evaluating the severity of IgAN.

KPNA2 promotes cell proliferation and tumorigenicity in epithelial ovarian carcinoma through upregulation of c-Myc and downregulation of FOXO3a.

Karyopherin alpha 2 (KPNA2), a member of the karyopherin family, has a central role in nucleocytoplasmic transport and is overexpressed in many cancers. Our previous study identified KPNA2 as significantly upregulated in epithelial ovarian carcinoma (EOC), correlating with poor survival of patients. However, the precise mechanism of this effect remains unclear. The aim of the present study was to examine the role of KPNA2 in the proliferation and tumorigenicity of EOC cells, and its clinical significance in tumor progression. Real-time quantitative RT-PCR analysis revealed high expression levels of KPNA2 in 162 out of 191 (84.8%) fresh EOC tissues, which was significantly correlated with International Federation of Gynecology and Obstetrics (FIGO) stage, differentiation, histological type, recurrence, and prognosis of EOC patients. Our results showed that upregulation of KPNA2 expression significantly increased the proliferation and tumorigenicity of EOC cells (EFO-21 and SK-OV3) in vitro and in vivo, by promoting cell growth rate, foci formation, soft agar colony formation, and tumor formation in nude mice. By contrast, knockdown of KPNA2 effectively suppressed the proliferation and tumorigenicity of these EOC cells in vitro and in vivo. Our results also indicated that the molecular mechanisms of the effect of KPNA2 in EOC included promotion of G1/S cell cycle transition through upregulation of c-Myc, enhanced transcriptional activity of c-Myc, activation of Akt activity, suppression of FOXO3a activity, downregulation of cyclin-dependent kinase (CDK) inhibitor p21Cip1 and p27Kip1, and upregulation of CDK regulator cyclin D1. Our results show that KPNA2 has an important role in promoting proliferation and tumorigenicity of EOC, and may represent a novel prognostic biomarker and therapeutic target for this disease.

The nuclear import factor importin α4 can protect against oxidative stress.

The importin (IMP) superfamily of nuclear transport proteins is essential to key developmental pathways, including in the murine testis where expression of the 6 distinct IMPα proteins is highly dynamic. Present predominantly from the spermatocyte stage onwards, IMPα4 is unique in showing a striking nuclear localization, a property we previously found to be linked to maintenance of pluripotency in embryonic stem cells and to the cellular stress response in cultured cells. Here we examine the role of IMPα4 in vivo for the first time using a novel transgenic mouse model in which we overexpress an IMPα4-EGFP fusion protein from the protamine 1 promoter to recapitulate endogenous testicular germ cell IMPα4 expression in spermatids. IMPα4 overexpression did not affect overall fertility, testis morphology/weight or spermatogenic progression under normal conditions, but conferred significantly (>30%) increased resistance to oxidative stress specifically in the spermatid subpopulation expressing the transgene. Consistent with a cell-specific role for IMPα4 in protecting against oxidative stress, haploid germ cells from IMPα4 null mice were significantly (c. 30%) less resistant to oxidative stress than wild type controls. These results from two unique and complementary mouse models demonstrate a novel protective role for IMPα4 in stress responses specifically within haploid male germline cells, with implications for male fertility and genetic integrity.

KPNA7, an oocyte- and embryo-specific karyopherin α subtype, is required for porcine embryo development.

Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin α/ß heterodimer functions to import cytoplasmic proteins that possess classical nuclear localisation signals into the nucleus. Seven karyopherinαsubtypes have been identified in mammals. The aim of this study was to determine the relative abundance of transcripts encoding seven karyopherinαsubtypes in porcine oocytes and embryos at discrete stages of cleavage development, and to determine the developmental requirements of karypopherinα7 (KPNA7), an oocyte and cleavage stage embryo-specific karyopherinαsubtype. We hypothesised that knockdown of KPNA7 would negatively affect porcine cleavage development. To test this hypothesis, in vitro matured and fertilised porcine oocytes were injected with a double-stranded interfering RNA molecule that targeted KPNA7; nuclei were counted in all embryos 6 days after fertilisation. Embryos injected with KPNA7-interfering RNAs possessed significantly lower cell numbers than their respective control groups (P<0.05). In vitro binding assays also suggest that KPNA7 may transport intracellular proteins that possess unique nuclear localisation signals. Our data suggest that embryos have differential requirements for individual karyopherinαsubtypes and that these karyopherinαsubtypes differentially transport intracellular cargo during cleavage development.

Nuclear transport receptor karyopherin-α2 promotes malignant breast cancer phenotypes in vitro.

Tumorigenesis and tumor progression are associated with dysfunction of the nuclear transport machinery at the level of import and export receptors (karyopherins). Recent studies have shown that the nuclear import factor karyopherin-α2 (KPNA2) is a novel prognostic marker for poor prognosis in human breast cancer. Based on the well-defined hallmarks of cancer progression, we performed a detailed in vitro characterization of the phenotypic effects caused by KPNA2 overexpression and KPNA2 silencing in benign and malignant human breast cells. KPNA2 overexpression clearly increased proliferation of MCF7 tumor cells and further led to a reduction of cell-matrix adhesion in benign MCF10A cells, whereas cell migration was significantly increased (P<0.0001) in both tumor models. Remarkably, these individual effects of KPNA2 overexpression on proliferation, cell-matrix adhesion and migration resulted in an increased colony spreading of benign MCF10A breast cells and malignant MCF7 tumor cells (P<0.001), which is a hallmark of cancer progression. Conversely, RNA interference-mediated KPNA2 silencing caused a complete inhibition of MCF7 tumor cell proliferation and migration (P<0.0001). In addition, in these experiments apoptosis was increased (P<0.05) and formation of tumor cell colonies was reduced (P<0.01). Thus, KPNA2 overexpression provoked increased aggressiveness of malignant MCF7 breast tumor cells and induced a shift in benign MCF10A breast cells toward a malignant breast cancer phenotype. In conclusion, we demonstrate for the first time in experimental tumor models that forced KPNA2 expression drives malignant features relevant for breast cancer progression, while its silencing is required for the remission of those progressive phenotypes. This study gives clear evidence that KPNA2 acts as a novel oncogenic factor in human breast cancer, in vitro.

Involvement of classical bipartite/karyopherin nuclear import pathway components in acrosomal trafficking and assembly during bovine and murid spermiogenesis.

This study arose from our finding that SubH2Bv, a histone H2B variant residing in the subacrosomal compartment of mammalian spermatozoa, contains a bipartite nuclear localization signal (bNLS) but in spite of this did not enter the spermatid nucleus. Instead, it associated with proacrosomic and acrosomic vesicles, which were targeted to the nuclear surface to form the acrosome. On this basis we proposed that SubH2Bv targets proacrosomic/acrosomic vesicles from the Golgi apparatus to the nuclear envelope by utilizing the classical bipartite/karyopherin alpha (KPNA) nuclear import pathway. To test the protein's nuclear targeting ability, SubH2Bv, with and without targeted mutations of the basic residues of bNLS, as well as bNLS alone, were transfected into mammalian cells as GFP-fusion proteins. Only the intact bNLS conferred nuclear entry. Subsequently, we showed that a KPNA, most likely KPNA6, occupies the same sperm head compartment and follows the same pattern of acrosomal association during spermiogenesis as SubH2Bv. Sperm head fractionation combined with Western blotting located this KPNA to the subacrosomal layer of the perinuclear theca, while immunocytochemistry of testicular sections showed that it associates with the surface of proacrosomic/acrosomic vesicles during acrosomal biogenesis. The identical sperm-localization and testicular-expression patterns between KPNA and SubH2Bv suggested a potential binding interaction between these proteins. This was supported by recombinant SubH2Bv affinity pull-down assays on germ cell extracts. The results of this study provide a compelling argument that these two nuclear homing proteins work in concert to direct the acrosomic vesicle to the nucleus. Their final residence in the subacrosomal layer of the perinuclear theca of spermatozoa indicates a role for SubH2Bv and KPNA in acrosomal-nuclear docking.

Distinct effects of importin α2 and α4 on Oct3/4 localization and expression in mouse embryonic stem cells.

The cellular repertoire of importin (IMP) proteins that mediates nuclear import of transcription factors and chromatin remodeling agents is critical to processes such as differentiation and transformation. This study identifies for the first time independent roles for specific IMPαs in murine embryonic stem cells (mESCs), showing that mESC differentiation is accompanied by dynamic changes in the levels of transcripts encoding the IMPs, IMPα3, IMPα4, IMPß1, and IPO5. Of these, only IMPα4 was maintained at higher levels in differentiating mESCs, correlating with the finding that IMPα4 overexpression induced a significant decrease in Oct3/4 protein levels compared to control transfections. In parallel, IMPα4 protein showed a unique and striking shift in subcellular localization from the nucleus to the cytoplasm during differentiation, which is consistent with activation of a role in nuclear import of differentiation factors. Overexpression of a dominant-negative IMPα2 isoform, when assessed against adjacent untransfected or IMPα2 transfected cells, led to both a significant reduction in endogenous Oct3/4 protein levels and inhibition of Oct3/4 nuclear localization, suggesting that IMPα2-mediated delivery of Oct3/4 to the nucleus contributes directly to maintenance of mESC pluripotency. These findings implicate IMPα2 and IMPα4 in specific but distinct roles in the fate choice between pluripotency and commitment to differentiation.