Effects of Ran-GTP/importin ß inhibition on the meiotic division of porcine oocytes.

The Ran-GTP/importin ß pathway has been implicated in a diverse array of mitotic functions in somatic mitosis; however, the possible meiotic roles of Ran-GTP/importin ß in mammalian oocyte meiosis are still not fully understood. In the present study, importazole (IPZ), a small molecule inhibitor of the interaction between Ran and importin ß was used to explore the potential meiotic roles of Ran-GTP/importin ß in porcine oocytes undergoing meiosis. After IPZ treatment, the extrusion rate of the first polar body (PB1) was significantly decreased, and a higher proportion of the oocytes were arrested at the germinal vesicle breakdown (GVBD) stage. Moreover, IPZ treatment led to severe defects in metaphase I (MI) spindle assembly and chromosome alignment during the germinal vesicle (GV)-to-MI stage, as well as failure of metaphase II (MII) spindle reassembly and homologous chromosome segregation during the MI-to-MII stage. Notably, IPZ treatment decreased TPX2 expression and abnormal subcellular localization. Furthermore, the expression levels of aurora kinase A (AURKA) and transforming acidic coiled-coil 3 (TACC3) were significantly reduced after IPZ treatment. Collectively, these data indicate that the interaction of Ran-GTP and importin ß is essential for proper spindle assembly and successful chromosome segregation during two consecutive meiotic divisions in porcine oocytes, and regulation of this complex might be related to its effect on the TPX2 signaling cascades.

Intracellular distribution of pseudorabies virus UL2 and detection of its nuclear import mechanism.

Pseudorabies virus (PRV) UL2 (pUL2) is a multifunctional protein, which is homologous with herpes simplex virus 1 early protein UL2 (hUL2) and crucial for the viral propagation. Yet, how pUL2 executes its roles in the viral life cycle remain inadequately understood. In order to uncover its effect on the procedure of PRV infection, investigation was performed to examine the subcellular distribution of pUL2 and establish its trafficking mechanism. In the present study, enhanced yellow fluorescent protein or Myc tag fused pUL2 was transiently overexpressed in transfected cells and exhibited an absolutely nuclear accumulation without the existence of other PRV proteins. Additionally, the nuclear trafficking of pUL2 was proved to rely on Ran-, transportin-1, importin ß1, importin α1, α3 and α5. Accordingly, these data will benefit the knowledge of pUL2-mediated biological effects in PRV infection cycle.

Identification of the molecular determinants for nuclear import of PRV EP0.

Pseudorabies virus (PRV) early protein EP0 is a homologue of the herpes simplex virus 1 (HSV-1) immediate-early protein ICP0, which is a multifunctional protein and important for HSV-1 infection. However, the definite function of EP0 during PRV infection is not clear. In this study, to determine if EP0 might localize to the nucleus, as it is shown for its homologue in HSV-1, the subcellular localization pattern and molecular determinants for the nuclear import of EP0 were investigated. EP0 was demonstrated to predominantly target the nucleus in both PRV infected- and plasmid-transfected cells. Furthermore, the nuclear import of EP0 was shown to be dependent on the Ran-, importin α1-, α3-, α7-, ß1- and transportin-1-mediated multiple pathways. Taken together, these data will open up new horizons for portraying the biological roles of EP0 in the course of PRV lytic cycle.

Characterization of the Nucleocytoplasmic Transport Mechanisms of Epstein-Barr Virus BFLF2.

BACKGROUND/AIMS: Epstein-Barr virus (EBV) BFLF2, the homologue of herpes simplex virus 1 (HSV-1) UL31, is crucial for the efficient viral DNA packaging and primary egress across the nuclear membrane. However, we still do not know its subcellular transport mechanisms. METHODS: Interspecies heterokaryon assays were utilized to detect the nucleocytoplasmic shuttling of BFLF2, and mutation analysis, plasmid transfection and fluorescence microscopy assays were performed to identify the functional nuclear localization sequence (NLS) and nuclear export sequence (NES) of BFLF2 in live cells. Furthermore, the nuclear import and export of BFLF2 were assessed by confocal microscopy, co-immunoprecipitation and immunoblot assays. RESULTS: BFLF2 was confirmed to shuttle between the nucleus and cytoplasm. Two predicted NESs were shown to be nonfunctional, yet we proved that the nuclear export of BFLF2 was mediated through transporter associated with antigen processing (TAP), but not chromosomal region maintenance 1 (CRM1) dependent pathway. Furthermore, one functional NLS, 22RRLMHPHHRNYTASKASAH40, was identified, and the aa22-23, aa22-25, aa28-30 and aa37-40 had an important role in the nuclear localization of BFLF2. Besides, the nuclear import of BFLF2 was demonstrated through Ran-, importin α7-, importin ß1- and transportin-1-dependent mechanism that does not require importin α1, α3 and α5. CONCLUSION: These works are of significance for the further study of the functions of BFLF2 during EBV infection, as well as for further insights into the design of new antiviral drug target and vaccine development against EBV.

Inside out: the role of nucleocytoplasmic transport in ALS and FTLD.

Neurodegenerative diseases are characterized by the presence of protein inclusions with a different protein content depending on the type of disease. Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are no exceptions to this common theme. In most ALS and FTLD cases, the predominant pathological species are RNA-binding proteins. Interestingly, these proteins are both depleted from their normal nuclear localization and aggregated in the cytoplasm. This key pathological feature has suggested a potential dual mechanism with both nuclear loss of function and cytoplasmic gain of function being at play. Yet, why and how this pathological cascade is initiated in most patients, and especially sporadic cases, is currently unresolved. Recent breakthroughs in C9orf72 ALS/FTLD disease models point at a pivotal role for the nuclear transport system in toxicity. To address whether defects in nuclear transport are indeed implicated in the disease, we reviewed two decades of ALS/FTLD literature and combined this with bioinformatic analyses. We find that both RNA-binding proteins and nuclear transport factors are key players in ALS/FTLD pathology. Moreover, our analyses suggest that disturbances in nucleocytoplasmic transport play a crucial initiating role in the disease, by bridging both nuclear loss and cytoplasmic gain of functions. These findings highlight this process as a novel and promising therapeutic target for ALS and FTLD.

Identification of molecular determinants for the nuclear import of pseudorabies virus UL31.

Herpes simplex virus 1 (HSV-1) UL31 is a multifunctional protein and important for HSV-1 infection. Pseudorabies virus (PRV) UL31 is a late protein homologous to HSV-1 UL31. Previous studies showed that PRV UL31 is predominantly localized to nucleus, however, the molecular determinants for its nuclear import were unclear to date. Here, by utilizing live cells fluorescent microscopy, UL31 fused with enhanced yellow fluorescent protein was transiently expressed in live cells and confirmed to exclusively target to the nucleus in the absence of other viral proteins. Furthermore, the nuclear import of UL31 was found to be dependent on the Ran-, importin α1-, α3-, α5-, α7-, ß1-and transportin-1-mediated pathway. Therefore, these results would open up new avenues for depicting the biological functions of UL31 during PRV infection.

Importin-7 mediates glucocorticoid receptor nuclear import and is impaired by oxidative stress, leading to glucocorticoid insensitivity.

Some patients with severe inflammatory disease fail to respond to glucocorticoids, and oxidative stress contributes to this insensitivity. Importin receptors are associated with nuclear translocation of the glucocorticoid receptor (GR), which is essential for glucocorticoid function. We hypothesized that importin-7 is central to GR nuclear translocation and glucocorticoid sensitivity. We investigated the effects of importin-7 siRNA on fluticasone propionate (FP)-induced GR nuclear localization and suppression of IL-1ß-induced CXCL8 and the effects of hydrogen peroxide (H2O2) plus IL-1ß costimulation on importin-7 expression, function, and glucocorticoid responsiveness in a human macrophagecell line (U937). H2O2 significantly reduced FP-induced GR nuclear localization (3.4±0.51- vs. 5.7±0.85-fold increase, P<0.05) and suppression of IL-1ß-induced CXCL8 (62.3±2.3 vs. 85.1±7.0%, P<0.05). Knockdown of importin-7 by 38.4 ± 11.5% (compared with control siRNA) significantly reduced FP-mediated GR nuclear localization (3.5±0.5- vs. 5.7±0.85-fold increase, P<0.05) and suppression of IL-1ß-induced CXCL8 expression (40.2±16.1 vs. 68.4±3.0%, P<0.05). H2O2 plus IL-1ß had no direct effect on importin-7 but caused a significant loss (61.2±12.6% compared with baseline) of nuclear RanGTP, an essential cofactor for importin-7-mediated nuclear import of cargo proteins. The importin-7 complex is essential for glucocorticoid function, and the expression of its cofactor RanGTP is reduced during oxidative stress-induced glucocorticoid insensitivity.

In vivo evaluation of mebendazole and Ran GTPase inhibition in breast cancer model system.

Aim: To investigate the therapeutic potential of mebendazole (MBZ)-loaded nanostructured lipid carriers (NLCs). Methodology: NLC-MBZ was prepared and characterized to evaluate the in vitro and in vivo anticancer effects and the inhibitory effect on RanGTP and its potential as an antimetastatic treatment in vivo. Results: NLC-MBZ exhibited a size and charge of 155 ± 20 nm and -27 ± 0.5 mV, respectively, with 90.7% encapsulation. Free MBZ and NLC-MBZ had a 50% inhibitory concentration of 610 and 305 nM, respectively, against MDA-MB-231 cell lines. NLC-MBZ decreased tumor size, suppressed tumor lung metastases, and lowered the expression of CDC25A, SKP2, RbX1 and Cullin1 while boosting the Rb proteins. Conclusion: NLC-MBZ displayed antiangiogenic potential and resulted in a reduced rate of lung metastasis in vivo.

[Box: see text].

Unraveling the impact of disrupted nucleocytoplasmic transport systems in C9orf72-associated ALS.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two adult-onset neurodegenerative diseases that are part of a common disease spectrum due to clinical, genetic, and pathological overlap. A prominent genetic factor contributing to both diseases is a hexanucleotide repeat expansion in a non-coding region of the C9orf72 gene. This mutation in C9orf72 leads to nuclear depletion and cytoplasmic aggregation of Tar DNA-RNA binding protein 43 (TDP-43). TDP-43 pathology is characteristic of the majority of ALS cases, irrespective of disease causation, and is present in ~50% of FTD cases. Defects in nucleocytoplasmic transport involving the nuclear pore complex, the Ran-GTPase cycle, and nuclear transport factors have been linked with the mislocalization of TDP-43. Here, we will explore and discuss the implications of these system abnormalities of nucleocytoplasmic transport in C9orf72-ALS/FTD, as well as in other forms of familial and sporadic ALS.

Matrix metalloproteinase 2 is a target of the RAN-GTP pathway and mediates migration, invasion and metastasis in human breast cancer.

RAS-related nuclear protein(RAN) is a nuclear shuttle and normally regulates events in the cell cycle. When overexpressed in cultured cells, it causes increases in cell migration/invasion in vitro and its overexpression is associated with early breast cancer patient deaths in vivo. However, the underlying mechanism is unknown. The effect of RAN overexpression on potential targets MMP2, ATF3, CXCR3 was investigated by Real-Time PCR/Western blots in the triple receptor negative breast cancer(TRNBC) cell line MDA-MB231 and consequent biological effects were measured by cell adhesion, cell migration and cell invasion assays. Results showed that knockdown of RAN lead to a reduction of MMP2 and its potential regulators ATF3 and CXCR3. Moreover, knockdown of ATF3 or CXCR3 downregulated MMP2 without affecting RAN, indicating that RAN regulates MMP2 through ATF3 and CXCR3. Knockdown of RAN and MMP2 reduced cell adhesion, cell migration and cell growth in agar, whilst overexpression of MMP2 reversed the knockdown of RAN. Furthermore, immunohistochemical staining for RAN and MMP2 are positively associated with each other in the same tumour and separately with patient survival times in breast cancer specimens, suggesting that a high level of RAN may be a pre-requisite for MMP2 overexpression and metastasis. Moreover, positive immunohistochemical staining for both RAN and MMP-2 reduces further patient survival times over that for either protein separately. Our results suggest that MMP2 expression can stratify progression of breast cancers with a high and low incidence of RAN, both RAN and MMP2 in combination can be used for a more accurate patient prognosis. SIMPLE SUMMARY: Ran is an important regulator of normal cell growth and behaviour. We have established in cell line models of breast cancer (BC) a molecular pathway between RAN and its protein-degrading effector MMP-2 and properties related to metastasis in culture. Using immunohistochemistry (IHC) staining of primary BCs, we have shown that RAN and MMP-2 are on their own significantly associated with patient demise from metastatic BC. Moreover, when staining for MMP-2 is added to that for RAN in the primary tumours, there is a significant decrease in patient survival time over that for either protein alone. Thus a combination of staining for RAN and MMP2 is an excellent marker for poor prognosis in breast cancer.

The Nuclear Mitotic Apparatus (NuMA) Protein: A Key Player for Nuclear Formation, Spindle Assembly, and Spindle Positioning.

The nuclear mitotic apparatus (NuMA) protein is well conserved in vertebrates, and dynamically changes its subcellular localization from the interphase nucleus to the mitotic/meiotic spindle poles and the mitotic cell cortex. At these locations, NuMA acts as a key structural hub in nuclear formation, spindle assembly, and mitotic spindle positioning, respectively. To achieve its variable functions, NuMA interacts with multiple factors, including DNA, microtubules, the plasma membrane, importins, and cytoplasmic dynein. The binding of NuMA to dynein via its N-terminal domain drives spindle pole focusing and spindle positioning, while multiple interactions through its C-terminal region define its subcellular localizations and functions. In addition, NuMA can self-assemble into high-ordered structures which likely contribute to spindle positioning and nuclear formation. In this review, we summarize recent advances in NuMA's domains, functions and regulations, with a focus on human NuMA, to understand how and why vertebrate NuMA participates in these functions in comparison with invertebrate NuMA-related proteins.

Ran-GTP Is Non-essential to Activate NuMA for Mitotic Spindle-Pole Focusing but Dynamically Polarizes HURP Near Chromosomes.

Spindle assembly is spatially regulated by a chromosome-derived Ran- GTP gradient. Previous work proposed that Ran-GTP activates spindle assembly factors (SAFs) around chromosomes by dissociating inhibitory importins from SAFs. However, it is unclear whether the Ran-GTP gradient equivalently activates SAFs that localize at distinct spindle regions. In addition, Ran's dual functions in interphase nucleocytoplasmic transport and mitotic spindle assembly have made it difficult to assess its mitotic roles in somatic cells. Here, using auxin-inducible degron technology in human cells, we developed acute mitotic depletion assays to dissect Ran's mitotic roles systematically and separately from its interphase function. In contrast to the prevailing model, we found that the Ran pathway is not essential for spindle assembly activities that occur at sites spatially separated from chromosomes, including activating NuMA for spindle-pole focusing or for targeting TPX2. On the other hand, Ran-GTP is required to localize HURP and HSET specifically at chromosome-proximal regions to set proper spindle length during prometaphase. We demonstrated that Ran-GTP and importin-ß coordinately promote HURP's dynamic microtubule binding-dissociation cycle, which maintains HURP near chromosomes during metaphase. Together, we propose that the Ran pathway acts on spindle assembly independently of its interphase functions in mitotic human cells but does not equivalently regulate all Ran-regulated SAFs. Ran-dependent spindle assembly is likely coupled with additional parallel pathways that activate SAFs distantly located from the chromosomes.

Nanoparticulate RNA delivery systems in cancer.

BACKGROUND: Drug delivery system is a common practice in cancer treatment. RNA interference-mediated post-transcriptional gene silencing holds promise as an approach to knockdown in the expression of target genes responsible for cancer cell growth and metastasis. RNA interference (RNAi) can be achieved by delivering small interfering RNA (siRNA) and short hairpin RNA (shRNA) to target cells. Since neither interfering RNAs can be delivered in naked form due to poor stability, an efficient delivery system is required that protects, guides, and delivers the siRNA and shRNA to target cells as part of cancer therapy (chemotherapy). RECENT FINDINGS: In this review, a discussion is presented about the different types of drug delivery system used to deliver siRNA and shRNA, together with an overview of the potential benefits associated with this sophisticated biomolecular therapy. Improved understanding of the different approaches used in nanoparticle (NP) fabrication, along with an enhanced appreciation of the biochemical properties of siRNA/shRNA, will assist in developing improved drug delivery strategies in basic and clinical research. CONCLUSION: These novel delivery techniques are able to solve the problems that form an inevitable part of delivering genes in more efficient manner and as part of more effective treatment protocols. The present review concludes that the nanoparticulate RNA delivery system has great possibility for cancer treatment along with several other proposed methods. Several NPs or nanocarriers are already in use, but the methods proposed here could fulfill the missing gap in cancer research. It is the future technology, which unravels the mystery of resolving genomic diseases that is, especially genomic instability and its signaling cascades.

Molecular anatomy of the subcellular localization and nuclear import mechanism of herpes simplex virus 1 UL6.

As an indispensable structure protein, the herpes simplex virus 1 (HSV-1) UL6 has been described to exert numerous roles in viral proliferation. However, its exact subcellular localization and subcellular transport mechanism is not well known. In the present study, by utilizing confocal fluorescent microscopy, UL6 was shown to mainly locate in the nucleus in enhanced yellow fluorescent protein or Flag tag fused expression plasmid-transfected cells or HSV-1-infected cells, whereas its predicted nuclear localization signal was nonfunctional. In addition, by exploiting dominant negative mutant and inhibitor of different nuclear import receptors, as well as co-immunoprecipitation and RNA interference assays, UL6 was established to interact with importin α1, importin α7 and transportin-1 to mediate its nuclear translocation under the help of Ran-mediated GTP hydrolysis. Accordingly, these results will advance the knowledge of UL6-mediated biological significances in HSV-1 infection cycle.

Mechanochemical Crosstalk Produces Cell-Intrinsic Patterning of the Cortex to Orient the Mitotic Spindle.

Proliferating animal cells are able to orient their mitotic spindles along their interphase cell axis, setting up the axis of cell division, despite rounding up as they enter mitosis. This has previously been attributed to molecular memory and, more specifically, to the maintenance of adhesions and retraction fibers in mitosis [1-6], which are thought to act as local cues that pattern cortical Gαi, LGN, and nuclear mitotic apparatus protein (NuMA) [3, 7-18]. This cortical machinery then recruits and activates Dynein motors, which pull on astral microtubules to position the mitotic spindle. Here, we reveal a dynamic two-way crosstalk between the spindle and cortical motor complexes that depends on a Ran-guanosine triphosphate (GTP) signal [12], which is sufficient to drive continuous monopolar spindle motion independently of adhesive cues in flattened human cells in culture. Building on previous work [1, 12, 19-23], we implemented a physical model of the system that recapitulates the observed spindle-cortex interactions. Strikingly, when this model was used to study spindle dynamics in cells entering mitosis, the chromatin-based signal was found to preferentially clear force generators from the short cell axis, so that cortical motors pulling on astral microtubules align bipolar spindles with the interphase long cell axis, without requiring a fixed cue or a physical memory of interphase shape. Thus, our analysis shows that the ability of chromatin to pattern the cortex during the process of mitotic rounding is sufficient to translate interphase shape into a cortical pattern that can be read by the spindle, which then guides the axis of cell division.

Intertwined control of the cell cycle and nucleocytoplasmic transport by the cyclin-dependent kinase Pho85 and RanGTPase Gsp1 in Saccharomyces cerevisiae.

Deciphering the molecular mechanisms that connect cell cycle progression and nucleocytoplasmic transport is of particular interest: this intertwined relationship, once understood, may provide useful insight on the diseases resulting from the malfunction of these processes. In the present study we report on findings that indicate a biochemical connection between the cell cycle regulator CDK Pho85 and Ran-GTPase Gsp1, an essential nucleocytoplasmic transport component. When Gsp1 cannot be phosphorylated by Pho85, the cell cycle progression is impaired. Accordingly, a nonphosphorylatable version of Gsp1 abnormally localizes to the nucleus, which impairs the nuclear transport of molecules, including key components of cell cycle progression. Furthermore, our results suggest that the physical interaction of Gsp1 and the Kap95 karyopherin, essential to the release of nuclear cargoes, is altered. Altogether, the present findings point to the involvement of a biochemical mechanism in the interlocked regulation of the cell cycle and nuclear transport.

Structural insight into TPX2-stimulated microtubule assembly.

During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements ('ridge' and 'wedge') in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces. These MT-interacting elements overlap with the binding site of importins on TPX2. Fluorescence microscopy-based in vitro reconstitution assays reveal that this interaction mode is critical for MT binding and facilitates MT nucleation. Together, our results suggest a molecular mechanism of how the Ran-GTP gradient can regulate TPX2-dependent MT formation.

Probing the nuclear import signal and nuclear transport molecular determinants of PRV ICP22.

BACKGROUND: Herpes simplex virus 1 (HSV-1) ICP22 is a multifunctional protein and important for HSV-1 replication. Pseudorabies virus (PRV) ICP22 (P-ICP22) is a homologue of HSV-1 ICP22 and is reported to be able to selectively modify the transcription of different kinetic classes of PRV genes, however, the subcellular localization, localization signal and molecular determinants for its transport to execute this function is less well understood. RESULTS: In this study, by utilizing live cells fluorescent microscopy, P-ICP22 fused to enhanced yellow fluorescent protein (EYFP) gene was transient expressed in live cells and shown to exhibit a predominantly nucleus localization in the absence of other viral proteins. By transfection of a series of P-ICP22 deletion mutants fused to EYFP, a bona fide nuclear localization signal (NLS) and its key amino acids (aa) of P-ICP22 was, for the first time, determined and mapped to aa 41-60 (PASTPTPPKRGRYVVEHPEY) and aa 49-50 (KR), respectively. Besides, the P-ICP22 was demonstrated to be targeted to the nucleus via Ran-, importin α1-, and α7-mediated pathway. CONCLUSIONS: Our findings reported herein disclose the NLS and molecular mechanism for nuclear transport of P-ICP22, these results will uncover new avenues for depicting the biological roles of P-ICP22 during PRV infection.

Ran GTPase promotes cancer progression via Met recepto-rmediated downstream signaling.

It has been shown previously that cancer cells with an activated oncogenic pathway, including Met activation, require Ran for growth and survival.Here, we show that knockdown of Ran leads to a reduction of Met receptor expression in several breast and lung cancer cell lines. This, in turn suppressed HGF expression and the Met-mediated activation of the Akt pathway, as well as cell adhesion, migration, and invasion. In a cell line model where Met amplification has previously been shown to contribute to gefitinib resistance, Ran knockdown sensitized cells to gefitinib-mediated inhibition of Akt and ERK1/2 phosphorylation and consequently reduced cell proliferation. We further demonstrate that Met reduction-mediated by knockdown of Ran, occurs at the post-transcriptional level, probably via a matrix metalloproteinase. Moreover, the level of immunoreactive Ran and Met are positively associated in human breast cancer specimens, suggesting that a high level of Ran may be a pre-requisite for Met overexpression. Interestingly, a high level of immunoreactive Ran dictates the prognostic significance of Met, indicating that the co-overexpression of Met and Ran may be associated with cancer progression and could be used in combination as a prognostic indicator.

Mechanisms of daughter cell-size control during cell division.

Daughter cell size is tightly regulated during cell division. In animal cells, the position of the anaphase spindle specifies the cell cleavage site to dictate the relative size of the daughter cells. Although spindle orientation is regulated by dynein-dependent cortical pulling forces exerted on astral microtubules in many cell types, it was unclear how these forces are precisely regulated to center or displace the spindle. Recently, intrinsic signals derived from chromosomes or spindle poles have been demonstrated to regulate dynein-dependent pulling forces in symmetrically dividing cells. Unexpectedly, myosin-dependent contractile forces have also been shown to control spindle position by altering the cellular boundaries during anaphase. In this review, I discuss how dynein- and myosin-dependent forces are coordinately regulated to control daughter cell size.