DEFECTIVE EMBRYO AND MERISTEMS genes are required for cell division and gamete viability in Arabidopsis.

The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1's primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.

Artesunate inhibits intestinal tumorigenesis through inhibiting wnt signaling.

Artesunate (ART) is a clinically approved antimalarial drug and was revealed as a candidate of colorectal cancer chemopreventive agents in our drug screening system. Here, we aimed to understand the suppressive effects of ART on intestinal tumorigenesis. In vitro, ART reduced T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter transcriptional activity. In vivo, ART inhibited intestinal polyp development. We found that ART reduces TCF1/TCF7 nuclear translocation by binding the Ras-related nuclear protein (RAN), suggesting that ART inhibits TCF/LEF transcriptional factor nuclear translocation by binding to RAN, thereby inhibiting Wnt signaling. Our results provide a novel mechanism through which artesunate inhibits intestinal tumorigenesis.

Proper Level of Cytosolic Disabled-1, Which Is Regulated by Dual Nuclear Translocation Pathways, Is Important for Cortical Neuronal Migration.

Disabled-1 (Dab1) is an essential intracellular protein in the Reelin pathway. It has a nuclear localization signal (NLS; hereafter referred to as "NLS1") and 2 nuclear export signals, and shuttles between the nucleus and the cytoplasm. In this study, we found that Dab1 has an additional unidentified NLS, and that the Dab1 NLS1 mutant could translocate to the nucleus in an unconventional ATP/temperature-dependent and cytoplasmic factor/RanGTP gradient-independent manner. Additional mutations in the NLS1 mutant revealed that K(67) and K(69) are important for the nuclear transport. Furthermore, an excess of the intracellular domain of the Reelin receptors inhibited the nuclear translocation of Dab1. An in utero electroporation study showed that a large amount of Dab1 in the cytoplasm in migrating neurons inhibited the migration, and that forced transport of Dab1 into the nucleus attenuated this inhibitory effect. In addition, rescue experiments using yotari, an autosomal recessive mutant of dab1, revealed that cells expressing Dab1 NLS1 mutant tend to distribute at more superficial positions than those expressing wild-type Dab1. Taken together, these findings suggest that Dab1 has at least 2 NLSs, and that the regulation of the subcellular localization of Dab1 is important for the proper migration of excitatory neurons.

Ran signaling in melanoma: implications for the development of alternative therapeutic strategies.

We performed a comparative study between two human metastatic melanoma cell lines (A375 and 526), and melanocytes (FOM78) by gene expression profiling and pathway analysis, using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) software. Genes involved in Ran signaling were significantly over-represented (p ≤ 0.001) and up-regulated in melanoma cells. A melanoma-associated molecular pathway was identified, where Ran, Aurora Kinase A (AurkA) and TERT were up-regulated, while c-myc and PTEN were down-regulated. A consistent high Ran and AurkA gene expression was detected in about 48% and 53%, respectively, of 113 tissue samples from metastatic melanoma patients. AurkA down-regulation was observed in melanoma cells, by Ran knockdown, suggesting AurkA protein is a Ran downstream target. Furthermore, AurkA inhibition, by exposure of melanoma cells to MLN8054, a specific AurKA inhibitor, induced apoptosis in both melanoma cell lines and molecular alterations in the IPA-identified molecular pathway. These alterations differed between cell lines, with an up-regulation of c-myc protein level observed in 526 cells and a slight reduction seen in A375 cells. Moreover, Ran silencing did not affect the A375 invasive capability, while it was enhanced in 526 cells, suggesting that Ran knockdown, by AurkA down-regulation, resulted in a Ran-independent enhanced melanoma cell invasion. Finally, AurK A inhibition induced a PTEN up-regulation and its action was independent of B-RAF mutational status. These findings provide insights relevant for the development of novel therapeutic strategies as well as for a better understanding of mechanisms underlying therapy resistance in melanoma.

The perfect storm? Histiocytoid cardiomyopathy and compound CACNA2D1 and RANGRF mutation in a baby.

A female baby suffered from a rare association between histiocytoid cardiomyopathy, left ventricular non-compaction, and Wolff-Parkinson-White syndrome causing severe and recurrent arrhythmic storms. Antiarrhythmic drugs, radiofrequency ablation of Purkinje tissue, and sympathetic denervation were ineffective. The implant of a cardiac defibrillator allowed her to survive till heart transplant. Compound mutation of CACNA2D1 and RANGRF genes were found. To the best of our knowledge, this is the first comprehensive description of the concurrence of these two mutations and histiocytoid cardiomyopathy.

Identification of a small molecule inhibitor of importin ß mediated nuclear import by confocal on-bead screening of tagged one-bead one-compound libraries.

In eukaryotic cells, proteins and RNAs are transported between the nucleus and the cytoplasm by nuclear import and export receptors. Over the past decade, small molecules that inhibit the nuclear export receptor CRM1 have been identified, most notably leptomycin B. However, up to now no small molecule inhibitors of nuclear import have been described. Here we have used our automated confocal nanoscanning and bead picking method (CONA) for on-bead screening of a one-bead one-compound library to identify the first such import inhibitor, karyostatin 1A. Karyostatin 1A binds importin ß with high nanomolar affinity and specifically inhibits importin α/ß mediated nuclear import at low micromolar concentrations in vitro and in living cells, without perturbing transportin mediated nuclear import or CRM1 mediated nuclear export. Surface plasmon resonance binding experiments suggest that karyostatin 1A acts by disrupting the interaction between importin ß and the GTPase Ran. As a selective inhibitor of the importin α/ß import pathway, karyostatin 1A will provide a valuable tool for future studies of nucleocytoplasmic trafficking.

Human Ran cysteine 112 oxidation by pervanadate regulates its binding to keratins.

We used a proteomic approach to identify proteins that associate with keratins 8 or 18 (K8/K18) in a pervanadate-dependent manner. Pervanadate triggers Ran-K8/K18 binding and a gel-migration-shift of Ran from 25 to 27 kDa, which does not occur upon exposure to H2O2 or vanadate or if pervanadate is excluded during cell solubilization. Generation of 27-kDa Ran is not related to hyperphosphorylation, is heat-insensitive, but occurs upon conversion of Ran cysteines to cysteic acid. The pervanadate-mediated Ran cysteine --> cysteic acid oxidation and its related gel migration shift affects other proteins including actin. Mutation of the three Ran cysteines (Cys-85, -112, and -120) showed that Ran Cys-112 oxidation generates 27-kDa Ran and accounts for its keratin binding. Proteasome inhibition accentuates Ran-keratin binding after cell exposure to pervanadate. Therefore, cell-free exposure to pervanadate causes cysteine to cysteic acid oxidation of Ran and several other proteins and Ran-K8/K18 association. In cells, stabilization of oxidized Ran by proteasome inhibition promotes Ran-keratin interaction. Keratin sequestration of oxidized Ran may provide a back-up protective mechanism in some cases of oxidative injury.

Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes.

Transport receptors of the importin beta superfamily account for many of the nuclear import and export events in eukaryotic cells. They mediate translocation through nuclear pore complexes, shuttle between nucleus and cytoplasm and co-operate with the RanGTPase system to regulate their interactions with cargo molecules in a compartment-specific manner. We used affinity chromatography on immobilized RanGTP to isolate further candidate nuclear transport receptors and thereby identified exportin 4 as the most distant member of the importin beta family so far. Exportin 4 appears to be conserved amongst higher eukaryotes, but lacks obvious orthologues in yeast. It mediates nuclear export of eIF-5A (eukaryotic translation initiation factor 5A) and possibly that of other cargoes. The export signal in eIF-5A appears to be complex and to involve the hypusine modification that is unique to eIF-5A. We discuss possible cellular roles for nuclear export of eIF-5A.

Dissecting the interactions between NTF2, RanGDP, and the nucleoporin XFXFG repeats.

We have used a range of complementary biochemical and biophysical methods to investigate the interactions between nuclear transport factor 2 (NTF2), the Ras family GTPase Ran, and XFXFG nucleoporin repeats that are crucial for nuclear trafficking. Microcalorimetry, microtiter plate binding, and fluorescence quenching in solution are all consistent with the binding constant for the NTF2-RanGDP interaction being in the 100 nM range, whereas the interaction between NTF2 and XFXFG repeat-containing nucleoporins such as Nsp1p is in the 1 microM range. Although the accumulation of NTF2 at the nuclear envelope is enhanced by RanGDP, we show that Ran binding does not alter the affinity of NTF2 for nucleoporins nor does the binding of nucleoporins alter the affinity of NTF2 for RanGDP. These results indicate that, instead, Ran increases the binding of NTF2 to nucleoporins by another mechanism, most probably by Ran itself binding to nucleoporins and NTF2 binding to this nuclear pore-associated Ran.

The Translocon-Associated Protein beta (TRAPbeta) in zebrafish embryogenesis. I. Enhanced expression of transcripts in notochord and hatching gland precursors.

The normal translocation of nascent polypeptides into the lumen of the endoplasmic reticulum (ER) is thought to be aided in part by a translocon-associated protein (TRAP) complex consisting of 4 protein subunits. The association of mature proteins with the ER and Golgi, or other intracellular locales, such as lysosomes, depends on the initial targeting of the nascent polypeptide to the ER membrane. A similar scenario must also exist for proteins destined for secretion. We have identified a member of the TRAP complex using a two hybrid screen to isolate proteins that bind to zebrafish (Danio) Ran binding protein 1. The polypeptide predicted from the largest open reading frame contains 183 amino acids with a 86 and 87% sequence identity to the TRAPbeta subunits in human and chicken, respectively. Sequence analysis identified a cleavable amino-terminal signal peptide in the zebrafish TRAPbeta subunit and a region of the protein spans the membrane of the endoplasmic reticulum. A reverse transcriptase-polymerase chain reaction assay showed that TRAPbeta mRNA is expressed in the developing zebrafish embryo. TRAPbeta mRNA is maternally supplied to the egg and is expressed constitutively throughout development and in the adult. This pattern of expression indicates that the message encoding part of the machinery targeting nascent polypeptides to the ER lumen is available at the onset of embryogenesis when the rate of translation increases exponentially over that occurring in the oocyte. In situ hybridization was used to test whether or not TRAPbeta transcripts might become localized and/or enriched in the developing embryo. Homogeneous staining is seen in the blastula and early gastrula stages. At mid-to-late gastrula stages, however, the message becomes enriched in the developing notochord and polster, or hatching gland rudiment. The TRAPbeta gene, mapped using the LN54 mouse-zebrafish radiation hybrid panel to linkage group 19, resides next to a gene (Z15451) which has sequence homology to notch2 and vascular endothelial growth factor. TRAPbeta, however, does not appear to belong to a group of genes which are syntenic with orthologues or paralogues on human chromosomes.