Exportins can inhibit major mitotic assembly events in vitro: membrane fusion, nuclear pore formation, and spindle assembly.

XENOPUS: egg extracts are a powerful in vitro tool for studying complex biological processes, including nuclear reconstitution, nuclear membrane and pore assembly, and spindle assembly. Extracts have been further used to demonstrate a moonlighting regulatory role for nuclear import receptors or importins on these cell cycle assembly events. Here we show that exportins can also play a role in these events. Addition of Crm1, Exportin-t, or Exportin-5 decreased nuclear pore assembly in vitro. RanQ69L-GTP, a constitutively active form of RanGTP, ameliorated inhibition. Both Crm1 and Exportin-t inhibited fusion of nuclear membranes, again counteracted by RanQ69L-GTP. In mitotic extracts, Crm1 and Exportin-t negatively impacted spindle assembly. Pulldowns from the extracts using Crm1- or Exportin-t-beads revealed nucleoporins known to be essential for both nuclear pore and spindle assembly, with RanQ69L-GTP decreasing a subset of these target interactions. This study suggests a model where exportins, like importins, can regulate major mitotic assembly events.

The UPR Activator ATF6 Responds to Proteotoxic and Lipotoxic Stress by Distinct Mechanisms.

The unfolded protein response (UPR) is induced by proteotoxic stress of the endoplasmic reticulum (ER). Here we report that ATF6, a major mammalian UPR sensor, is also activated by specific sphingolipids, dihydrosphingosine (DHS) and dihydroceramide (DHC). Single mutations in a previously undefined transmembrane domain motif that we identify in ATF6 incapacitate DHS/DHC activation while still allowing proteotoxic stress activation via the luminal domain. ATF6 thus possesses two activation mechanisms: DHS/DHC activation and proteotoxic stress activation. Reporters constructed to monitor each mechanism show that phenobarbital-induced ER membrane expansion depends on transmembrane domain-induced ATF6. DHS/DHC addition preferentially induces transcription of ATF6 target lipid biosynthetic and metabolic genes over target ER chaperone genes. Importantly, ATF6 containing a luminal achromatopsia eye disease mutation, unresponsive to proteotoxic stress, can be activated by fenretinide, a drug that upregulates DHC, suggesting a potential therapy for this and other ATF6-related diseases including heart disease and stroke.

Reprint of "Nuclear transport factors: global regulation of mitosis".

The unexpected repurposing of nuclear transport proteins from their function in interphase to an equally vital and very different set of functions in mitosis was very surprising. The multi-talented cast when first revealed included the import receptors, importin alpha and beta, the small regulatory GTPase RanGTP, and a subset of nuclear pore proteins. In this review, we report that recent years have revealed new discoveries in each area of this expanding story in vertebrates: (a) The cast of nuclear import receptors playing a role in mitotic spindle regulation has expanded: both transportin, a nuclear import receptor, and Crm1/Xpo1, an export receptor, are involved in different aspects of spindle assembly. Importin beta and transportin also regulate nuclear envelope and pore assembly. (b) The role of nucleoporins has grown to include recruiting the key microtubule nucleator ­ the γ-TuRC complex ­ and the exportin Crm1 to the mitotic kinetochores of humans. Together they nucleate microtubule formation from the kinetochores toward the centrosomes. (c) New research finds that the original importin beta/RanGTP team have been further co-opted by evolution to help regulate other cellular and organismal activities, ranging from the actual positioning of the spindle within the cell perimeter, to regulation of a newly discovered spindle microtubule branching activity, to regulation of the interaction of microtubule structures with specific actin structures. (d) Lastly, because of the multitudinous roles of karyopherins throughout the cell cycle, a recent large push toward testing their potential as chemotherapeutic targets has begun to yield burgeoning progress in the clinic.

Nuclear transport factors: global regulation of mitosis.

The unexpected repurposing of nuclear transport proteins from their function in interphase to an equally vital and very different set of functions in mitosis was very surprising. The multi-talented cast when first revealed included the import receptors, importin alpha and beta, the small regulatory GTPase RanGTP, and a subset of nuclear pore proteins. In this review, we report that recent years have revealed new discoveries in each area of this expanding story in vertebrates: (a) The cast of nuclear import receptors playing a role in mitotic spindle regulation has expanded: both transportin, a nuclear import receptor, and Crm1/Xpo1, an export receptor, are involved in different aspects of spindle assembly. Importin beta and transportin also regulate nuclear envelope and pore assembly. (b) The role of nucleoporins has grown to include recruiting the key microtubule nucleator - the γ-TuRC complex - and the exportin Crm1 to the mitotic kinetochores of humans. Together they nucleate microtubule formation from the kinetochores toward the centrosomes. (c) New research finds that the original importin beta/RanGTP team have been further co-opted by evolution to help regulate other cellular and organismal activities, ranging from the actual positioning of the spindle within the cell perimeter, to regulation of a newly discovered spindle microtubule branching activity, to regulation of the interaction of microtubule structures with specific actin structures. (d) Lastly, because of the multitudinous roles of karyopherins throughout the cell cycle, a recent large push toward testing their potential as chemotherapeutic targets has begun to yield burgeoning progress in the clinic.

Analysis of the initiation of nuclear pore assembly by ectopically targeting nucleoporins to chromatin.

Nuclear pore complexes (NPCs) form the gateway to the nucleus, mediating virtually all nucleocytoplasmic trafficking. Assembly of a nuclear pore complex requires the organization of many soluble sub-complexes into a final massive structure embedded in the nuclear envelope. By use of a LacI/LacO reporter system, we were able to assess nucleoporin (Nup) interactions, show that they occur with a high level of specificity, and identify nucleoporins sufficient for initiation of the complex process of NPC assembly in vivo. Eleven nucleoporins from different sub-complexes were fused to LacI-CFP and transfected separately into a human cell line containing a stably integrated LacO DNA array. The LacI-Nup fusion proteins, which bound to the array, were examined for their ability to recruit endogenous nucleoporins to the intranuclear LacO site. Many could recruit nucleoporins of the same sub-complex and a number could also recruit other sub-complexes. Strikingly, Nup133 and Nup107 of the Nup107/160 subcomplex and Nup153 and Nup50 of the nuclear pore basket recruited a near full complement of nucleoporins to the LacO array. Furthermore, Nup133 and Nup153 efficiently targeted the LacO array to the nuclear periphery. Our data support a hierarchical, seeded assembly pathway and identify Nup133 and Nup153 as effective "seeds" for NPC assembly. In addition, we show that this system can be applied to functional studies of individual nucleoporin domains as well as to specific nucleoporin disease mutations. We find that the R391H cardiac arrhythmia/sudden death mutation of Nup155 prevents both its subcomplex assembly and nuclear rim targeting of the LacO array.

Analysis of nuclear reconstitution, nuclear envelope assembly, and nuclear pore assembly using Xenopus in vitro assays.

The large and complex eukaryotic nucleus is the arbiter of DNA replication, RNA transcription, splicing, and ribosome assembly. With the advent of in vitro nuclear reconstitution extracts derived from Xenopus eggs in the 1980s, it became possible to assemble multiple nuclei in vitro around added DNA or chromatin substrates. Such reconstituted nuclei contain a nuclear lamina, double nuclear membranes, nuclear pores, and are competent for DNA replication and nuclear import. In vitro nuclear reconstitution has allowed the assembly of "wild-type" and "biochemically mutant" nuclei in which the impact of individual components can be assessed. Here, we describe protocols for preparation of the nuclear reconstitution extract, nuclear reconstitution in vitro, assessment of nuclear membrane integrity, and a more specialized assay for nuclear pore assembly into preformed pore-free nuclear intermediates.

Transportin acts to regulate mitotic assembly events by target binding rather than Ran sequestration.

The nuclear import receptors importin ß and transportin play a different role in mitosis: both act phenotypically as spatial regulators to ensure that mitotic spindle, nuclear membrane, and nuclear pore assembly occur exclusively around chromatin. Importin ß is known to act by repressing assembly factors in regions distant from chromatin, whereas RanGTP produced on chromatin frees factors from importin ß for localized assembly. The mechanism of transportin regulation was unknown. Diametrically opposed models for transportin action are as follows: 1) indirect action by RanGTP sequestration, thus down-regulating release of assembly factors from importin ß, and 2) direct action by transportin binding and inhibiting assembly factors. Experiments in Xenopus assembly extracts with M9M, a superaffinity nuclear localization sequence that displaces cargoes bound by transportin, or TLB, a mutant transportin that can bind cargo and RanGTP simultaneously, support direct inhibition. Consistently, simple addition of M9M to mitotic cytosol induces microtubule aster assembly. ELYS and the nucleoporin 107-160 complex, components of mitotic kinetochores and nuclear pores, are blocked from binding to kinetochores in vitro by transportin, a block reversible by M9M. In vivo, 30% of M9M-transfected cells have spindle/cytokinesis defects. We conclude that the cell contains importin ß and transportin "global positioning system"or "GPS" pathways that are mechanistically parallel.

Nuclear transport: beginning to gel?

The massive nuclear pore complex mediates nucleocytoplasmic traffic ranging from a single histone to a viral genome. To date, dissecting mechanism has been more an exercise in prediction than biochemical certainty. A recent study combines recombinant proteins with nuclei reconstituted in vitro to test predictions in a startlingly productive manner.

Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis.

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.

Transportin mediates nuclear entry of DNA in vertebrate systems.

Delivery of DNA to the cell nucleus is an essential step in many types of viral infection, transfection, gene transfer by the plant pathogen Agrobacterium tumefaciens and in strategies for gene therapy. Thus, the mechanism by which DNA crosses the nuclear pore complex (NPC) is of great interest. Using nuclei reconstituted in vitro in Xenopus egg extracts, we previously studied DNA passage through the nuclear pores using a single-molecule approach based on optical tweezers. Fluorescently labeled DNA molecules were also seen to accumulate within nuclei. Here we find that this import of DNA relies on a soluble protein receptor of the importin family. To identify this receptor, we used different pathway-specific cargoes in competition studies as well as pathway-specific dominant negative inhibitors derived from the nucleoporin Nup153. We found that inhibition of the receptor transportin suppresses DNA import. In contrast, inhibition of importin beta has little effect on the nuclear accumulation of DNA. The dependence on transportin was fully confirmed in assays using permeabilized HeLa cells and a mammalian cell extract. We conclude that the nuclear import of DNA observed in these different vertebrate systems is largely mediated by the receptor transportin. We further report that histones, a known cargo of transportin, can act as an adaptor for the binding of transportin to DNA.

Transportin regulates major mitotic assembly events: from spindle to nuclear pore assembly.

Mitosis in higher eukaryotes is marked by the sequential assembly of two massive structures: the mitotic spindle and the nucleus. Nuclear assembly itself requires the precise formation of both nuclear membranes and nuclear pore complexes. Previously, importin alpha/beta and RanGTP were shown to act as dueling regulators to ensure that these assembly processes occur only in the vicinity of the mitotic chromosomes. We now find that the distantly related karyopherin, transportin, negatively regulates nuclear envelope fusion and nuclear pore assembly in Xenopus egg extracts. We show that transportin-and importin beta-initiate their regulation as early as the first known step of nuclear pore assembly: recruitment of the critical pore-targeting nucleoporin ELYS/MEL-28 to chromatin. Indeed, each karyopherin can interact directly with ELYS. We further define the nucleoporin subunit targets for transportin and importin beta and find them to be largely the same: ELYS, the Nup107/160 complex, Nup53, and the FG nucleoporins. Equally importantly, we find that transportin negatively regulates mitotic spindle assembly. These negative regulatory events are counteracted by RanGTP. We conclude that the interplay of the two negative regulators, transportin and importin beta, along with the positive regulator RanGTP, allows precise choreography of multiple cell cycle assembly events.

Nucleoporin levels regulate cell cycle progression and phase-specific gene expression.

The Nup107-160 complex, the largest subunit of the nuclear pore, is multifunctional. It mediates mRNA export in interphase, and has roles in kinetochore function, spindle assembly, and postmitotic nuclear pore assembly. We report here that the levels of constituents of the Nup107-160 complex are coordinately cell cycle-regulated. At mitosis, however, a member of the complex, Nup96, is preferentially downregulated. This occurs via the ubiquitin-proteasome pathway. When the levels of Nup96 are kept high, a significant delay in G1/S progression occurs. Conversely, in cells of Nup96(+/-) mice, which express low levels of Nup96, cell cycle progression is accelerated. These lowered levels of Nup96 yield specific defects in nuclear export of certain mRNAs and protein expression, among which are key cell cycle regulators. Thus, Nup96 levels regulate differential gene expression in a phase-specific manner, setting the stage for proper cell cycle progression.

Capture of AT-rich chromatin by ELYS recruits POM121 and NDC1 to initiate nuclear pore assembly.

Assembly of the nuclear pore, gateway to the genome, from its component subunits is a complex process. In higher eukaryotes, nuclear pore assembly begins with the binding of ELYS/MEL-28 to chromatin and recruitment of the large critical Nup107-160 pore subunit. The choreography of steps that follow is largely speculative. Here, we set out to molecularly define early steps in nuclear pore assembly, beginning with chromatin binding. Point mutation analysis indicates that pore assembly is exquisitely sensitive to the change of only two amino acids in the AT-hook motif of ELYS. The dependence on AT-rich chromatin for ELYS binding is borne out by the use of two DNA-binding antibiotics. AT-binding Distamycin A largely blocks nuclear pore assembly, whereas GC-binding Chromomycin A(3) does not. Next, we find that recruitment of vesicles containing the key integral membrane pore proteins POM121 and NDC1 to the forming nucleus is dependent on chromatin-bound ELYS/Nup107-160 complex, whereas recruitment of gp210 vesicles is not. Indeed, we reveal an interaction between the cytoplasmic domain of POM121 and the Nup107-160 complex. Our data thus suggest an order for nuclear pore assembly of 1) AT-rich chromatin sites, 2) ELYS, 3) the Nup107-160 complex, and 4) POM121- and NDC1-containing membrane vesicles and/or sheets, followed by (5) assembly of the bulk of the remaining soluble pore subunits.

Xenopus importin beta validates human importin beta as a cell cycle negative regulator.

BACKGROUND: Human importin beta has been used in all Xenopus laevis in vitro nuclear assembly and spindle assembly studies. This disconnect between species raised the question for us as to whether importin beta was an authentic negative regulator of cell cycle events, or a dominant negative regulator due to a difference between the human and Xenopus importin beta sequences. No Xenopus importin beta gene was yet identified at the time of those studies. Thus, we first cloned, identified, and tested the Xenopus importin beta gene to address this important mechanistic difference. If human importin beta is an authentic negative regulator then we would expect human and Xenopus importin beta to have identical negative regulatory effects on nuclear membrane fusion and pore assembly. If human importin beta acts instead as a dominant negative mutant inhibitor, we should then see no inhibitory effect when we added the Xenopus homologue. RESULTS: We found that Xenopus importin beta acts identically to its human counterpart. It negatively regulates both nuclear membrane fusion and pore assembly. Human importin beta inhibition was previously found to be reversible by Ran for mitotic spindle assembly and nuclear membrane fusion, but not nuclear pore assembly. During the present study, we observed that this differing reversibility varied depending on the presence or absence of a tag on importin beta. Indeed, when untagged importin beta, either human or Xenopus, was used, inhibition of nuclear pore assembly proved to be Ran-reversible. CONCLUSION: We conclude that importin beta, human or Xenopus, is an authentic negative regulator of nuclear assembly and, presumably, spindle assembly. A difference in the Ran sensitivity between tagged and untagged importin beta in pore assembly gives us mechanistic insight into nuclear pore formation.

Global histone acetylation induces functional genomic reorganization at mammalian nuclear pore complexes.

The nuclear localization of genes is intimately tied to their transcriptional status in Saccharomyces cerevisiae, with populations of both active and silent genes interacting with components of the nuclear envelope. We investigated the relationship between the mammalian nuclear pore and the human genome by generating high-resolution, chromosome-wide binding maps of human nucleoporin 93 (Nup93) in the presence and absence of a potent histone deacetylase inhibitor (HDACI). Here, we report extensive genomic reorganization with respect to the nuclear pore following HDACI treatment, including the recruitment of promoter regions, euchromatin-rich domains, and differentially expressed genes. In addition to biochemical mapping, we visually demonstrate the physical relocalization of several genomic loci with respect to the nuclear periphery. Our studies show that inhibiting HDACs leads to significant changes in genomic organization, recruiting regions of transcriptional regulation to mammalian nuclear pore complexes.

Centrin 2 localizes to the vertebrate nuclear pore and plays a role in mRNA and protein export.

Centrins in vertebrates have traditionally been associated with microtubule-nucleating centers such as the centrosome. Unexpectedly, we found centrin 2 to associate biochemically with nucleoporins, including the Xenopus laevis Nup107-160 complex, a critical subunit of the vertebrate nuclear pore in interphase and of the kinetochores and spindle poles in mitosis. Immunofluorescence of Xenopus cells and in vitro reconstituted nuclei indeed revealed centrin 2 localized at the nuclear pores. Use of the mild detergent digitonin in immunofluorescence also allowed centrin 2 to be clearly visualized at the nuclear pores of human cells. Disruption of nuclear pores using RNA interference of the pore assembly protein ELYS/MEL-28 resulted in a specific decrease of centrin 2 at the nuclear rim of HeLa cells. Functionally, excess expression of either the N- or C-terminal calcium-binding domains of human centrin 2 caused a dominant-negative effect on both mRNA and protein export, leaving protein import intact. The mRNA effect mirrors that found for the Saccharomyes cerevisiae centrin Cdc31p at the yeast nuclear pore, a role until now thought to be unique to yeast. We conclude that in vertebrates, centrin 2 interacts with major subunits of the nuclear pore, exhibits nuclear pore localization, and plays a functional role in multiple nuclear export pathways.

ELYS is a dual nucleoporin/kinetochore protein required for nuclear pore assembly and proper cell division.

Nuclear pores span the nuclear envelope and act as gated aqueous channels to regulate the transport of macromolecules between the nucleus and cytoplasm, from individual proteins and RNAs to entire viral genomes. By far the largest subunit of the nuclear pore is the Nup107-160 complex, which consists of nine proteins and is critical for nuclear pore assembly. At mitosis, the Nup107-160 complex localizes to kinetochores, suggesting that it may also function in chromosome segregation. To investigate the dual roles of the Nup107-160 complex at the pore and during mitosis, we set out to identify binding partners by immunoprecipitation from both interphase and mitotic Xenopus egg extracts and mass spectrometry. ELYS, a putative transcription factor, was discovered to copurify with the Nup107-160 complex in Xenopus interphase extracts, Xenopus mitotic extracts, and human cell extracts. Indeed, a large fraction of ELYS localizes to the nuclear pore complexes of HeLa cells. Importantly, depletion of ELYS by RNAi leads to severe disruption of nuclear pores in the nuclear envelope, whereas lamin, Ran, and tubulin staining appear normal. At mitosis, ELYS targets to kinetochores, and RNAi depletion from HeLa cells leads to an increase in cytokinesis defects. Thus, we have identified an unexpected member of the nuclear pore and kinetochore that functions in both pore assembly at the nucleus and faithful cell division.

Topology of yeast Ndc1p: predictions for the human NDC1/NET3 homologue.

The nuclear pore complex is the predominant structure in the nuclear envelope that spans the double nuclear membranes of all eukaryotes. Yeasts have one additional organelle that is also embedded in the nuclear envelope: the spindle pole body, which functions as the microtubule organizing center. The only protein known to localize to and be important in the assembly of both of these yeast structures is the integral membrane protein, Ndc1p. However, no homologues of Ndc1p had been characterized in metazoa. Here, we identify and analyze NDC1 homologues that are conserved throughout evolution. We show that the overall topology of these homologues is conserved. Each contains six transmembrane segments in its N-terminal half and has a large soluble C-terminal half of approximately 300 amino acids. Charge distribution analysis infers that the N- and C-termini are exposed to the cytoplasm. Limited proteolysis of yeast Ndc1p in cellular membranes confirms the orientation of its C-terminus. Although it is not known whether vertebrate NDC1 protein localizes to nuclear pores like its yeast counterpart, the human homologue contains three FG repeats in the C-terminus, a feature of many nuclear pore proteins. Moreover, a small region containing mutations that affect assembly of the nuclear pore in yeast is highly conserved throughout evolution. Lastly, we bring together data from another study to demonstrate that the human homologue of NDC1 is the known inner nuclear membrane protein, NET3.

The Nup107-160 nucleoporin complex is required for correct bipolar spindle assembly.

The Nup107-160 complex is a critical subunit of the nuclear pore. This complex localizes to kinetochores in mitotic mammalian cells, where its function is unknown. To examine Nup107-160 complex recruitment to kinetochores, we stained human cells with antisera to four complex components. Each antibody stained not only kinetochores but also prometaphase spindle poles and proximal spindle fibers, mirroring the dual prometaphase localization of the spindle checkpoint proteins Mad1, Mad2, Bub3, and Cdc20. Indeed, expanded crescents of the Nup107-160 complex encircled unattached kinetochores, similar to the hyperaccumulation observed of dynamic outer kinetochore checkpoint proteins and motors at unattached kinetochores. In mitotic Xenopus egg extracts, the Nup107-160 complex localized throughout reconstituted spindles. When the Nup107-160 complex was depleted from extracts, the spindle checkpoint remained intact, but spindle assembly was rendered strikingly defective. Microtubule nucleation around sperm centrosomes seemed normal, but the microtubules quickly disassembled, leaving largely unattached sperm chromatin. Notably, Ran-GTP caused normal assembly of microtubule asters in depleted extracts, indicating that this defect was upstream of Ran or independent of it. We conclude that the Nup107-160 complex is dynamic in mitosis and that it promotes spindle assembly in a manner that is distinct from its functions at interphase nuclear pores.

Importin beta: conducting a much larger cellular symphony.

Importin beta, once thought to be exclusively a nuclear transport receptor, is emerging as a global regulator of diverse cellular functions. Importin beta acts positively in multiple interphase roles: in nuclear import, as a chaperone for highly charged nuclear proteins, and as a potential motor adaptor for movement along microtubules. In contrast, importin beta plays a negative regulatory role in mitotic spindle assembly, centrosome dynamics, nuclear membrane formation, and nuclear pore assembly. In most of these, importin beta is counteracted by its regulator, Ran-GTP. In light of this, the recent discovery of Ran's involvement in spindle checkpoint control suggested a potential new arena for importin beta action, although it is also possible that one of importin beta's relatives, the karyopherin family of proteins, manages this checkpoint. Lastly, importin beta plays a role in transducing damage signals from the axons of injured neurons back to the cell body.