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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.

Leucine-rich nuclear export signals (NESs) are recognized by the NES receptor exportin 1 and are central to the export of multiple shuttling proteins and RNAs. The export of messenger RNA in vertebrates was, however, thought to occur by a different pathway, because inhibition by injection of a synthetic Rev NES conjugate could not be demonstrated. Here we find that peptide conjugates composed of the NES of either protein kinase A inhibitor protein (PKI) or the HIV-1 Rev protein, when coupled to human serum albumin, are potent inhibitors of mRNA and small nuclear RNA export. These results provide direct evidence that mRNA export in vertebrates depends on interactions between an NES and its cognate NES receptors. PKI NES conjugates are significantly more efficient at inhibiting RNA export than are REV NES conjugates, indicating that different NESs may have different abilities to promote protein and RNA export. Surprisingly, an expected control conjugate containing the mutant Rev NES sequence M10 strongly inhibited the export of intronless dihydrofolate reductase mRNA. Nuclear injection of NES peptide conjugates led to mislocalization to the nucleus of 10-20% of the cytoplasmic Ran GTPase-binding protein (RanBP1) indicating that RanBP1 shuttles between the nucleus and the cytoplasm via an NES pathway. These results demonstrate that in vertebrates the export of mRNA, like that of small nuclear RNA, 5S rRNA, and transport factors such as RanBP1, employs NES-mediated molecular machinery.

Nuclear import involves pore docking, NLS recognition, translocation through pore, and release from the side of the pore. Ran, the small GTPase, is needed for nuclear import. RNAs leave the nucleus through the nuclear pore, and work by the Hope and Cullen laboratories has delineated the critical features of a nuclear export signal (NES). In the search for an export receptor, a screening method has successfully identified mutations in known transport-related factors. It is believed that the exportin 1, the export receptor, recognizes an NES as an export signal.

Nuclear RNA transcription is silenced when eukaryotic cells enter mitosis. Until recently, this repression was thought to derive solely from the condensation of interphase chromatin into mitotic chromosomes. Recent studies, however, have shown that changes in chromatin structure and occupancy of promoter elements by both general and gene-specific transcription factors also play a role in transcriptional silencing. In addition, studies with simplified systems reveal that reversible phosphorylation of the basal transcriptional machinery represses transcription at mitosis.

A study of the mechanism of annulate lamellae (AL) formation and the assembly of nucleoprotein into pore complexes using biochemical assay models shows that AL formation and incorporation of pore proteins into membranes is simultaneous and time-temperature dependent process. Electron microscopy shows that annulate lamellae are abundant in pore protein-containing fractions and the default pathway assembles pore and nuclear membrane components into AL when nuclear formation is abnormal.

Nuclear transport of small proteins involves a nuclear localization signal (NLS). A 97-kDa accessory protein helps bind a protein with NLS to NLS receptors such as importin, a 54/56 kDa receptor or SRP1 in the cytosol. Yeast NUP1 and NUP2 mediate the binding of the NLS protein-NLS receptor-P96 pore in a step that involves a Ran/TC4-B-2 dimer complex, following the binding step. The dual localization of the cytosolic factors NLS receptor, hsc protein and Ran/TC4 in the cytoplasm and nucleus indicates that they may enter the nucleus during the import of proteins into the nucleus or later.

Crude extracts of Xenopus eggs which have the capacity for nuclear assembly near chromatin templates or naked DNA templates require cytosolic and membrane fractions as well as a gelatinous pellet fraction, identified as glycogen. Pre-incubation of glycogen with lambda-DNA and cytosol before membrane addition are a pre-requisite for nuclear assembly. However, nuclear formation is inhibited by the hydrolysis of glycogen.

Abstract 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.

A study was conducted to examine the correlation 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). Results showed that inhibition of HDACs lead to significant changes in genomic organization, recruiting regions of transcriptional regulation to mammalian nuclear pore complexes.