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

egg extracts are a powerful 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 on these cell cycle assembly events. Here we show that can also play a role in these events.

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.

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.

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.

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

The nuclear import receptors importin β 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 β is known to act by repressing assembly factors in regions distant from chromatin, whereas RanGTP produced on chromatin frees factors from importin β for localized assembly. The mechanism of transportin regulation was unknown.

Constant regulation of both the MPF amplification loop and the Greatwall-PP2A pathway is required for metaphase II arrest and correct entry into the first embryonic cell cycle.

Recent results indicate that regulating the balance between cyclin-B-Cdc2 kinase, also known as M-phase-promoting factor (MPF), and protein phosphatase 2A (PP2A) is crucial to enable correct mitotic entry and exit. In this work, we studied the regulatory mechanisms controlling the cyclin-B-Cdc2 and PP2A balance by analysing the activity of the Greatwall kinase and PP2A, and the different components of the MPF amplification loop (Myt1, Wee1, Cdc25) during the first embryonic cell cycle. Previous data indicated that the Myt1-Wee1-Cdc25 equilibrium is tightly regulated at the G2-M and M-G1 phase transitions; however, no data exist regarding the regulation of this balance during M phase and interphase.

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.

Pin1 stabilizes Emi1 during G2 phase by preventing its association with SCF(betatrcp).

The anaphase-promoting complex (APC) early mitotic inhibitor 1 (Emi1) is required to induce S- and M-phase entries by stimulating the accumulation of cyclin A and cyclin B through APC(Cdh1/cdc20) inhibition. In this report, we show that Emi1 proteolysis can be induced by cyclin A/cdk (cdk for cyclin-dependent kinase). Paradoxically, Emi1 is stable during G2 phase, when cyclin A/cdk, Plx1 and SCF(betatrcp) (SCF for Skp1-Cul1-Fbox protein)--which play a role in its degradation--are active.

Ubiquitin-mediated protein degradation in Xenopus egg extracts.

Events controlling cell division are governed by the degradation of different regulatory proteins by the ubiquitin-dependent pathway. In this pathway, the attachment of a polyubiquitin chain to a substrate by an ubiquitin-ligase targets this substrate for degradation. Xenopus egg extracts present many advantages for the study of the cell cycle, including the availability of a large quantity of material synchronized at a particular phase of the cell cycle.

The anaphase-promoting complex: a key factor in the regulation of cell cycle.

Events controlling cell division are governed by the degradation of different regulatory proteins by the ubiquitin-dependent pathway. In this pathway, the attachment of a polyubiquitin chain to a substrate by an ubiquitin-ligase targets this substrate for degradation by the 26S proteasome. Two different ubiquitin ligases play an important role in the cell cycle: the SCF (Skp1/Cullin/F-box) and the anaphase-promoting complex (APC).

Genes regulated in neurons undergoing transcription-dependent apoptosis belong to signaling pathways rather than the apoptotic machinery.

Neuronal apoptosis has been shown to require de novo RNA/protein synthesis. However, very few genes whose expression is necessary for inducing apoptosis have been identified so far. To systematically identify such genes, we have used genome-scale, long oligonucleotide microarrays and characterized the gene expression profile of cerebellar granule neurons in the early phase of apoptosis elicited by KCl deprivation.

Kinetochore localization of spindle checkpoint proteins: who controls whom?

The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, we have studied the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway.

Xkid is degraded in a D-box, KEN-box, and A-box-independent pathway.

During mitosis, the Xenopus chromokinesin Kid (Xkid) provides the polar ejection forces needed at metaphase for chromosome congression, and its degradation is required at anaphase to induce chromosome segregation. Despite the fact that the degradation of Xkid at anaphase seems to be a key regulatory factor to induce chromosome movement to the poles, little is known about the mechanisms controlling this proteolysis. We investigated here the degradation pathway of Xkid.

The D-Box-activating domain (DAD) is a new proteolysis signal that stimulates the silent D-Box sequence of Aurora-A.

We have demonstrated previously that Xenopus Aurora-A is degraded at late mitosis by the APC/Fizzy-Related in a D-Box-dependent manner. Here we demonstrate that, although Aurora-B possesses the same D-Box as Aurora-A, Aurora-B is not degraded by this ubiquitin ligase. We have constructed a chimera Aurora-A/B with the N-terminus of Aurora-A and the C-terminus of Aurora-B and we have examined its degradation by APC/Fizzy-Related.