Extra Spindle Pole Research Articles

Kif2a depletion generates chromosome segregation and pole coalescence defects in animal caps and inhibits gastrulation of the Xenopus embryo.

Kif2a is a member of the kinesin-13 microtubule depolymerases, which tightly regulate microtubule dynamics for many cellular processes. We characterized Kif2a depletion in Xenopus animal caps and embryos. Kif2a depletion generates defects in blastopore closure. These defects are rescued by removing the animal cap, suggesting that Kif2a-depleted animal caps are not compliant enough to allow gastrulation movements. Gastrulation defects are not rescued by a Kif2a mutated in an Aurora kinase phosphorylation site, suggesting that the phenotypes are caused by problems in mitosis. During animal cap mitoses, Kif2a localizes to the spindle poles and centromeres. Depletion of Kif2a generated multipolar spindles in stage 12 embryos. Kif2a-depleted animal caps have anaphase lagging chromosomes in stage 9 and 10 embryos and subsequent cytokinesis failure. Later divisions have greater than two centrosomes, generating extra spindle poles. Kif2a-depleted embryos are also defective at coalescing extra spindle poles into a bipolar spindle. The gastrulation and mitotic phenotypes can be rescued by either human Kif2a or Kif2b, which suggests that the two homologues redundantly regulate mitosis in mammals. These studies demonstrate that defects in mitosis can inhibit large-scale developmental movements in vertebrate tissues.

The Caspase-Related Protease Separase (EXTRA SPINDLE POLES) Regulates Cell Polarity and Cytokinesis inArabidopsis

Vesicle trafficking plays an important role in cell division, establishment of cell polarity, and translation of environmental cues to developmental responses. However, the molecular mechanisms regulating vesicle trafficking remain poorly understood. Here, we report that the evolutionarily conserved caspase-related protease separase (extra spindle poles [ESP]) is required for the establishment of cell polarity and cytokinesis in Arabidopsis thaliana. At the cellular level, separase colocalizes with microtubules and RabA2a (for RAS genes from rat brainA2a) GTPase-positive structures. Separase facilitates polar targeting of the auxin efflux carrier PIN-formed2 (PIN2) to the rootward side of the root cortex cells. Plants with the radially swollen4 (rsw4) allele with compromised separase activity, in addition to mitotic failure, display isotropic cell growth, perturbation of auxin gradient formation, slower gravitropic response in roots, and cytokinetic failure. Measurements of the dynamics of vesicle markers on the cell plate revealed an overall reduction of the delivery rates of KNOLLE and RabA2a GTPase in separase-deficient roots. Furthermore, dissociation of the clathrin light chain, a protein that plays major role in the formation of coated vesicles, was slower in rsw4 than in the control. Our results demonstrate that separase is a key regulator of vesicle trafficking, which is indispensable for cytokinesis and the establishment of cell polarity.

A NUP98-positive acute myeloid leukemia with a t(11;12)(p15;q13) without HOXC cluster gene involvement

We report a case of adult acute myeloid leukemia with a new t(11;12)(p15;q13) underlying a NUP98 rearrangement without HOXC cluster gene involvement. We designed a specific double-color double-fusion FISH assay to discriminate between this t(11;12)(p15;q13) and those producing NUP98-HOXC11 or NUP98-HOXC13. Our fluorescence in situ hybridization (FISH) showed that putative candidate partners mapping 600 kilobases centromeric to HOXC were RARG (retinoic acid receptor γ), MFSD5 (major facilitator superfamily domain containing 5), and ESPL1 (extra spindle pole bodies homolog 1). It is noteworthy that so far only ESPL1 has been implicated in human cancers. This FISH assay is useful for diagnostic screening of NUP98-positive leukemias.

A Rae1-Containing Ribonucleoprotein Complex Is Required for Mitotic Spindle Assembly

Centrosome-independent microtubule polymerization around chromosomes has been shown to require a local gradient of RanGTP, which discharges mitotic cargoes from the nuclear import receptor importin beta. Here, we have used an activity-based assay in Xenopus egg extracts to purify the mRNA export protein Rae1 as a spindle assembly factor regulated by this pathway. Rae1 is a microtubule-associated protein that binds directly to importin beta. Depletion of Rae1 from extracts or cells severely inhibits mitotic spindle assembly. A purified Rae1 complex stabilizes microtubules in egg extracts in a RanGTP/importin beta-regulated manner. Interestingly, Rae1 exists in a large ribonucleoprotein complex, which requires RNA for its activity to control microtubule dynamics in vitro. Furthermore, we provide evidence that RNA associates with the mitotic spindle and that it plays a direct, translation-independent role in spindle assembly. Our studies reveal an unexpected function for RNA in spindle morphogenesis.

"It takes two to tango": understanding how centrosome duplication is regulated throughout the cell cycle.

The essence of successful mitosis is the generation of two genetically identical daughter cells. This requires the assembly of a strictly bipolar mitotic apparatus that will ensure that all daughter chromosomes are segregated to opposite sides of the cell before the completion of mitosis. The agent of the higher animal cell for the establishment of this essential “twoness” is the pair of centrosomes that organize the spindle poles during mitosis (Mazia 1987). Before the cell enters mitosis, the single interphase centrosome duplicates or reproduces exactly once, and the daughter centrosomes form the two poles of the spindle after breakdown of the nuclear envelope. Failure of the cell to precisely control this duplication of the centrosome can have disastrous consequences. If the centrosome fails to duplicate before the onset of mitosis, most somatic cells eventually return to interphase without dividing, which causes polyploidy. If the centrosome duplicates more than once in a cell cycle, then a multipolar spindle may be assembled and the chromosomes may be unequally distributed to the daughter cells, leading to genetic imbalances that produce cells with aggressive growth characteristics (for reviews, see Orr-Weaver and Weinberg 1998; Brinkley 2001). In fact, the cells of many lethal human tumors are genetically unstable and have abnormally high numbers of centrosomes (Lingle et al. 1998; Pihan et al. 1998; Carroll et al. 1999). Extra centrosomes are a problem because the condition is not remediated; cells do not have a checkpoint that aborts mitosis in response to extra spindle poles (Sluder et al. 1997). In this review, we focus on the mechanisms that control centrosome duplication and coordinate it with nuclear events during the cell cycle.