14-3-3 interacts with the kinesin-14 Ncd and prevents it from binding microtubules. Aurora B provides a spatial cue that releases Ncd from 14-3-3 around chromosomes, allowing Ncd to selectively bind the spindle microtubules in the large volume of oocytes.

Tissue homeostasis requires accurate control of cell proliferation, differentiation and chromosome segregation. Drosophila sas-4 and aurA mutants present brain tumours with extra neuroblasts (NBs), defective mitotic spindle assembly and delayed mitosis due to activation of the spindle assembly checkpoint (SAC). Here we inactivate the SAC in aurA and sas-4 mutants to determine whether the generation of aneuploidy compromises NB proliferation. Inactivation of the SAC in the sas-4 mutant impairs NB proliferation and disrupts euploidy. By contrast, disrupting the SAC in the aurA mutant does not prevent NB amplification, tumour formation or chromosome segregation. The monitoring of Mad2 and cyclin B dynamics in live aurA NBs reveals that SAC satisfaction is not coupled to cyclin B degradation. Thus, the NBs of aurA mutants present delayed mitosis, with accurate chromosome segregation occurring in a SAC-independent manner. We report here the existence of an Aurora A-dependent mechanism promoting efficient, timed cyclin B degradation.

Phosphorylation of a dynactin subunit is important for its release from mitotic spindles.

BackgroundCDK11p58 is a mitotic protein kinase, which has been shown to be required for different mitotic events such as centrosome maturation, chromatid cohesion and cytokinesis.Methodology/Principal FindingsIn addition to these previously described roles, our study shows that CDK11p58 inhibition induces a failure in the centriole duplication process in different human cell lines. We propose that this effect is mediated by the defective centrosomal recruitment of proteins at the onset of mitosis. Indeed, Plk4 protein kinase and the centrosomal protein Cep192, which are key components of the centriole duplication machinery, showed reduced levels at centrosomes of mitotic CDK11-depleted cells. CDK11p58, which accumulates only in the vicinity of mitotic centrosomes, directly interacts with the centriole-associated protein kinase Plk4 that regulates centriole number in cells. In addition, we show that centriole from CDK11 defective cells are not able to be over duplicated following Plk4 overexpression.Conclusion/SignificanceWe thus propose that CDK11 is required for centriole duplication by two non-mutually-exclusive mechanisms. On one hand, the observed duplication defect could be caused indirectly by a failure of the centrosome to fully maturate during mitosis. On the other hand, CDK11p58 could also directly regulate key centriole components such as Plk4 during mitosis to trigger essential mitotic centriole modifications, required for centriole duplication during subsequent interphase.

Romé and Ohkura show that the kinesin-6 Subito/MKlp2 mediates a novel oocyte-specific microtubule nucleation pathway, which is essential for assembling spindle microtubules complementarily with the Augmin pathway.

No abstract

Studies using Drosophila have played pivotal roles in advancing our understanding of molecular mechanisms of mitosis throughout the past decades, due to the short generation time and advanced genetic research of this organism. Drosophila is also an excellent model to study female meiosis in oocytes. Pathways such as the acentrosomal assembly of the meiotic spindle in oocytes are conserved from fly to humans. Collecting and manipulating large Drosophila oocytes for microscopy and biochemistry are both time and cost efficient, offering advantages over mouse or human oocytes. Therefore, Drosophila oocytes serve as an excellent platform for molecular studies of female meiosis using a combination of genetics, microscopy, and biochemistry. Here we describe key methods to observe the formation of the meiotic spindle either in fixed or in live oocytes. Moreover, biochemical methods are described to identify protein-protein interactions in vivo.

1The meiotic spindle in oocytes is assembled in the absence of centrosomes, the major 2 microtubule nucleation sites in mitotic and male meiotic cells. A crucial, yet unresolved 3 question in meiosis is how spindle microtubules are generated without centrosomes and 4 only around chromosomes in the large volume of oocytes. Here we report a novel oocyte-5 specific microtubule nucleation pathway that is essential for assembling most spindle 6 microtubules complementarily with the Augmin pathway, and sufficient for triggering 7 microtubule assembly in oocytes. This pathway is mediated by the kinesin-6 Subito/MKlp2, 8 which recruits the γ-tubulin complex to the spindle equator to nucleate microtubules in 9 Drosophila oocytes. Away from chromosomes, Subito interaction with the γ-tubulin complex 10 is suppressed by its N-terminal region to prevent ectopic microtubule assembly in oocytes. 11We further demonstrate that the Subito complex from ovaries can nucleate microtubules 12 from pure tubulin dimers in vitro. Taken together, microtubule nucleation regulated by 13Subito drives spatially restricted spindle assembly in oocytes.14 15 not peer-reviewed)