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Accurate genome inheritance during cell division relies on a complex chromosome segregation mechanism. This process occurs once all the kinetochores of sister chromatids are attached to microtubules emanating from the opposite poles of the mitotic spindle. To control the precision of this mechanism, the Chromosome Passenger Complex (CPC) actively identifies and corrects improper microtubule attachments. The depolymerase activity of the kinesin KIF2C/MCAK at the kinetochores is involved in this process. CCDC69 is a poorly characterized protein, primarily identified as a regulator of central spindle assembly, whose overexpression prompts rapid microtubule depolymerization. Here, we show that CCDC69 is a cell-cycle regulated protein belonging to the Microtubule-associated Tumor Suppressor (MTUS) superfamily, and even slight deregulation of its expression induces severe early mitotic phenotypes. Myristoylation anchors CCDC69 at the plasma membrane, thus protecting microtubule network integrity. We found that CCDC69 microtubule depolymerization activity relies on KIF2C, with a fraction of CCDC69 localizing to the centromere. Importantly, we demonstrated that CCDC69 regulates the stability of the CPC by safeguarding its members from degradation during mitosis. In summary, our findings underscore CCDC69's essential role as a mitotic regulator, which is crucial for maintaining the fidelity of chromosome segregation., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The timing of the M-phase is precisely controlled by a CDC6-dependent mechanism inhibiting the mitotic histone H1 kinase. Here, we describe the differential regulation of the dynamics of this mitotic kinase activity by exogenous cyclin A or cyclin B in the Xenopus laevis cycling extracts. We show that the experimental increase in cyclin A modifies only the level of histone H1 kinase activity, while the cyclin B increase modifies two parameters: histone H1 kinase activity and the timing of its full activation, which is accelerated. On the other hand, the cyclin A depletion significantly delays full activation of histone H1 kinase. However, when CDC6 is added to such an extract, it inhibits cyclin B-associated histone H1 kinase, but does not modify the mitotic timing in the absence of cyclin A. Further, we show via p9 co-precipitation with Cyclin-Dependent Kinases (CDKs), that both CDC6 and the bona fide CDK1 inhibitor Xic1 associate with the mitotic CDKs. Finally, we show that the Xic1 temporarily separates from the mitotic CDKs complexes during the peak of histone H1 kinase activity. These data show the differential coordination of the M-phase progression by cyclin A- and cyclin B-dependent CDKs, confirm the critical role of the CDC6-dependent histone H1 kinase inhibition in this process, and show that CDC6 acts differentially through the cyclin B- and cyclin A-associated CDKs. This CDC6- and cyclins-dependent mechanism likely depends on the precisely regulated association of Xic1 with the mitotic CDKs complexes. We postulate that: i. the dissociation of Xic1 from the CDKs complexes allows the maximal activation of CDK1 during the M-phase, ii. the switch between cyclin A- and cyclin B-CDK inhibition upon M-phase initiation may be responsible for the diauxic growth of mitotic histone H1 kinase activity.

Protein phase separation or coacervation has emerged as a potential mechanism to regulate biological functions. We have shown that coacervation of a mostly unstructured protein, BuGZ, promotes assembly of spindle and its matrix. BuGZ in the spindle matrix binds and concentrates tubulin to promote microtubule (MT) assembly. It remains unclear, however, whether BuGZ could regulate additional proteins to promote spindle assembly. In this study, we report that BuGZ promotes Aurora A (AurA) activation in vitro. Depletion of BuGZ in cells reduces the amount of phosphorylated AurA on spindle MTs. BuGZ also enhances MCAK phosphorylation. The two zinc fingers in BuGZ directly bind to the kinase domain of AurA, which allows AurA to incorporate into the coacervates formed by BuGZ in vitro. Importantly, mutant BuGZ that disrupts the coacervation activity in vitro fails to promote AurA phosphorylation in Xenopus laevis egg extracts. These results suggest that BuGZ coacervation promotes AurA activation in mitosis., (© 2018 Huang et al.)

Harmonious embryo development requires precise coordination between the timing of the cell cycle and the developmental program. Cyclin accumulation determines the timing of the cell cycle M-phase entry and its degradation determines the timing of the M-phase exit. It is well known that CDK1 and PP2A also govern M-phase entry. However, it is unknown how this kinase and phosphatase regulate the precise timing of events at the beginning of the M-phase and how they cooperate with cyclin metabolism. Here we use Xenopus laevis one-cell embryo cell-free extract experiments to answer this question critical for understanding the regulation of embryo development. Using, separately, low concentrations of the chemical inhibitor of CDK1, RO3306 (RO), or the inhibitor of phosphatases, okadaic acid (OA), we show that moderately diminished CDK1 or PP2A activities results in a delay and an acceleration respectively, of M-phase entry. Simultaneous diminution of CDK1 and PP2A activities results in an intermediate timing of M-phase entry, prolongs the duration of M-phase and diminishes the dynamics of cyclin B2 degradation. We thus show, for the first time, that equilibrium between CDK1 and PP2A specifies the timing of M-phase entry and exit and regulates the dynamics of cyclin B degradation upon M-phase exit in Xenopus laevis first embryonic mitosis.

Targeting protein for Xklp2 (TPX2) activates the Ser/Thr kinase Aurora A in mitosis and targets it to the mitotic spindle [1, 2]. These effects on Aurora A are mediated by the N-terminal domain of TPX2, whereas a C-terminal fragment has been reported to affect microtubule nucleation [3]. Using the Xenopus system, we identified a novel role of TPX2 during mitosis. Injection of TPX2 or its C terminus (TPX2-CT) into blastomeres of two-cell embryos led to potent cleavage arrest. Despite cleavage arrest, TPX2-injected embryos biochemically undergo multiple rounds of DNA synthesis and mitosis, and arrested blastomeres have abnormal spindles, clustered centrosomes, and an apparent failure of cytokinesis. In Xenopus S3 cells, transfection of TPX2-FL causes spindle collapse, whereas TPX2-CT blocks pole segregation, resulting in apposing spindle poles with no evident displacement of Aurora A. Analysis of TPX2-CT deletion peptides revealed that only constructs able to interact with the class 5 kinesin-like motor protein Eg5 induce the spindle phenotypes. Importantly, injection of Eg5 into TPX2-CT-arrested blastomeres causes resumption of cleavage. These results define a discrete domain within the C terminus of TPX2 that exerts a novel Eg5-dependent function in spindle pole segregation.

Proper chromosome segregation is required to maintain the appropriate number of chromosomes from one cell generation to another and to prevent aneuploidy, which is mainly found in solid cancers. A correct mitotic spindle is necessary to accomplish such a process. Aurora kinases play critical roles in chromosome segregation and cell division; their deregulation impairs spindle assembly, checkpoint function and cell division causing chromosome mis-segregation. These kinases have been implicated in tumorigenesis. Aurora-A (AurA), in particular has been identified as a cancer-susceptibility gene, is overexpressed in a number of tumors and is required for G2/M transition and spindle assembly. ASAP is a novel spindle-associated protein, the deregulation of which induces severe mitotic defects. We show here that ASAP is a novel substrate of AurA kinase. We have identified serine 625 as the major phosphorylation site for AurA in vivo and localized the phosphorylated form of ASAP to centrosomes from late G2 to telophase, and around the midbody during cytokinesis. AurA depletion induces a proteasome-dependent degradation of ASAP. ASAP depletion induces spindle defects rescued by the expression of the phosphorylation-mimetic mutant ASAP-S625E and not by the non-phosphorylatable mutant ASAP-S625A. Microinjection of mono-specific S625 phospho-antibodies also impaired spindle formation and mitosis. These results strongly indicate that the phosphorylation of ASAP on S625 by AurA is required for bipolar spindle assembly and is essential for a correct mitotic progression. All together, these results suggest that we have identified a novel AurA substrate, pointing out ASAP as a new potential target for antitumoral drugs.

A remarkable study published in this issue of Cell reveals a key role of Aurora A protein kinase in G2/M progression. To achieve this role, Aurora A acts in conjunction with the LIM protein Ajuba, which functions as an activating factor.

Successful cell division requires that daughter cells inherit not only a complete set of chromosomes, but also only one centrosome, and similar amounts of organelles and cytoplasmic components. The different mitotic processes are driven by cell cycle-regulated protein kinases and phosphatases and their fidelity is closely monitored by a number of checkpoint mechanisms. Histone H3 is phosphorylated during mitosis, but the kinases involved were not known until recently. Recent work has revealed that Aurora kinases are required for mitotic phosphorylation of histone H3 and of its centromeric variant CENP-A. This finding has stimulated functional studies of the role(s) of Aurora kinases and H3 phosphorylation during mitosis, which are reviewed in this chapter.

In mammalian cells, the separation of centrosomes is a prerequisite for bipolar mitotic spindle assembly. We have investigated the respective contribution of the two cytoskeleton components, microtubules and actin filaments, in this process. Distances between centrosomes have been measured during cell cycle progression in Xenopus laevis XL2 cultured cells in the presence or absence of either network. We considered two stages in centrosome separation: the splitting stage, when centrosomes start to move apart (minimum distance of 1 microm), and the elongation stage (from 1 to 7 microm). In interphase, depolymerisation of microtubules by nocodazole significantly inhibited the splitting stage, while the elongation stage was, on the contrary, facilitated. In mitosis, while nocodazole treatment completely blocked spindle assembly, in prophase, we observed that 55% of the centrosomes separated, versus 94% in the control. Upon actin depolymerisation by latrunculin, splitting of the interphase centrosome was blocked, and cells entered mitosis with unseparated centrosomes. Cells compensated for this separation delay by increasing the length of both prophase and prometaphase stages to allow for centrosome separation until a minimal distance was reached. Then the cells passed through anaphase, performing proper chromosome separation, but cytokinesis did not occur, and binuclear cells were formed. Our results clearly show that the actin microfilaments participate in centrosome separation at the G2/M transition and work in synergy with the microtubules to accelerate centrosome separation during mitosis.

Disruption of the function of the A-type Aurora kinase of Drosophila by mutation or RNAi leads to a reduction in the length of astral microtubules in syncytial embryos, larval neuroblasts, and cultured S2 cells. In neuroblasts, it can also lead to loss of an organized centrosome and its associated aster from one of the spindle poles, whereas the centrosome at the other pole has multiple centrioles. When centrosomes are present at the poles of aurA mutants or aurA RNAi spindles, they retain many antigens but are missing the Drosophila counterpart of mammalian transforming acidic coiled coil (TACC) proteins, D-TACC. We show that a subpopulation of the total Aurora A is present in a complex with D-TACC, which is a substrate for the kinase. We propose that one of the functions of Aurora A kinase is to direct centrosomal organization such that D-TACC complexed to the MSPS/XMAP215 microtubule-associated protein may be recruited, and thus modulate the behavior of astral microtubules.

Several serine-threonine kinases related to the Ipl1p kinase in budding yeast, termed aurora kinases, have been cloned recently. Their characterization revealed them to be important regulators of mitotic functions, including (i) the separation of the centrosome, (ii) assembly of the spindles, and (iii) segregation of the chromosomes. The Perspective by Descamps and Prigent delves into the latest observations on aurora kinases in humans and the specific roles of each kinase within the process of mitosis.

During the past five years, a growing number of serine-threonine kinases highly homologous to the Saccharomyces cerevisiae Ipl1p kinase have been isolated in various organisms. A Drosophila melanogaster homologue, aurora, was the first to be isolated from a multicellular organism. Since then, several related kinases have been found in mammalian cells. They localise to the mitotic apparatus: in the centrosome, at the poles of the bipolar spindle or in the midbody. The kinases are necessary for completion of mitotic events such as centrosome separation, bipolar spindle assembly and chromosome segregation. Extensive research is now focusing on these proteins because the three human homologues are overexpressed in various primary cancers. Furthermore, overexpression of one of these kinases transforms cells. Because of the myriad of kinases identified, we suggest a generic name: Aurora/Ipl1p-related kinase (AIRK). We denote AIRKs with a species prefix and a number, e.g. HsAIRK1.