EVI5 protein associates with the INCENP-aurora B kinase-survivin chromosomal passenger complex and is involved in the completion of cytokinesis

EVI5 has been shown to be a novel centrosomal protein in interphase cells. In this report, we demonstrate using immunofluorescence microscopy that EVI5 has a dynamic distribution during mitosis, being associated with the mitotic spindle through anaphase and remaining within the midzone and midbody until completion of cytokinesis. Knockdown of EVI5 using siRNA results in a multinucleate phenotype, which is consistent with an essential role for this protein in the completion of cytokinesis. The EVI5 protein also undergoes posttranslational modifications during the cell cycle, which involve phosphorylation in early mitosis and proteolytic cleavage during late mitosis and cytokinesis. Since the subcellular distribution of the EVI5 protein was similar to that characteristic of chromosomal passenger proteins during the terminal stages of cytokinesis, we used immunoprecipitation and GST pull-down approaches to demonstrate that EVI5 is associated with the aurora B kinase protein complex (INCENP, aurora B kinase and survivin). Together, these data provide evidence that EVI5 is an essential component of the protein machinery facilitating the final stages of cell septation at the end of mitosis.     

Xenopus Shugoshin 2 regulates the spindle assembly pathway mediated by the chromosomal passenger complex

Shugoshins (Sgo) are conserved proteins that act as protectors of centromeric cohesion and as sensors of tension for the machinery that eliminates improper kinetochore-microtubule attachments. Most vertebrates contain two Sgo proteins, but their specific functions are not always clear. Xenopus laevis Sgo1, XSgo1, protects centromeric cohesin from the prophase dissociation pathway. Here, we report the identification of XSgo2 and show that it does not regulate cohesion. Instead, we find that it participates in bipolar spindle assembly. Both Sgo proteins interact physically with the Chromosomal Passenger Complex (CPC) containing Aurora B, a key regulator of mitosis, but the functional consequences of such interaction are distinct. XSgo1 is required for proper localization of the CPC while XSgo2 positively contributes to its activation and the subsequent phosphorylation of at least one key substrate for bipolar spindle assembly, the microtubule depolymerizing kinesin MCAK (Mitotic Centromere-Associated Kinesin). Thus, the two Xenopus Sgo proteins have non-overlapping functions in chromosome segregation. Our results further suggest that this functional specificity could rely on the association of XSgo1 and XSgo2 with different regulatory subunits of the PP2A complex.     

Inhibitor-3 ensures bipolar mitotic spindle attachment by limiting association of SDS22 with kinetochore-bound protein phosphatase-1

Faithful chromosome segregation during mitosis is tightly regulated by opposing activities of Aurora B kinase and protein phosphatase-1 (PP1). PP1 function at kinetochores has been linked to SDS22, but the exact localization of SDS22 and how it affects PP1 are controversial. Here, we confirm that SDS22 is required for PP1 activity, but show that SDS22 does not normally localize to kinetochores. Instead, SDS22 is kept in solution by formation of a ternary complex with PP1 and inhibitor-3 (I3). Depletion of I3 does not affect the amount of PP1 at kinetochores but causes quantitative association of SDS22 with PP1 on KNL1 at the kinetochore. Such accumulation of SDS22 at kinetochores interferes with PP1 activity and inhibits Aurora B threonine-232 dephosphorylation, which leads to increased Aurora B activity in metaphase and persistence in anaphase accompanied with segregation defects. We propose a model in which I3 regulates an SDS22-mediated PP1 activation step in solution that precedes SDS22 dissociation and transfer of PP1 to kinetochores, and which is required for PP1 to efficiently antagonize Aurora B.     

The chromosomal passenger complex controls the function of endosomal sorting complex required for transport-III Snf7 proteins during cytokinesis

Cytokinesis controls the proper segregation of nuclear and cytoplasmic materials at the end of cell division. The chromosomal passenger complex (CPC) has been proposed to monitor the final separation of the two daughter cells at the end of cytokinesis in order to prevent cell abscission in the presence of DNA at the cleavage site, but the precise molecular basis for this is unclear. Recent studies indicate that abscission could be mediated by the assembly of filaments comprising components of the endosomal sorting complex required for transport-III (ESCRT-III). Here, we show that the CPC subunit Borealin interacts directly with the Snf7 components of ESCRT-III in both Drosophila and human cells. Moreover, we find that the CPC's catalytic subunit, Aurora B kinase, phosphorylates one of the three human Snf7 paralogues-CHMP4C-in its C-terminal tail, a region known to regulate its ability to form polymers and associate with membranes. Phosphorylation at these sites appears essential for CHMP4C function because their mutation leads to cytokinesis defects. We propose that CPC controls abscission timing through inhibition of ESCRT-III Snf7 polymerization and membrane association using two concurrent mechanisms: interaction of its Borealin component with Snf7 proteins and phosphorylation of CHMP4C by Aurora B.     

The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly

Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1-GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.     

Salt-inducible kinase 3 is a novel mitotic regulator and a target for enhancing antimitotic therapeutic-mediated cell death

Many mitotic kinases are both critical for maintaining genome stability and are important targets for anticancer therapies. We provide evidence that SIK3 (salt-inducible kinase 3), an AMP-activated protein kinase-related kinase, is important for mitosis to occur properly in mammalian cells. Downregulation of SIK3 resulted in an extension of mitosis in both mouse and human cells but did not affect the DNA damage checkpoint. Time-lapse microscopy and other approaches indicated that mitotic exit but not mitotic entry was delayed. Although repression of SIK3 alone simply delayed mitotic exit, it was able to sensitize cells to various antimitotic chemicals. Both mitotic arrest and cell death caused by spindle poisons were enhanced after SIK3 depletion. Likewise, the antimitotic effects due to pharmacological inhibition of mitotic kinases including Aurora A, Aurora B, and polo-like kinase 1 were enhanced in the absence of SIK3. Finally, in addition to promoting the sensitivity of a small-molecule inhibitor of the mitotic kinesin Eg5, SIK3 depletion was able to overcome cells that developed drug resistance. These results establish the importance of SIK3 as a mitotic regulator and underscore the potential of SIK3 as a druggable antimitotic target.     

Spatial regulation of Aurora A activity during mitotic spindle assembly requires RHAMM to correctly localize TPX2

Construction of a mitotic spindle requires biochemical pathways to assemble spindle microtubules and structural proteins to organize these microtubules into a bipolar array. Through a complex with dynein, the receptor for hyaluronan-mediated motility (RHAMM) cross-links mitotic microtubules to provide structural support, maintain spindle integrity, and correctly orient the mitotic spindle. Here, we locate RHAMM to sites of microtubule assembly at centrosomes and non-centrosome sites near kinetochores and demonstrate that RHAMM is required for the activation of Aurora kinase A. Silencing of RHAMM delays the kinetics of spindle assembly, mislocalizes targeting protein for XKlp2 (TPX2), and attenuates the localized activation of Aurora kinase A with a consequent reduction in mitotic spindle length. The RHAMM–TPX2 complex requires a C-terminal basic leucine zipper in RHAMM and a domain that includes the nuclear localization signal in TPX2. Together, our findings identify RHAMM as a critical regulator for Aurora kinase A signaling and suggest that RHAMM ensures bipolar spindle assembly and mitotic progression through the integration of biochemical and structural pathways.     

染色体移动复合物的结构、定位与功能

染色体移动复合物主要由蛋白激酶Aurora B、内层着丝粒蛋白、存活蛋白及蛋白Borealin组成。它在细胞分裂的不同阶段,能及时精确地定位到相关部位并作用于相应底物;具有调节染色质组蛋白磷酸化,控制姐妹染色单体的粘着、分离,参与分裂纺锤体组装及其对染色体的捕捉,纠正动粒与微管间不适当附着,将染色体精确分配到子细胞及促进胞浆分离等重要功能。文章简要介绍了染色体移动复合物的结构成分,在染色体臂部、内层着丝粒及纺锤体中区的定位过程,及其定位在不同部位的相应功能。     

Skp2 is required for Aurora B activation in cell mitosis and spindle checkpoint

The Aurora B kinase plays a critical role in cell mitosis and spindle checkpoint. Here, we showed that the ubiquitin E3-ligase protein Skp2, also as a cell-cycle regulatory protein, was required for the activation of Aurora B and its downstream protein. When we restored Skp2 knockdown Hela cells with Skp2 and Skp2-LRR E3 ligase dead mutant we found that Skp2 could rescue the defect in the activation of Aurora B, but the mutant failed to do so. Furthermore, we discovered that Skp2 could interact with Aurora B and trigger Aurora B Lysine (K) 63-linked ubiquitination. Finally, we demonstrated the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which was Aurora B dependent. Our results identified a novel ubiquitinated substrate of Skp2, and also indicated that Aurora B ubiquitination might serve as an important event for Aurora B activation in cell mitosis and spindle checkpoint.     

A dual role for Bub1 in the spindle checkpoint and chromosome congression

The spindle checkpoint ensures faithful chromosome segregation by linking the onset of anaphase to the establishment of bipolar kinetochore-microtubule attachment. The checkpoint is mediated by a signal transduction system comprised of conserved Mad, Bub and other proteins. In this study, we use live-cell imaging coupled with RNA interference to investigate the functions of human Bub1. We find that Bub1 is essential for checkpoint control and for correct chromosome congression. Bub1 depletion leads to the accumulation of misaligned chromatids in which both sister kinetochores are linked to microtubules in an abnormal fashion, a phenotype that is unique among Mad and Bub depletions. Bub1 is similar to the Aurora B/Ipl1p kinase in having roles in both the checkpoint and microtubule binding. However, human Bub1 and Aurora B are recruited to kinetochores independently of each other and have an additive effect when depleted simultaneously. Thus, Bub1 and Aurora B appear to function in parallel pathways that promote formation of stable bipolar kinetochore-microtubule attachments.     

Automated tracking of mitotic spindle pole positions shows that LGN is required for spindle rotation but not orientation maintenance

Spindle orientation defines the plane of cell division and, thereby, the spatial position of all daughter cells. Here, we develop a live cell microscopy-based methodology to extract spindle movements in human epithelial cell lines and study how spindles are brought to a pre-defined orientation. We show that spindles undergo two distinct regimes of movements. Spindles are first actively rotated toward the cells’ long-axis and then maintained along this pre-defined axis. By quantifying spindle movements in cells depleted of LGN, we show that the first regime of rotational movements requires LGN that recruits cortical dynein. In contrast, the second regime of movements that maintains spindle orientation does not require LGN, but is sensitive to 2ME2 that suppresses microtubule dynamics. Our study sheds first insight into spatially defined spindle movement regimes in human cells, and supports the presence of LGN and dynein independent cortical anchors for astral microtubules.     

Polo-like kinase-activating kinases

The events of cell division are regulated by a complex interplay between kinases and phosphatases. Cyclin-dependent kinases (Cdks), polo-like kinases (Plks) and Aurora kinases play central roles in this process. Polo kinase (Plk1 in humans) regulates a wide range of events in mitosis and cytokinesis. To ensure the accuracy of these processes, polo activity itself is subject to complex regulation. Phosphorylation of polo in its T loop (or activation loop) increases its kinase activity several-fold. It has been shown that Aurora A kinase, with its co-factor Bora, activates Plk1 in G₂, and that this is essential for recovery from cell cycle arrest induced by DNA damage. In a recent article published in PLoS Biology, we report that Drosophila polo is activated by Aurora B kinase at centromeres, and that this is crucial for polo function in regulating chromosome dynamics in prometaphase. Our results suggest that this regulatory pathway is conserved in humans. Here, we propose a model for the collaboration between Aurora B and polo in the regulation of kinetochore attachment to microtubules in early mitosis. Moreover, we suggest that Aurora B could also function to activate Polo/Plk1 in cytokinesis. Finally, we discuss recent findings and open questions regarding the activation of polo and polo-like kinases by different kinases in mitosis, cytokinesis and other processes.     

Mast, a conserved microtubule-associated protein required for bipolar mitotic spindle organization

Through mutational analysis in Drosopjila we have identified the gene multiple asters (mast), which encodes a new 165 kDa protein. mast mutant neuroblasts are highly polyploid and show severe mitotic abnormalities including the formation of mono- and multi-polar spindles organized by an irregular number of microtubule-organizing centres of abnormal size and shape. The mast gene product is evolutionarily conserved since homologues were identified from yeast to man, revealing a novel protein family. Antibodies against Mast and analysis of tissue culture cells expressing an enhanced green fluorescent protein-Mast fusion protein show that during mitosis, this protein localizes to centrosomes, the mitotic spindle, centromeres and spindle midzone. Microtubule-binding assays indicate that Mast is a microtubule-associated protein displaying strong affinity for polymerized microtubules. The defects observed in the mutant alleles and the intracellular localization of the protein suggest that Mast plays an essential role in centrosome separation and organization of the bipolar mitotic spindle.     

有丝分裂激酶Aurora B的研究进展

真核生物细胞通过有丝分裂将遗传物质均等地分配到两个子细胞中,从而维持基因组的稳定性。有丝分裂的每一环节都需要精准而细致的调控,这依赖于一系列调节机制,尤其需要多个相关激酶的共同协调。Aurora B是一个关键的有丝分裂调控激酶,伴随有丝分裂的进行,其先后在染色体臂、内着丝粒、中央纺锤体、中体上动态分布。与其高度时空动态性相一致的是,Aurora B在有丝分裂的多个环节,如姐妹染色体粘连、动粒微管连接、纺锤体检验点和胞质分裂过程中都发挥着一系列重要功能。本文将概述近年来Aurora B激酶功能与调控方面的研究进展。     

NuSAP,a novel microtubule-associated protein involved in mitotic spindle organization

Here, we report on the identification of nucleolar spindle-associated protein (NuSAP), a novel 55-kD vertebrate protein with selective expression in proliferating cells. Its mRNA and protein levels peak at the transition of G2 to mitosis and abruptly decline after cell division. Microscopic analysis of both fixed and live mammalian cells showed that NuSAP is primarily nucleolar in interphase, and localizes prominently to central spindle microtubules during mitosis. Direct interaction of NuSAP with microtubules was demonstrated in vitro. Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain. In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis. In addition, many NuSAP-depleted interphase cells had deformed nuclei. Both overexpression and knockdown of NuSAP impaired cell proliferation. These results suggest a crucial role for NuSAP in spindle microtubule organization.     

Polo-like kinase-activating kinases: Aurora A,Aurora B and what else?

The events of cell division are regulated by a complex interplay between kinases and phosphatases. Cyclin-dependent kinases (Cdks), polo-like kinases (Plks) and Aurora kinases play central roles in this process. Polo kinase (Plk1 in humans) regulates a wide range of events in mitosis and cytokinesis. To ensure the accuracy of these processes, polo activity itself is subject to complex regulation. Phosphorylation of polo in its T loop (or activation loop) increases its kinase activity several-fold. It has been shown that Aurora A kinase, with its co-factor Bora, activates Plk1 in G₂, and that this is essential for recovery from cell cycle arrest induced by DNA damage. In a recent article published in PLoS Biology, we report that Drosophila polo is activated by Aurora B kinase at centromeres, and that this is crucial for polo function in regulating chromosome dynamics in prometaphase. Our results suggest that this regulatory pathway is conserved in humans. Here, we propose a model for the collaboration between Aurora B and polo in the regulation of kinetochore attachment to microtubules in early mitosis. Moreover, we suggest that Aurora B could also function to activate Polo/Plk1 in cytokinesis. Finally, we discuss recent findings and open questions regarding the activation of polo and polo-like kinases by different kinases in mitosis, cytokinesis and other processes.     

Kizuna is a novel mitotic substrate for CDC25B phosphatase

CDC25 dual-specificity phosphatases play a central role in cell cycle control through the activation of Cyclin-Dependent Kinases (CDKs). Expression during mitosis of a stabilized CDC25B mutant (CDC25B-DDA), which cannot interact with the F-box protein βTrCP for proteasome-dependent degradation, causes mitotic defects and chromosome segregation errors in mammalian cells. We found, using the same CDC25B mutant, that stabilization and failure to degrade CDC25B during mitosis lead to the appearance of multipolar spindle cells resulting from a fragmentation of pericentriolar material (PCM) and abolish mitotic Plk1-dependent phosphorylation of Kizuna (Kiz), which is essential for the function of Kiz in maintaining spindle pole integrity. Thus, in mitosis Kiz is a new substrate of CDC25B whose dephosphorylation following CDC25B stabilization leads to the formation of multipolar spindles. Furthermore, endogenous Kiz and CDC25B interact only in mitosis, suggesting that Kiz phosphorylation depends on a balance between CDC25B and Plk1 activities. Our data identify a novel mitotic substrate of CDC25B phosphatase that plays a key role in mitosis control.     

Cell death in leukemia: passenger protein regulation by topoisomerase inhibitors

Etoposide is a potent inducer of mitotic catastrophe; a type of cell death resulting from aberrant mitosis. It is important in p53 negative cells where p53 dependent apoptosis and events at the G1 and G2 cell cycle checkpoints are compromised. Passenger proteins regulate many aspects of mitosis and siRNA interference or direct inhibition of Aurora B kinase results in mitotic catastrophe. However, there is little available data of clinical relevance in leukaemia models. Here, in p53 negative K562 myeloid leukemia cells, etoposide-induced mitotic catastrophe is shown to be time and/or concentration dependent. Survivin and Aurora remained bound to chromosomes. Survivin and Aurora were also associated with Cdk1 and were shown to form complexes, which in pull down experiments, included INCENP. There was no evidence of Aurora B kinase suppression. These data suggests etoposide will complement Aurora B kinase inhibitors currently in clinical trials for cancer.