HEP-2细胞染色体不稳定性与kinetochore变异

染色体非整倍性畸变是恶性肿瘤细胞的显著细胞遗传学特征,但其诱发染色体数目不稳定的机制一直尚未阐明。本研究从与染色体分离直接相关的动粒(kinetochore)角度,采用kine-tochore-NOR同步银染技术对HEP-2细胞染色体kinetochore变异进行分析,以探讨恶性肿瘤细胞染色体数目变异的形成机制。实验共分析HEP-2细胞分裂相308个,计染色体16 962条,正常对照个体外周血细胞分裂相300个,计染色体13 800条。结果表明:与正常人外周血细胞相比,HEP-2细胞染色体kinetochore缺失和kinetochore迟滞复制频率显著升高(P<0.01),而kinetochore-NOR融合频率二者没有显著差异(P>0.05),这些结果提示kinetochore缺失和kinetochore迟滞复制可能是HEP-2细胞染色体非整倍性变异起源的诱因之一。此外,我们还在某些HEP-2细胞染色体上观察到多重kinetochore现象,并认为染色体多重kinetochore可能是恶性肿瘤细胞染色体结构畸变产生的一个新的途径。

Protein kinase TTK interacts and co-localizes with CENP-E to the kinetochore of human cells

Spindle checkpoint is an important biochemical signaling cascade during mitosis which monitors the fidelity of chromosome segregation, and is mediated by protein kinases Mpsl and Bubl/BubRl. Our recent studies show that kinesin-related motor protein CENP-E interacts with BubRl and participates in spindle checkpoint signaling. To elucidate the molecular mechanisms underlying spindle checkpoint signaling, we carried out proteomic dissection of human cell kinetochore and revealed protein kinase TTK, human homologue of yeast Mpsl. Our studies show that TTK is localized to the kinetochore of human cells, and interacts with CENP-E, suggesting that TTK may play an important role in chromosome segregation during mitosis.

Phosphorylation of CENP-R by Aurora B regulates kinetochore-microtubule attachment for accurate chromosome segregation

Error-free mitosis depends on accurate chromosome attachment to spindle microtubules via a fine structure called the centromere that is epigenetically specified by the enrichment of CENP-A nucleosomes.Centromere maintenance during mitosis requires CENP-A-mediated deposition of constitutive centromere-associated network that establishes the inner kinetochore and connects centromeric chromatin to spindle microtubules during mitosis.Although previously proposed to be an adaptor of retinoic acid receptor,here,we show that CENP-R synergizes with CENP-OPQU to regulate kinetochore-microtubule attachment stability and ensure accurate chromosome segregation in mitosis.We found that a phospho-mimicking mutation of CENP-R weakened its localization to the kinetochore,suggesting that phosphorylation may regulate its localization.Perturbation of CENP-R phosphorylation is shown to prevent proper kinetochore-microtubule attachment at metaphase.Mechanistically,CENP-R phosphorylation disrupts its binding with CENP-U.Thus,we speculate that Aurora B-mediated CENP-R phosphorylation promotes the correction of improper kinetochore-microtubule attachment in mitosis.As CENP-R is absent from yeast,we reasoned that metazoan evolved an elaborate chromosome stability control machinery to ensure faithful chromosome segregation in mitosis.

CENP-K and CENP-H may form coiled-coils in the kinetochores

Kinetochores are large proteinaceous structure on the surface of chromosomes’ primary constriction during mitosis. They link chromosomes to spindle microtubules and also regulate the spindle assem- bly checkpoint, which is crucial for correct chromosome segregation in all eukaryotes. The better known core networks of kinetochores include the KMN network (K, KNL1; M, Mis12 complex; N, Ndc80 complex)and CCAN (constitutive centromere-associated network). However, the detailed molecular mechanism of the kinetochore protein network remains unclear. This study demonstrates that CENP-H and CENP-K form quite stable subcomplex by TAP (tandem affinity purification) with HEK 293 cells which express TAP-CENP-K, with the ratio of purified CENP-H and CENP-K being close to 1︰1 even with high salt. Bioinformatic analysis suggests that CENP-H and CENP-K are enriched with coiled-coil regions. This implies that CENP-H and CENP-K form heterodimeric coiled-coils. Furthermore, the func- tional regions which form the complex are respectively located on their N- and C-terminals, but the association between the C-terminals is more complex. It is possible that this is the first identified het- erodimeric coiled-coils within the inner kinetochore, which is directly involved in the attachment be- tween kinetochores and the spindle microtubules.

SW626细胞染色体动粒变异对其染色体不稳定的影响

目的探讨恶性肿瘤细胞染色体不稳定性的可能机制。方法采用本实验室建立的kinetochore-NOR同步银染技术,对SW626细胞染色体kinetochore变异进行分析。结果与正常人外周血细胞相比,SW626细胞染色体kineto-chore缺失[132(0.89%)vs26(0.18%)]、kinetochore迟滞复制[82(0.55%)vs14(0.10%)]和kinetochore-NOR融合[153(1.03%)vs51(0.37%)]频率显著升高(P<0.01),而不对称kinetochore频率二者差异性不显著(P>0.05)。此外,在某些SW626细胞染色体上还观察到多重kinetochores现象。结论 kinetochore缺失、kinetochore迟滞复制和kinetochore-NOR融合可能是SW626细胞染色体非整倍性变异起源的诱因之一;染色体多重kinetochores可能是恶性肿瘤细胞染色体结构畸变产生的重要途径之一。

Sgo1 interacts with CENP-A to guide accurate chromosome segregation in mitosis

Shugoshin-1(Sgo1)is necessary for maintaining sister centromere cohesion and ensuring accurate chromosome segregation during mitosis.It has been reported that the localization of Sgo1 at the centromere is dependent on Bub1-mediated phosphorylation of histone H2A at T120.However,it remains uncertain whether other centromeric proteins play a role in regulating the localization and function of Sgo1 during mitosis.Here,we show that CENP-A interacts with Sgo1 and determines the localization of Sgo1 to the centromere during mitosis.Further biochemical characterization revealed that lysine and arginine residues in the C-terminal domain of Sgo1 are critical for binding CENP-A.Interestingly,the replacement of these basic amino acids with acidic amino acids perturbed the localization of Sgo1 and Aurora B to the centromere,resulting in aberrant chromosome segregation and premature chromatid separation.Taken together,these findings reveal a previously unrecognized but direct link between Sgo1 and CENP-A in centromere plasticity control and illustrate how the Sgo1–CENP-A interaction guides accurate cell division.

High temperature increases centromeremediated genome elimination frequency and enhances haploid induction in Arabidopsis

Double haploid production is the most effective way to create true-breeding lines in a single generation.In Arabidopsis,haploid induction via mutation of the centromere-specific histone H3(cenH3)has been shown when the mutant is outcrossed to the wild-type,and the wild-type genome remains in the haploid progeny.However,factors that affect haploid induction are still poorly understood.Here,we report that a mutant of the cenH3 assembly factor Kinetochore Null2(KNL2)can be used as a haploid inducer when pollinated by the wild-type.We discovered that short-term temperature stress of the knl2 mutant increased the efficiency of haploid induction 10-fold.We also demonstrated that a point mutation in the CENPC-k motif of KNL2 is sufficient to generate haploid-inducing lines,suggesting that haploidinducing lines in crops can be identified in a naturally occurring or chemically induced mutant population,avoiding the generic modification(GM)approach at any stage.Furthermore,a cenh3-4 mutant functioned as a haploid inducer in response to short-term heat stress,even though it did not induce haploids under standard conditions.Thus,we identified KNL2 as a new target gene for the generation of haploid-inducer lines and showed that exposure of centromeric protein mutants to high temperature strongly increases their haploid induction efficiency.

Dentromere Size and Its Relationship to Haploid Formation in Plants

Wide species crosses often result in uniparental genome elimination and visible failures in centromere func- tion. Crosses involving lines with mutated forms of the CENH3 histone variant that organizes the centromere/ kinetochore interface have been shown to have similar effects, inducing haploids at high frequencies. Here, we propose a simple centromere size model that endeavors to explain both observations. It is based on the idea of a quantitative centromere architecture where each centromere in an individual is the same size, and the average size is dictated by a natural equilibrium between bound and unbound CENH3 （and its chaperones or binding proteins）. While centromere size is determined by the cellular milieu, centromere positions are heritable and defined by the interactions of a small set of proteins that bind to both DNA and CENH3. Lines with defective or mutated CENH3 have a lower loading capacity and support smaller centromeres. In cases where a line with small or defective centromeres is crossed to a line with larger or normal centromeres, the smaller/defective centromeres are selectively degraded or not maintained, resulting in chromosome loss from the small-centromere parent. The model is testable and generalizable, and helps to explain the coun- terintuitive observation that inducer lines do not induce haploids when crossed to themselves.

Functions of spindle check-point and its relationship to chromosome instability

It is generally believed that the equal distribution of genetic materials to two daughter cells during mitosis is the key to cell health and development. During the dynamic process, spindle checkpoint plays a very important role in chromosome movements and final sister chromatid separation. The equal and precise segregation of chromosomes contributes to the genomic stability while aberrant separations result in chromosome instability that causes pathogenesis of certain diseases such as Down’s syndrome and cancers. Kinetochore and its regulatory proteins consist of the spindle checkpoint and determine the spatial and temporal orders of chromosome segregation.

Mps1 dimerization and multisite interactions with Ndc80 complex enable responsive spindle assembly checkpoint signaling

Error-free mitosis depends on accurate chromosome attachment to spindle microtubules,which is monitored by the spindle assembly checkpoint(SAC)signaling.As an upstream factor of SAC,the precise and dynamic kinetochore localization of Mps1 kinase is critical for initiating and silencing SAC signaling.However,the underlying molecular mechanism remains elusive.Here,we demonstrated that the multisite interactions between Mps1 and Ndc80 complex(Ndc80C)govern Mps1 kinetochore targeting.Importantly,we identified direct interaction between Mps1 tetratricopeptide repeat domain and Ndc80C.We further identified that Mps1 C-terminal fragment,which contains the protein kinase domain and C-tail,enhances Mps1 kinetochore localization.Mechanistically,Mps1 C-terminal fragment mediates its dimerization.Perturbation of C-tail attenuates the kinetochore targeting and activity of Mps1,leading to aberrant mitosis due to compromised SAC function.Taken together,our study highlights the importance of Mps1 dimerization and multisite interactions with Ndc80C in enabling responsive SAC signaling.

Mitotic dynamics

A new model for mitotic dynamics of eukaryotic cells is proposed. In the kinetochore mo-tor-midzone motor model two kinds of motors, the kinetochore motors and the midzone motors, play important roles in chromosome movement. Using this model the chromosome congression during prometaphase, the chromosome oscillation during metaphase and the chromatid segregation during anaphase are described in a unified way.

Crystal structure of a PP2A B56-BubR1 complex and its implications for PP2A substrate recruitment and localization

Protein phosphatase 2A （PP2A） accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a cat- alytic C subunit, and one of the regulatory/targeting B subunits. How the B subunit controls PP2A localization and substrate specificity, which is a crucial aspect of PP2A regulation, remains poorly understood. The kine- tochore is a critical site for PP2A functioning, where PP2A orchestrates chromosome segregation through its interactions with BubR1. The PP2A-BubR1 interac- tion plays important roles in both spindle checkpoint silencing and stable microtubule-kinetochore attach- ment. Here we present the crystal structure of a PP2A B56-BubRI complex, which demonstrates that a con- served BubRl LxxlxE motif binds to the concave side of the B56 pseudo-HEAT repeats. The BubR1 motif binds to a groove formed between B56 HEAT repeats 3 and 4, which is quite distant from the B56 binding surface for PP2A catalytic C subunit and thus is unlikely to affect PP2A activity. In addition, the BubR1 binding site on B56 is far from the B56 binding site of shugoshin, another kinetochore PP2A-binding protein, and thus BubR1 and shugoshin can potentially interact with PP2A-B56 simultaneously. Our structural and biochemical analysis indicates that other proteins with the LxxlxE motif may also bind to the same PP2A B56 surface. Thus, our structure of the PP2A B56-BubR1 complex provides important insights into how the B56 subunit directs the recruitment of PP2A to specific targets.

Spatial separation of phosphatase and kinase activity within the Bub complex is required for proper mitosis

The Bub1 and BubR1 kinetochore proteins support proper chromosome segregation and mitotic checkpoint activity. Bub1 and BubR1 are paralogs with Bub1 being a kinase, while BubR1 localizes the PP2A-B56 protein phosphatase to kinetochores in humans. Whether this spatial separation of kinase and phosphatase activity is important is unclear as some organisms integrate both activities into one Bub protein. Here, we engineer human Bub1 and BubR1 proteins integrating kinase and phosphatase activities into one protein and show that these do not support normal mitotic progression. A Bub1–PP2A-B56 complex can support chromosome alignment but results in impairment of the checkpoint due to dephosphorylation of the Mad1 binding site in Bub1. Furthermore, a chimeric BubR1 protein containing the Bub1 kinase domain induces delocalized H2ApT120 phosphorylation, resulting in the reduction of centromeric hSgo2 and chromosome segregation errors. Collectively, these results argue that the spatial separation of kinase and phosphatase activities within the Bub complex is required for balancing its functions in the checkpoint and chromosome alignment.