Studies of the Kinetochore Proteins of the Regenerating Liver and the Liver Cells of Rats at Different Stages of Development

The kinetochore composition of rat liver cells was studied by indirect immunofluorescence andimmunoblotting using human anti-kinetochore/centromere autoantibodies(ACAs).Besides threemajor antigens(50kD,42 kD and 34 kD),ACAs used in this study could also identify those of 32-30 kD and 20 kD in newborn rat liver cells,90 kD in old rat liver cells,37 kD and 32-30 kD inregenerating liver cells.These results indicate that some kinetochore antigen(s)may be related to cellproliferation or specific for different stages of development.

Mitosin/CENP-F is a conserved kinetochore protein subjected to cyto plasmic dynein-mediated poleward transport

Mitosin/CENP-F is a human nuclear protein transiently associated with the outer kinetochore plate in M phase and is involved in M phase progression. LEK1 and CMF1, which are its murine and chicken orthologs, however, are implicated in muscle differentiation and reportedly not distributed at kinetochores.We therefore conducted several assays to clarify this issue. The typical centromere staining patterns were observed in mitotic cells from both human primary culture and murine, canine, and mink cell lines. A C-terminal portion of LEK1 also conferred centromere localization. Our analysis further suggests conserved kinetochore localization of mammalian mitosin orthologs. Moreover, mitosin was associated preferentially with kinetochores of unaligned chromosomes. It was also constantly transported from kinetochores to spindle poles by cytoplasmic dynein. These properties resemble those of other kinetochore proteins important for the spindle checkpoint, thus implying a role of mitosin in this checkpoint. Therefore, mitosin family may serve as multifunctional proteins involved in both mitosis and differentiation.

Kinetochore dynein generates a poleward pulling force to facilitate congression and full chromosome alignment

For proper chromosome segregation, all kinetochores must achieve bipolar microtubule （MT） attachment and subsequently align at the spindle equator before anaphase onset. The MT minus end-directed motor dynein/dynactin binds kinetoehores in prometaphase and has long been implicated in chromosome congression. Unfortunately, inactivation of dynein usually disturbs spindle organization, thus hampering evaluation of its kinetochore roles. Here we specifically eliminated kinetochore dynein/dynactin by RNAi-mediated depletion of ZW10, a protein essential for kinetochore localization of the motor. Time-lapse microscopy indicated markedly-reduced congression efficiency, though congressing chromosomes displayed similar velocities as in control cells. Moreover, cells frequently failed to achieve full chromosome alignment, despite their normal spindles. Confocal microcopy revealed that the misaligned kinetochores were monooriented or unattached and mostly lying outside the spindle, suggesting a difficulty to capture MTs from the opposite pole. Kinetoehores on monoastral spindles were dispersed farther away from the pole and exhibited only mild oscillation. Furthermore, inactivating dynein by other means generated similar phenotypes. Therefore, kinetochore dynein produces on monooriented kinetochores a poleward pulling force, which may contribute to efficient bipolar attachment by facilitating their proper microtubule captures to promote congression as well as full chromosome alignment.

Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis

Cyclin B1 is a key regulatory protein controlling cell cycle progression in vertebrates. Cyclin B1 binds CDK1, a cy-clin-dependent kinase catalytic subunit, forming a complex that orchestrates mitosis through phosphorylation of key proteins. Cyclin B1 regulates both the activation of CDK1 and its subcellular localization, which may be critical for substrate selection. Here, we demonstrate that cyclin B1 is concentrated on the outer plate of the kinetochore during prometaphase. This localization requires the cyclin box region of the protein. Cyclin B1 is displaced from individual kinetochores to the spindle poles by microtubule attachment to the kinetochores, and this displacement is dependent on the dynein/dynactin complex. Depletion of cyclin B1 by vector-based siRNA causes inefficient attachment between kinetochores and microtubules, and chromosome alignment defects, and delays the onset of anaphase. We conclude that cyclin B1 accumulates at kinetochores during prometaphase, where it contributes to the correct attachment of mi- crotubules to kinetochores and efficient alignment of the chromosomes, most likely through localized phosphorylation of specific substrates by cyclin B1-CDK1. Cyclin B1 is then transported from each kinetochore as microtubule attachment is completed, and this relocalization may redirect the activity of cyclin B1-CDK1 and contribute to inactivation of the spindle assembly checkpoint.

MPF Induction of Microtubule Assembly at Interphase Kinetochore of CHO Cells

Properties of the interphase microtubule organizing centers (MTOCs) of CHO cells were investigated using indirect immunofluorescence staining technique.Microtubule proteins were isolated from swine brain by three cycles of assembly-disassembly- 6S tubulin was purified by chromatograph y through cellulose phosphate p-11 column. Rabbits were immunized with the purified 6S tubulin and the anti-6S tubulin antiserum thus prepared was used in the indirect immunofluorescence staining of nuclei With double thymidine block method, we obtained cells at G1/S, S and G2 phases of cell cycle and isolated nuclei from cells of each pltase, Immunofluorescence staining of MTOCs could hardly he detected in the Triton X-100 treated cells nor in nuclei of any phase after preincubation with purified 6S tubulin alone. Yet,if the Triton X-100 treated cells or nuclei were incubated, in the presence of 5 mM ATP, with maturation promoting factor (MPF)isolated from mature oocytes of Xenopus for 40 minutes before the addition of the 6S tubulin, specifically stained MTOCs were clearly demonstrated in S and G2 nuclei Furthermore, microbubules could be observed radiating from the MTOCs in late S and G2 nuclei. However, alkaline pHospHatase treatment of the nuclei which had been incubated with the activated MPF prohibited the staining of MTOCs, and MPF isolated from premature oocytes failed to contribute to the immunostaining of MTOCs. Our results thus suggest that the transformation of kinetochores into MTOCs during cell cycle requires activated MPF and phosphorylation of proteins.

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。

Phosphorylation of human Sgo1 by NEK2A is essential for chromosome congression in mitosis

Chromosome segregation in mitosis is orchestrated by the interaction of the kinetochore with spindle microtubules. Our recent study shows that NEK2A interacts with MAD 1 at the kinetochore and possibly functions as a novel integrator of spindle checkpoint signaling. However, it is unclear how NEK2A regulates kinetochore-microtubule attachment in mitosis. Here we show that NEK2A phosphorylates human Sgo 1 and such phosphorylation is essential for faithful chromosome congression in mitosis. NEK2A binds directly to HsSgol in vitro and co-distributes with HsSgol to the kinetochore of mitotic cells. Our in vitro phosphorylation experiment demonstrated that HsSgo 1 is a substrate of NEK2A and the phosphorylation sites were mapped to Ser^14 and Ser^507 as judged by the incorporation of 32^P. Although such phosphorylation is not required for assembly of HsSgo 1 to the kinetochore, expression of non-phosphorylatable mutant HsSgo 1 perturbed chromosome congression and resulted in a dramatic increase in microtubule attachment errors, including syntelic and monotelic attachments. These findings reveal a key role for the NEK2A-mediated phosphorylation ofHsSgo 1 in orchestrating dynamic kinetochore-microtubule interaction. We propose that NEK2A-mediated phosphorylation of human Sgo 1 provides a link between centromeric cohesion and spindle microtubule attachment at the kinetochores.

Dynamic distribution of TTK in HeLa cells: insights from an ultrastructural study

Entry into mitosis is driven by signaling cascades of mitotic kinases.Our recent studies show that TTK,a kinetochore-associated protein kinase,interacts with CENP-E,a mitotic kinesin located to corona fiber ofkinetochore.Using immunoelectron microscopy,here we show that TTK is present at the nuclear pore adjacent complex of interphase HeLa cells.Upon nuclear envelope fragmentation,TTK targets to the outermostregion of the developing kinetochores ofmonoorient chromosome as well as to spindle poles.After stable attachment,throughout chromosome congression,TTK is a constituent of the corona fibers,extending up to 90 nm away from the kinetochore outer plate.Upon metaphase alignment,TTK departs from the kinetochore and migrates toward the centrosomes.Taken together,this evidence strongly supports a model in which TTK functions in spindle checkpoint signaling cascades at both kinetochore and centrosome.

The characteristics of the spindle fiber attachments (SFAs) enriched fraction from mouse nuclei

The characteristics of the particulate mouse centromere enriched fraction from isolated nuclei obtained in our laboratory were investigated by indirect immunoflu-orescence, test of the activity of microtubule organizing center(MTOC), SDS-PAGE, and fluorescence in situ hybridization. Most of the particles of the fraction are complexes of DNA and kinetochore proteins and show MTOC activity. The DNA isolated from the fraction can hybridize with DNA in the regions of the primary constrictions of all chromosomes of ascites cells. The kinetochore proteins isolated from the fraction are mainly those with molecular weight of 55 KD and 59 KD. Results suggested that the fraction obtained is a centromere enriched nuclear fraction as indicated in our previous report.

Aurora B promotes the CENP-T–CENP-W interaction to guide accurate chromosome segregation in mitosis

Accurate chromosome segregation in mitosis depends on kinetochores that connect centromeric chromatin to spindle microtubules.Centromeres are captured by individual microtubules via a kinetochore constitutive centromere-associated network(CCAN)during chromosome segregation.CCAN contains 16 subunits,including CENP-W and CENP-T.However,the molecular recognition and mitotic regulation of the CCAN assembly remain elusive.Here,we revealed that CENP-W binds to the histone fold domain and an uncharacterized N-terminal region of CENP-T.Aurora B phosphorylates CENP-W at threonine 60,which enhances the interaction between CENP-W and CENP-T to ensure robust metaphase chromosome alignment and accurate chromosome segregation in mitosis.These findings delineate a conserved signaling cascade that integrates protein phosphorylation with CCAN integrity for the maintenance of genomic stability.

PLK1 phosphorylation of ZW10 guides accurate chromosome segregation in mitosis

Stable transmission of genetic information during cell division requires faithful chromosome segregation.Mounting evidence has demonstrated that polo-like kinase 1(PLK1)dynamics at kinetochores control correct kinetochore–microtubule attachments and subsequent silencing of the spindle assembly checkpoint.However,the mechanisms underlying PLK1-mediated silencing of the spindle checkpoint remain elusive.Here,we identified a regulatory mechanism by which PLK1-elicited zeste white 10(ZW10)phosphorylation regulates spindle checkpoint silencing in mitosis.ZW10 is a cognate substrate of PLK1,and the phosphorylation of ZW10 at Ser12 enables dynamic ZW10–Zwint1 interactions.Inhibition of ZW10 phosphorylation resulted in misaligned chromosomes,while persistent expression of phospho-mimicking ZW10 mutant caused premature anaphase,in which sister chromatids entangled as cells entered anaphase.These findings reveal the previously uncharacterized PLK1–ZW10 interaction through which dynamic phosphorylation of ZW10 fine-tunes accurate chromosome segregation in mitosis.

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.