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...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.展开更多
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, ...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.展开更多
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/dynact...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.展开更多
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 phosphor...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.展开更多
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 cyc...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.展开更多
Background:Lung adenocarcinoma(LUAD),the most prevalent histological subtype of lung cancer,remains a leading cause of cancer-related mortality due to late diagnosis,metastasis,and therapy resistance.The aim of the st...Background:Lung adenocarcinoma(LUAD),the most prevalent histological subtype of lung cancer,remains a leading cause of cancer-related mortality due to late diagnosis,metastasis,and therapy resistance.The aim of the study is to investigate the role of Kinetochore Scaffold 1(KNL1)in promoting LUAD progression and its underlying molecular regulatory mechanisms.Methods:KNL1 mRNA expression levels across 33 cancer types were analyzed using bioinformatics analysis based on the TCGA database.Immunohistochemistry(IHC)was used to assess KNL1 expression in LUAD and normal tissues.Stable KNL1-knockdown and KNL1-overexpressing LUAD cell lines were established using lentiviral infection.Western blotting(WB)was used to measure epithelial-mesenchymal transition(EMT)markers and ferroptosis-related protein expression.Cell migration was evaluated via scratch wound healing assays.The thiobarbituric acid(TBA)method was employed for the detection of malondialdehyde.a fluorescent probe was utilized to determine ferrous ion content.WB determined the phosphorylation ratios of AMP-activated protein kinase(AMPK)and mammalian target of rapamycin(mTOR)proteins.Results:1.KNL1 was highly expressed in 31 cancer types,including LUAD.Kaplan-Meier curves showed significantly shorter median survival in patients with high KNL1 expression.IHC confirmed upregulated KNL1 expression in LUAD tissues.2.KNL1 overexpression significantly promoted LUAD cell migration and increased mesenchymal marker expression,whereas KNL1 knockdown exerted opposite effects.3.KNL1 overexpression significantly reduced MDA content and Fe^(2+)levels in RSL3-treated LUAD cells while increasing the expression of key ferroptosis defense proteins;conversely,it markedly increased the accumulation of MDA and Fe^(2+)and downregulated these proteins.KNL1 overexpression significantly increased phosphorylated AMPK(p-AMPK)expression but decreased phosphorylated mTOR(p-mTOR)expression in RSL3-treated LUAD cells;conversely,it inhibited p-AMPK expression and activated p-mTOR.Conclusion:KNL1 promotes lung adenocarcinoma progression by suppressing ferroptosis through regulation of the AMPK-mTOR signaling pathway.展开更多
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 th...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.展开更多
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 maint...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.展开更多
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 ...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.展开更多
Dear Editor,Chromosome segregation in eukary-otes relies on intricate interactions between spindle microtubules and centromeres(Liu et al,2020).Recently,using cryo-electron microscopy(cryo-EM)alongside functional anal...Dear Editor,Chromosome segregation in eukary-otes relies on intricate interactions between spindle microtubules and centromeres(Liu et al,2020).Recently,using cryo-electron microscopy(cryo-EM)alongside functional analyses,we and other groups unveiled the molecular basis of how human kinetochore constitutive centromere-associated network(CCAN)interacts with duplex DNA and facilitates accurate chromosome segregation(Pesenti et al,2022;Tian et al.,2022;Yatskevich et al,.2022).While these investigations offer a foundational understanding of the physical and chemical interdependency of CCAN constituents,the functional significance of individual contacts within this complex remains to be fully elucidated.展开更多
Objective ZW10 interacting kinetochore protein(ZWINT)has been demonstrated to play a pivotal role in the growth,invasion,and migration of cancers.Nevertheless,whether the expression levels of ZWINT are significantly c...Objective ZW10 interacting kinetochore protein(ZWINT)has been demonstrated to play a pivotal role in the growth,invasion,and migration of cancers.Nevertheless,whether the expression levels of ZWINT are significantly correlated with clinicopathological characteristics and prognostic outcomes of patients with breast cancer remains elusive.This study systematically investigated the clinical significance of ZWINT expression in breast cancer through integrated molecular subtyping and survival analysis.Methods We systematically characterized the spatial expression pattern of ZWINT across various breast cancer subtypes and assessed its prognostic significance using an integrated bioinformatics approach that involved multi-omics analysis.The approach included the Breast Cancer Gene-Expression Miner v5.1(bc-GenExMiner v5.1),TNMplot,MuTarget,PrognoScan database,and Database for Annotation,Visualization,and Integrated Discovery(DAVID).Results Our analysis revealed consistent upregulation of ZWINT mRNA and protein expression across distinct clinicopathological subtypes of breast cancer.ZWINT overexpression demonstrated significant co-occurrence with truncating mutations in cadherin 1(CDH1)and tumor protein p53(TP53),suggesting potential functional crosstalk in tumor progression pathways.The overexpression of ZWINT correlated with adverse clinical outcomes,showing 48%increased mortality risk(overall survival:HR 1.48,95%CI 1.23–1.79),66%higher recurrence probability(relapse-free survival:1.66,95%CI 1.50–1.84),and 63%elevated metastasis risk(distant metastasis-free survival:HR 1.63,95%CI 1.39–1.90).Multivariate Cox regression incorporating TNM staging and molecular subtypes confirmed ZWINT as an independent prognostic determinant(P<0.001,Harrell’s C-index=0.7827),which was validated through bootstrap resampling(1000 iterations).Conclusion ZWINT may serve as a potential biomarker for prognosis and a possible therapeutic target alongside TP53/CDH1 in breast cancer.展开更多
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 ...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.展开更多
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 ofkinetoch...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.展开更多
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 organizi...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.展开更多
Dear Editor,During mitosis,the precise separation of chromosomes relies on the accurate connection between kinetochores and microtubules(Liu et al.,2020).Centromere protein C(CENP-C)is a central hub for kinetochore as...Dear Editor,During mitosis,the precise separation of chromosomes relies on the accurate connection between kinetochores and microtubules(Liu et al.,2020).Centromere protein C(CENP-C)is a central hub for kinetochore assembly and its cell-cycle function depends on post-translational modifications.Previous yeast and Caenorhabditis elegans studies suggest that the mitotic kinase polo-like kinase 1(PLK1)binds to the PEST-rich domain of CENP-C(Hinshaw et al.,2023;Taylor et al.,2023).Yeast CENP-C can be co-phosphorylated at 11 sites by DDK and CDC5(yeast PLK1),facilitating the assembly of the inner kinetochore,the constitutive centromere-associated network(CCAN).However,these phosphorylation sites are not conserved in humans(Hinshaw et al.,2023),raising the question of whether PLK1 regulates human inner kinetochore assembly via CENP-C.展开更多
Dear Editor,The kinetochore is a supermolecular complex that is assembled at each centromere in eukaryotes and functions as the attachment point for spindle microtubules to ensure accurate chromosome segregation durin...Dear Editor,The kinetochore is a supermolecular complex that is assembled at each centromere in eukaryotes and functions as the attachment point for spindle microtubules to ensure accurate chromosome segregation during mitosis.Both kinetochore assembly and attachment to spindle microtubules require robust activity of the chromosomal passenger complex(CPC),which is composed of Aurora B,Borealin,inner centromere protein(INCENP),and Survivin[1].展开更多
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/...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.展开更多
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 imp...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.展开更多
基金This work was supported by the National Natural Science Foundation of China
文摘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.
基金supported by grants 97JC14006 from Shanghai Committee of Science and Technology(No.30025021,39970160,and 39500030)from the Natural Science Foundation of ChinaKSCX2-2-02 from Chinese Academy of Sciences.
文摘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.
基金Acknowledgments The authors thank Qiongping Huang, Lirong Liu, and Wei Bian for technical assistance. We are grateful to Drs G Chan （Cross Cancer Institute, University of Alberta, Edmonton Alberta, Canada） for antibodies to human ZW 10 and Rod, KH Choo （Murdoch Children＇s Research Institute, Royal Children＇s Hospital, Melbourne, Australia） for anti-CREST serum, and E Fuchs （Rockefeller University, USA） for mRFP cDNA. This work was supported by the National Science Foundation of China （30330330, 30421005, and 30623003）, Ministry of Science and Technology of China （2005CB522703 and 2007CB914501）, and the Shanghai Municipal Council for Science and Technology （S048014317, 06DZ22032, and 058014578）.
文摘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.
文摘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.
文摘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.
基金supported by grants from Natural Science Foundation of Hunan Province(No.2022JJ50158).
文摘Background:Lung adenocarcinoma(LUAD),the most prevalent histological subtype of lung cancer,remains a leading cause of cancer-related mortality due to late diagnosis,metastasis,and therapy resistance.The aim of the study is to investigate the role of Kinetochore Scaffold 1(KNL1)in promoting LUAD progression and its underlying molecular regulatory mechanisms.Methods:KNL1 mRNA expression levels across 33 cancer types were analyzed using bioinformatics analysis based on the TCGA database.Immunohistochemistry(IHC)was used to assess KNL1 expression in LUAD and normal tissues.Stable KNL1-knockdown and KNL1-overexpressing LUAD cell lines were established using lentiviral infection.Western blotting(WB)was used to measure epithelial-mesenchymal transition(EMT)markers and ferroptosis-related protein expression.Cell migration was evaluated via scratch wound healing assays.The thiobarbituric acid(TBA)method was employed for the detection of malondialdehyde.a fluorescent probe was utilized to determine ferrous ion content.WB determined the phosphorylation ratios of AMP-activated protein kinase(AMPK)and mammalian target of rapamycin(mTOR)proteins.Results:1.KNL1 was highly expressed in 31 cancer types,including LUAD.Kaplan-Meier curves showed significantly shorter median survival in patients with high KNL1 expression.IHC confirmed upregulated KNL1 expression in LUAD tissues.2.KNL1 overexpression significantly promoted LUAD cell migration and increased mesenchymal marker expression,whereas KNL1 knockdown exerted opposite effects.3.KNL1 overexpression significantly reduced MDA content and Fe^(2+)levels in RSL3-treated LUAD cells while increasing the expression of key ferroptosis defense proteins;conversely,it markedly increased the accumulation of MDA and Fe^(2+)and downregulated these proteins.KNL1 overexpression significantly increased phosphorylated AMPK(p-AMPK)expression but decreased phosphorylated mTOR(p-mTOR)expression in RSL3-treated LUAD cells;conversely,it inhibited p-AMPK expression and activated p-mTOR.Conclusion:KNL1 promotes lung adenocarcinoma progression by suppressing ferroptosis through regulation of the AMPK-mTOR signaling pathway.
基金This work was supported in part by the National Natural Science Foundation of China(Grant No.39925018)the Key Project of the Chinese Academy of Sciences(Grant No.KSCX2-2-01).
文摘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.
基金Ministry of Science and Technology of China(MOST)grants(2017YFA0503600)National Natural Science Foundation of China(NSFC)grants(91854203,31621002,91853115,21922706,92153302,32090040,22177106,31871359,92053104,32100612,22137007,and 31970655)+3 种基金Ministry of Education(IRT_17R102,20113402130010,and YD2070006001)Strategic Priority Research Program of the Chinese Academy of Sciences(XDB19040000)Anhui Provincial Natural Science Foundation Grant(2108085J15 and 1908085MC64)Fundamental Research Funds for the Central Universities(WK2070000066 and WK2070000194).
文摘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.
基金Supported by the National Key Scientific Program(Grant No.2006CB910100).
文摘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.
基金supported by grants from the Ministry of Science and Technology of China and the National Natural Science Foundation of China(2022YFA1303100,32090040,92254302,2022YFA0806800,92153302,W2411017,32400593,92253301,22137007,and 92053104)the Plansfor Major Provincial Science&Technology Projects of Anhui Province(202303a0702003).F.A.,L.Z.,X.L.,and X.Y.conceived the project and designed the experiments.O.A.D.and X.G.performed the chemical screening.O.A.D.and F.Y.performed the experiments and analyzed the data.O.A.D.,L.Z.,and X.Y.wrote the manuscript.X.L.and X.Y.supervised the project.
文摘Dear Editor,Chromosome segregation in eukary-otes relies on intricate interactions between spindle microtubules and centromeres(Liu et al,2020).Recently,using cryo-electron microscopy(cryo-EM)alongside functional analyses,we and other groups unveiled the molecular basis of how human kinetochore constitutive centromere-associated network(CCAN)interacts with duplex DNA and facilitates accurate chromosome segregation(Pesenti et al,2022;Tian et al.,2022;Yatskevich et al,.2022).While these investigations offer a foundational understanding of the physical and chemical interdependency of CCAN constituents,the functional significance of individual contacts within this complex remains to be fully elucidated.
基金supported by the Research Project of Maternal and Child Health Hospital of Hubei Province(No.2023SFYM008)Key Project of Hubei Provincial Natural Science Foundation(No.JCZRLH202500304).
文摘Objective ZW10 interacting kinetochore protein(ZWINT)has been demonstrated to play a pivotal role in the growth,invasion,and migration of cancers.Nevertheless,whether the expression levels of ZWINT are significantly correlated with clinicopathological characteristics and prognostic outcomes of patients with breast cancer remains elusive.This study systematically investigated the clinical significance of ZWINT expression in breast cancer through integrated molecular subtyping and survival analysis.Methods We systematically characterized the spatial expression pattern of ZWINT across various breast cancer subtypes and assessed its prognostic significance using an integrated bioinformatics approach that involved multi-omics analysis.The approach included the Breast Cancer Gene-Expression Miner v5.1(bc-GenExMiner v5.1),TNMplot,MuTarget,PrognoScan database,and Database for Annotation,Visualization,and Integrated Discovery(DAVID).Results Our analysis revealed consistent upregulation of ZWINT mRNA and protein expression across distinct clinicopathological subtypes of breast cancer.ZWINT overexpression demonstrated significant co-occurrence with truncating mutations in cadherin 1(CDH1)and tumor protein p53(TP53),suggesting potential functional crosstalk in tumor progression pathways.The overexpression of ZWINT correlated with adverse clinical outcomes,showing 48%increased mortality risk(overall survival:HR 1.48,95%CI 1.23–1.79),66%higher recurrence probability(relapse-free survival:1.66,95%CI 1.50–1.84),and 63%elevated metastasis risk(distant metastasis-free survival:HR 1.63,95%CI 1.39–1.90).Multivariate Cox regression incorporating TNM staging and molecular subtypes confirmed ZWINT as an independent prognostic determinant(P<0.001,Harrell’s C-index=0.7827),which was validated through bootstrap resampling(1000 iterations).Conclusion ZWINT may serve as a potential biomarker for prognosis and a possible therapeutic target alongside TP53/CDH1 in breast cancer.
基金We thank members of our group for insightful discussion during the course of this study.This work was supported by grants from Chinese Academy of Science(KSCX1-YW-R65,KSCX2-YW-H10)National Basic Research Program of China(2002CB713700)+4 种基金Hi-Tech Research and Development Program of China(2001AA215331)Chinese Minister of Education(20020358051 to XY,PCSIRT0413 to XD)National Natural Science Foundation of China(39925018,30270293 to XY,30500183 to XD,30600222 to JY)National Institutes of Health(USA)(DK56292,CA92080)to XY(a Georgia Cancer Coalition Eminent Scholar)JY was supported by China Postdoctor(2005037560).
文摘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.
基金supported by grants from the Chinese Outstanding Young Scientist Award(39925018)the Chinese Academy of Science(KSCX2-2-01)+1 种基金the Chinese 973 project(2002CB713700)the American Cancer Society(RPG59282)to XY.
文摘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.
基金China National"863 Project"for Biotechmoloigt Development
文摘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.
基金supported by grants from the Ministry of Science and Technology of China and the National Natural Science Foundation of China(2022YFA1303100,32090040,92254302,W2411017,2022YFA0806800,and 92153302)the Plans for Major Provincial Science&Technology Projects of Anhui Province(202303a0702003)+2 种基金the Ministry of Education(IRT_17R102)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB19040000)the Fundamental Research Funds for the Central Universities(KB9100000007,KB9100000006,and KB9100000013).
文摘Dear Editor,During mitosis,the precise separation of chromosomes relies on the accurate connection between kinetochores and microtubules(Liu et al.,2020).Centromere protein C(CENP-C)is a central hub for kinetochore assembly and its cell-cycle function depends on post-translational modifications.Previous yeast and Caenorhabditis elegans studies suggest that the mitotic kinase polo-like kinase 1(PLK1)binds to the PEST-rich domain of CENP-C(Hinshaw et al.,2023;Taylor et al.,2023).Yeast CENP-C can be co-phosphorylated at 11 sites by DDK and CDC5(yeast PLK1),facilitating the assembly of the inner kinetochore,the constitutive centromere-associated network(CCAN).However,these phosphorylation sites are not conserved in humans(Hinshaw et al.,2023),raising the question of whether PLK1 regulates human inner kinetochore assembly via CENP-C.
基金supported by MOST-NSFC grants(2022YFA1303100,32450537,2022YFA1302700,32090040,W2411017,92254302,2022YFA0806800,92153302,91953000,31621002,and 2017YFA0503600)Plans for Major Provincial Science&Technology Projects of Anhui Province(202303-a0702003)+4 种基金the Ministry of Education(IRT_17R102)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB19040000)the Fundamental Research Funds for the Central Universities(KB9100000007,KB9100000006,and KB9100000013)the Anhui Provincial Natural Science Foundation(2408085MC050)the USTC start-up fund(KY9990000167).
文摘Dear Editor,The kinetochore is a supermolecular complex that is assembled at each centromere in eukaryotes and functions as the attachment point for spindle microtubules to ensure accurate chromosome segregation during mitosis.Both kinetochore assembly and attachment to spindle microtubules require robust activity of the chromosomal passenger complex(CPC),which is composed of Aurora B,Borealin,inner centromere protein(INCENP),and Survivin[1].
文摘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.
基金This work was supported partly by the State Funds for Outstanding Young Scientists (Granted No. 39925018)the Chinese Academy of Sciences (Grant No. KSCX2-2-01).
文摘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.
