In mitosis,accurate chromosome segregation depends on the kinetochore,a supermolecular machinery that couples dynamic spin-dle microtubules to centromeric chromatin.However,the structure–activity relationship of the ...In mitosis,accurate chromosome segregation depends on the kinetochore,a supermolecular machinery that couples dynamic spin-dle microtubules to centromeric chromatin.However,the structure–activity relationship of the constitutive centromere-associated network(CCAN)during mitosis remains uncharacterized.Building on our recent cryo-electron microscopic analyses of human CCAN structure,we investigated how dynamic phosphorylation of human CENP-N regulates accurate chromosome segregation.Our mass spectrometric analyses revealed mitotic phosphorylation of CENP-N by CDK1,which modulates the CENP-L–CENP-N interaction for accurate chromosome segregation and CCAN organization.Perturbation of CENP-N phosphorylation is shown to prevent proper chromosome alignment and activate the spindle assembly checkpoint.These analyses provide mechanistic insight into a previously undefined link between the centromere–kinetochore network and accurate chromosome segregation.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 cell division depends on the accurate assembly of the spindle midzone from dynamic spindle microtubules to ensure chromatid segregation during metaphase-anaphase transition.However,the mechanism underlying ...Error-free cell division depends on the accurate assembly of the spindle midzone from dynamic spindle microtubules to ensure chromatid segregation during metaphase-anaphase transition.However,the mechanism underlying the key transition from the mitotic spindle to central spindle before anaphase onset remains elusive.Given the prevalence of chromosome instability phenotype in gastric tumorigenesis,we developed a strategy to model context-dependent cell division using a combination of light sheet microscope and 3D gastric organoids.Light sheet microscopic image analyses of 3D organoids showed that CENP-E inhibited cells undergoing aberrant metaphase-anaphase transition and exhibiting chromosome segregation errors during mitosis.Highresolution real-time imaging analyses of 2D cell culture revealed that CENP-E inhibited cells undergoing central spindle splitting and chromosome instability phenotype.Using biotinylated syntelin as an affinity matrix,we found that CENP-E forms a complex with PRC1 in mitotic cells.Chemical inhibition of CENP-E in metaphase by syntelin prevented accurate central spindle assembly by perturbing temporal assembly of PRC1 to the midzone.Thus,CENP-E-mediated PRC1 assembly to the central spindle constitutes a temporal switch to organize dynamic kinetochore microtubules into stable midzone arrays.These findings reveal a previously uncharacterized role of CENP-E in temporal control of central spindle assembly.Since CENP-E is absent from yeast,we reasoned that metazoans evolved an elaborate central spindle organization machinery to ensure accurate sister chromatid segregation during anaphase and cytokinesis.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
Evolution has enabled living cells to adopt their structural and functional complexity by organizing intricate cellular compartments,such as membrane-bound and membraneless organelles(MLOs),for spatiotemporal catalysi...Evolution has enabled living cells to adopt their structural and functional complexity by organizing intricate cellular compartments,such as membrane-bound and membraneless organelles(MLOs),for spatiotemporal catalysis of physiochemical reactions essential for cell plasticity control.Emerging evidence and view support the notion that MLOs are built by multivalent interactions of biomolecules via phase separation and transition mechanisms.In healthy cells,dynamic chemical modifications regulate MLO plasticity,and reversible phase separation is essential for cell homeostasis.Emerging evidence revealed that aberrant phase separation results in numerous neurodegenerative disorders,cancer,and other diseases.In this review,we provide molecular underpinnings on(i)mechanistic understanding of phase separation,(ii)unifying structural and mechanistic principles that underlie this phenomenon,(iii)various mechanisms that are used by cells for the regulation of phase separation,and(iv)emerging therapeutic and other applications.展开更多
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...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.展开更多
Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules.Our recent studies show that mitotic motor CENP-E cooperates with SKAP and forms a link be...Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules.Our recent studies show that mitotic motor CENP-E cooperates with SKAP and forms a link between kinetochore core MIS13 complexand spindle microtubule plus-ends to achieve accurate chromosome alignment in mitosis. However, it remains elusive how SKAP regulates kinetochore attachment from lateral association to end-on attachment during metaphase alignment. Here, we identify a novel interaction between Aurora B and SKAP that orchestrates accurate interaction between the kinetochore and dynamic spindle microtubules. Interestingly, SKAP spontaneously phase-separates in vitro via weak, multivalent interactions into droplets with fast internaldynamics. SKAP and Aurora B form heterogeneous coacervates in vitro, which recapitulate the dynamics and behavior of SKAP cometsin vivo. Importantly, SKAP interaction with Aurora B via phase separation is essential for accurate chromosome segregation and alignment. Based on those findings, we reason that SKAP–Aurora B interaction via phase separation constitutes a dynamic pool of Aurora Bactivity during the lateral to end-on conversion of kinetochore–microtubule attachments to achieve faithful cell division.展开更多
Faithful segregation of mitotic chromosomes requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle microtubules and kinetochores. Although the mechanisms under...Faithful segregation of mitotic chromosomes requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying PLK1 activation have been extensively studied, the regulatory mechanisms that couple PLK1 activity to accurate chromosome segregation are not well understood. In particular, PLK1 is implicated in stabilizing kinetochore–microtubule attachments, but how kinetochore PLK1 activity is regulated to avoid hyperstabilized kinetochore–microtubules in mitosis remains elusive. Here, we show that kinetochore PLK1 kinase activity is modulated by SET7/9 via lysine methylation during early mitosis. The SET7/9-elicited dimethylation occurs at the Lys191 of PLK1, which tunes down its activity by limiting ATP utilization. Overexpression of the non-methylatable PLK1 mutant or chemical inhibition of SET7/9 methyltransferase activity resulted in mitotic arrest due to destabilized kinetochore–microtubule attachments. These data suggest that kinetochore PLK1 is essential for stable kinetochore–microtubule attachments and methylation by SET7/9 promotes dynamic kinetochore–microtubule attachments for accurate error correction. Our findings define a novel homeostatic regulation at the kinetochore that integrates protein phosphorylation and methylation with accurate chromosome segregation for maintenance of genomic stability.展开更多
基金supported by the National Key R&D Program of China (2022YFA1303100, 2022YFA0806800)the National Natural Science Foundation of China (32090040, 31621002, 31871359, 32170733, 92153302)+1 种基金the Ministry of Education (IRT_17R102, 20113402130010, YD2070006001)the Fundamental Research Funds for the Central Universities (WK2070000194)
基金Acknowledgments
We thank members of our groups for insightful discussion during the course of this study. This work was initiated by the chemical biology grant PGX-t from the Proteomics Research Laboratory, and supported in part by Chinese Academy of Sciences Grants （KSCX2-YW-H-10 and KSCX2-YW-R195）, 973 projects （2002CB713700 2010CB912103）, National Natural Science Foundation of China （90913016）, and Georgia Cancer Coalition Eminent Scholar Award.
基金supported by grants from the Ministry of Science and Technology of the People’s Republic of China and the National Natural Science Foundation of China(2022YFA1303100,2022YFA0806800,92153302,32090040,92254302,21922706,91853115 to X.L.,2017YFA0503600,31621002,U1532109,91853133 to J.Z.,32170733,2017YFA0102900,31871359 to Z.D.,32000858 to T.T.)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB37010105 to J.Z.and XDB19040000 to X.L.)+3 种基金the Ministry of Education(IRT_17R102,20113402130010,YD2070006001 to X.L.)Anhui Provincial Natural Science Foundation(2108085J15 to Z.D.,2008085QC145 to T.T.)the Fundamental Research Funds for the Central Universities(WK2070000171 to T.T.)the University of Science and Technology of China Research Funds of the Double First-Class Initiative(YD2070002015 to X.Z.)。
文摘In mitosis,accurate chromosome segregation depends on the kinetochore,a supermolecular machinery that couples dynamic spin-dle microtubules to centromeric chromatin.However,the structure–activity relationship of the constitutive centromere-associated network(CCAN)during mitosis remains uncharacterized.Building on our recent cryo-electron microscopic analyses of human CCAN structure,we investigated how dynamic phosphorylation of human CENP-N regulates accurate chromosome segregation.Our mass spectrometric analyses revealed mitotic phosphorylation of CENP-N by CDK1,which modulates the CENP-L–CENP-N interaction for accurate chromosome segregation and CCAN organization.Perturbation of CENP-N phosphorylation is shown to prevent proper chromosome alignment and activate the spindle assembly checkpoint.These analyses provide mechanistic insight into a previously undefined link between the centromere–kinetochore network and accurate chromosome segregation.
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This work was supported in part by the National NaturalScience Foundation of China(31430054,31320103904,31621002,31671405,31601097,91854203,91753000,and91853115)'Strategic Priority Research Program'of the ChineseAcademy of Sciences(XDB19000000)+2 种基金the National Key Researchand Development Program of China(2017YFA0503600 and2016YFA-0100500)MOE Innovative Team project(IRT_17R102)and the US National Institutes of Health(CA164133,DK56292,and DK115812).
文摘Error-free cell division depends on the accurate assembly of the spindle midzone from dynamic spindle microtubules to ensure chromatid segregation during metaphase-anaphase transition.However,the mechanism underlying the key transition from the mitotic spindle to central spindle before anaphase onset remains elusive.Given the prevalence of chromosome instability phenotype in gastric tumorigenesis,we developed a strategy to model context-dependent cell division using a combination of light sheet microscope and 3D gastric organoids.Light sheet microscopic image analyses of 3D organoids showed that CENP-E inhibited cells undergoing aberrant metaphase-anaphase transition and exhibiting chromosome segregation errors during mitosis.Highresolution real-time imaging analyses of 2D cell culture revealed that CENP-E inhibited cells undergoing central spindle splitting and chromosome instability phenotype.Using biotinylated syntelin as an affinity matrix,we found that CENP-E forms a complex with PRC1 in mitotic cells.Chemical inhibition of CENP-E in metaphase by syntelin prevented accurate central spindle assembly by perturbing temporal assembly of PRC1 to the midzone.Thus,CENP-E-mediated PRC1 assembly to the central spindle constitutes a temporal switch to organize dynamic kinetochore microtubules into stable midzone arrays.These findings reveal a previously uncharacterized role of CENP-E in temporal control of central spindle assembly.Since CENP-E is absent from yeast,we reasoned that metazoans evolved an elaborate central spindle organization machinery to ensure accurate sister chromatid segregation during anaphase and cytokinesis.
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 in part by grants from the National Natural Science Foundation of China(32090040,31621002,21922706,81630080,31430054,91854203,91853115,and 31671405)the National Key Research and Development Program of China(2017YFA0503600 and 2016YFA0100500)+4 种基金the Ministry of Education of China(IRT_17R102)the Strategic Priority Research Program of Chinese Academy of Sciences(XDB19000000)the Fundamental Research Funds for the Central Universities(KB2070000023 and WK2070000194)National Institutes of Health Grants(CA164133,DK115812,and DK56292)We thank our laboratory members for stimulating discussion。
文摘Evolution has enabled living cells to adopt their structural and functional complexity by organizing intricate cellular compartments,such as membrane-bound and membraneless organelles(MLOs),for spatiotemporal catalysis of physiochemical reactions essential for cell plasticity control.Emerging evidence and view support the notion that MLOs are built by multivalent interactions of biomolecules via phase separation and transition mechanisms.In healthy cells,dynamic chemical modifications regulate MLO plasticity,and reversible phase separation is essential for cell homeostasis.Emerging evidence revealed that aberrant phase separation results in numerous neurodegenerative disorders,cancer,and other diseases.In this review,we provide molecular underpinnings on(i)mechanistic understanding of phase separation,(ii)unifying structural and mechanistic principles that underlie this phenomenon,(iii)various mechanisms that are used by cells for the regulation of phase separation,and(iv)emerging therapeutic and other applications.
基金supported by the National Key R&D Program of China(2017YFA 0102900 and 2017 YFA 0503600)the National Natural Science Fondation of China(31671407 and 31871359 to Z.D.+4 种基金31621002,31430054,91854203,and 31320103904 to X.Y.31301099 and 21672201 to X.G.31471275 to D.W.),Strategic Priority Research Program of the Chinese Academy of Sciences(XDB19040000)Chinese Academy of Sciences Center for Excellence in Molecular Cell Science(2015 HSC-UE010)MOE Innovative Team(IRT_17R102).
文摘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.
基金supported in part by grants from the National Natural Science Foundation of China(32090040,31621002,21922706,91854203,91853115,81630080,31430054,31471275,31871359,31970655,and 31671405)the National Key Research and Development Program of China(2017YFA0503600,2016YFA0100500,and 2017YFA0102900)+3 种基金the Ministry of Education(IRT_17R102)The Strategic Priority Research Program of Chinese Academy of Sciences(XDB19000000)the Fundamental Research Funds for the Central Universities(WK2070000066 and WK2070000194)National Institutes of Health Grants(CA164133,DK115812,and DK56292).
文摘Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules.Our recent studies show that mitotic motor CENP-E cooperates with SKAP and forms a link between kinetochore core MIS13 complexand spindle microtubule plus-ends to achieve accurate chromosome alignment in mitosis. However, it remains elusive how SKAP regulates kinetochore attachment from lateral association to end-on attachment during metaphase alignment. Here, we identify a novel interaction between Aurora B and SKAP that orchestrates accurate interaction between the kinetochore and dynamic spindle microtubules. Interestingly, SKAP spontaneously phase-separates in vitro via weak, multivalent interactions into droplets with fast internaldynamics. SKAP and Aurora B form heterogeneous coacervates in vitro, which recapitulate the dynamics and behavior of SKAP cometsin vivo. Importantly, SKAP interaction with Aurora B via phase separation is essential for accurate chromosome segregation and alignment. Based on those findings, we reason that SKAP–Aurora B interaction via phase separation constitutes a dynamic pool of Aurora Bactivity during the lateral to end-on conversion of kinetochore–microtubule attachments to achieve faithful cell division.
基金This work was supported in part by the National Natural Science Foundation of China(31430054,31320103904,31621002,31671405,91854203,91853115,21922706,31671407,31871359,31601097,and 21672201)the National Key Research and Development Program of China(2017YFA0503600 and 2016YFA0100500)+2 种基金Strategic Priority Research Program of the Chinese Academy of Sciences(XDB19000000)Chinese Academy of Sciences Center for Excellence in Molecular Cell Science(2015HSC-UE010)MOE Innovative Team(IRT_17R102),and the US National Institutes of Health(CA164133and DK26929).
文摘Faithful segregation of mitotic chromosomes requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying PLK1 activation have been extensively studied, the regulatory mechanisms that couple PLK1 activity to accurate chromosome segregation are not well understood. In particular, PLK1 is implicated in stabilizing kinetochore–microtubule attachments, but how kinetochore PLK1 activity is regulated to avoid hyperstabilized kinetochore–microtubules in mitosis remains elusive. Here, we show that kinetochore PLK1 kinase activity is modulated by SET7/9 via lysine methylation during early mitosis. The SET7/9-elicited dimethylation occurs at the Lys191 of PLK1, which tunes down its activity by limiting ATP utilization. Overexpression of the non-methylatable PLK1 mutant or chemical inhibition of SET7/9 methyltransferase activity resulted in mitotic arrest due to destabilized kinetochore–microtubule attachments. These data suggest that kinetochore PLK1 is essential for stable kinetochore–microtubule attachments and methylation by SET7/9 promotes dynamic kinetochore–microtubule attachments for accurate error correction. Our findings define a novel homeostatic regulation at the kinetochore that integrates protein phosphorylation and methylation with accurate chromosome segregation for maintenance of genomic stability.
