纺锤体检验点(spindle checkpoint)是一个重要的细胞分裂生化调节通路,可监督染色体正确分离和传代.着丝粒相关蛋白E(centromere-associated protein E,CENP-E)是一个分子量为312kD的微管马达驱动蛋白,可以衔接纺锤体微管与动点并参与...纺锤体检验点(spindle checkpoint)是一个重要的细胞分裂生化调节通路,可监督染色体正确分离和传代.着丝粒相关蛋白E(centromere-associated protein E,CENP-E)是一个分子量为312kD的微管马达驱动蛋白,可以衔接纺锤体微管与动点并参与纺锤体检验点调控.为研究CENP-E的作用机理,以其动点结合区域为诱饵蛋白,用酵母双杂交技术从人HeLa细胞cDNA文库中筛选出了Nuf2蛋白.体外的pull-down实验和体内的免疫共沉淀实验表明,Nuf2蛋白通过其卷曲螺旋(coiled-coil)功能域特异结合CENP-E的C末端区域,间接免疫荧光显示Nuf2与CENP-E共定位于细胞有丝分裂期染色体的动点.由此推论,CENP-E通过Nuf2的直接作用参与构筑动点-微管界面,进而参与细胞有丝分裂纺锤体检验点信号转导通路,为染色体正确分离发挥调控作用.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
Enhancer of rudimentary homolog(ERH)is a small,highly conserved protein among eukaryotes.Since its discovery nearly 20 years ago,its molecular function has remained enigmatic.It has been implicated to play a role in t...Enhancer of rudimentary homolog(ERH)is a small,highly conserved protein among eukaryotes.Since its discovery nearly 20 years ago,its molecular function has remained enigmatic.It has been implicated to play a role in transcriptional regulation and in cell cycle.We recently showed that ERH binds to the Sm complex and is required for the mRNA splicing of the mitotic motor protein CENP-E.Fur-thermore,cancer cells driven by mutations in the KRAS oncogene are particularly sensitive to RNAi-mediated suppression of ERH function,and ERH expression is inversely correlated with survival in colorectal cancer pa-tients whose tumors harbor KRAS mutation.These recent fi ndings indicate that ERH plays an important role in cell cycle through its mRNA splicing activity and is critically required for genomic stability and cancer cell survival.展开更多
The kinetochore is structurally composed offour layers. We know that three microtubule-based motorproteins such as CENP-E, dynein, and MCAK are located at the outmost region of the kinetochore. Experimentation ofthese...The kinetochore is structurally composed offour layers. We know that three microtubule-based motorproteins such as CENP-E, dynein, and MCAK are located at the outmost region of the kinetochore. Experimentation ofthese motor functions betters our understanding of mitotic regulation, and chromosome movements in particular.With real-time studies of chromosome movements in livecells, we hope to illustrate the molecular mechanisms under-lying mitotic regulation.展开更多
Kinesin superfamily of microtubule- based motor orchestrates a variety of cellular proc- esses. Recent availability of mammalian genomes has enabled analyses of kinesins on the whole ge- nome. Here we present a novel ...Kinesin superfamily of microtubule- based motor orchestrates a variety of cellular proc- esses. Recent availability of mammalian genomes has enabled analyses of kinesins on the whole ge- nome. Here we present a novel full-length kinesin prediction program (FKPP) for mammalian kinesin gene discovery based on a comparative genomics approach. Contrary to previous predictions of 94 kinesins, we identify a total of 134 potentially kinesin genes from mammalian genomes, including 45 from mouse, 45 from rat and 44 from human. In addition, FKPP synthesizes 25 potentially full-length mam- malian kinesins based on the partial sequences in the database. Surprisingly, FKPP reveals that full-length human CENP-E contains 2701 aa rather than 2663 aa in the database. Experimentation using sequence specific antibody and cDNA sequencing of human CENP-E validates the accuracy of FKPP. Given the remarkable computing efficiency and accuracy of FKPP, we reclassify the mammalian kinesin super- family. Since current databases contain many in- complete sequences, FKPP may provide a novel approach for molecular delineation of kinesins and other protein families.展开更多
文摘纺锤体检验点(spindle checkpoint)是一个重要的细胞分裂生化调节通路,可监督染色体正确分离和传代.着丝粒相关蛋白E(centromere-associated protein E,CENP-E)是一个分子量为312kD的微管马达驱动蛋白,可以衔接纺锤体微管与动点并参与纺锤体检验点调控.为研究CENP-E的作用机理,以其动点结合区域为诱饵蛋白,用酵母双杂交技术从人HeLa细胞cDNA文库中筛选出了Nuf2蛋白.体外的pull-down实验和体内的免疫共沉淀实验表明,Nuf2蛋白通过其卷曲螺旋(coiled-coil)功能域特异结合CENP-E的C末端区域,间接免疫荧光显示Nuf2与CENP-E共定位于细胞有丝分裂期染色体的动点.由此推论,CENP-E通过Nuf2的直接作用参与构筑动点-微管界面,进而参与细胞有丝分裂纺锤体检验点信号转导通路,为染色体正确分离发挥调控作用.
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This work is supported by the Liver Disease Prevention and Treatment Research Foundation of Taiwan to M.Z.W.by the Intramural Pro-gram of the U.S.National Cancer Institute to J.L..
文摘Enhancer of rudimentary homolog(ERH)is a small,highly conserved protein among eukaryotes.Since its discovery nearly 20 years ago,its molecular function has remained enigmatic.It has been implicated to play a role in transcriptional regulation and in cell cycle.We recently showed that ERH binds to the Sm complex and is required for the mRNA splicing of the mitotic motor protein CENP-E.Fur-thermore,cancer cells driven by mutations in the KRAS oncogene are particularly sensitive to RNAi-mediated suppression of ERH function,and ERH expression is inversely correlated with survival in colorectal cancer pa-tients whose tumors harbor KRAS mutation.These recent fi ndings indicate that ERH plays an important role in cell cycle through its mRNA splicing activity and is critically required for genomic stability and cancer cell survival.
文摘The kinetochore is structurally composed offour layers. We know that three microtubule-based motorproteins such as CENP-E, dynein, and MCAK are located at the outmost region of the kinetochore. Experimentation ofthese motor functions betters our understanding of mitotic regulation, and chromosome movements in particular.With real-time studies of chromosome movements in livecells, we hope to illustrate the molecular mechanisms under-lying mitotic regulation.
基金supported by the National Natural Science Foundation of China(Grant Nos.39925018,30121001,30270293&90508002)the Chinese Academy of Sciences(Grant No.KSCX2-2-01)+3 种基金the Chinese 973 Project(Grant No.2002CB713700)the Chinese 863 Project(Grant No.2001AA215331)Chinese Minister of Education(Grant No.20020358051)American Cancer Society(Grant No.RPG-99-173-01).
文摘Kinesin superfamily of microtubule- based motor orchestrates a variety of cellular proc- esses. Recent availability of mammalian genomes has enabled analyses of kinesins on the whole ge- nome. Here we present a novel full-length kinesin prediction program (FKPP) for mammalian kinesin gene discovery based on a comparative genomics approach. Contrary to previous predictions of 94 kinesins, we identify a total of 134 potentially kinesin genes from mammalian genomes, including 45 from mouse, 45 from rat and 44 from human. In addition, FKPP synthesizes 25 potentially full-length mam- malian kinesins based on the partial sequences in the database. Surprisingly, FKPP reveals that full-length human CENP-E contains 2701 aa rather than 2663 aa in the database. Experimentation using sequence specific antibody and cDNA sequencing of human CENP-E validates the accuracy of FKPP. Given the remarkable computing efficiency and accuracy of FKPP, we reclassify the mammalian kinesin super- family. Since current databases contain many in- complete sequences, FKPP may provide a novel approach for molecular delineation of kinesins and other protein families.
