【作者】 蔡欣；

【导师】 姚雪彪；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2008， 博士

【摘要】 染色体的不稳定性或异常分离被广泛认为是癌症发生的标志性事件,细胞有丝分裂过程中染色体的正确运动与分离是由纺锤体微管和动点的相互作用来协调实现的。我们早期的研究发现,中心体激酶Nek2A在有丝分裂时期中有新颖的动点定位,敲除Nek2A将导致染色体的不稳定性,然而Nek2A如何参与纺锤体检验点信号途径还不甚清楚。Hec1是动点-微管核心衔接点Ndc80复合物的主要组分,但Hec1在癌症细胞中高表达的原因一直未能攻克。我们最新的研究发现,Nek2A通过磷酸化Hec1调控其与动点内层结构蛋白质CENP-H之间的相互作用,我们拟从Hec1在有丝分裂过程中的地位和精细调控入手,来揭示其与癌症发生的关联以及Nek2A介导的新颖的动点组装途径。我们证实了Spc24-Spc25亚复合物直接结合CENP-H的N端1-105 aa,Hec1通过C端超卷曲螺旋和亮氨酸富集区（461-642 aa）结合在CENP-H的中心包含超卷曲螺旋的106-148 aa区域,合理分配了CENP-H提供的装配平台。Nek2A在有丝分裂过程中能磷酸化Hec1的Ser¹⁶⁵位点并借此调控Hec1和CENP-H之间的相互作用。颇有意思的是,Nek2A介导的磷酸化调控并不决定Hec1的动点定位。然而,在过表达模拟Hec1非磷酸化突变体的中期细胞中出现部分富集极区的滞后染色体和大量syntelic错误连接,进一步观察发现,后期个别染色体不能完全分离,姐妹染色单体之间保持桥联。我们把所有的表型归结为Nek2A的动态磷酸化影响Hec1的微管亲和力差异,并通过体外微管共沉淀实验得以验证。综上所述我们提出了新颖的分子模型,我们认为Nek2A介导的磷酸化引起Hec1的N端球状区域发生取向改变,一方面通过Hec1-CENP-H相互作用驱使Ndc80复合物收敛承接臂,促进动点紧凑和应力传承,另一方面上调Hec1的微管亲和力和结合数量,迅速建立动点.微管双极连接,从而将动点组装、微管连接和纺锤体检验点信号通路有机整合。为了进一步揭示有丝分裂过程中Ndc80复合物的过渡型结构特性及其验证我们提出的“时序组装”假设,我们应用了新颖的SNAP实时标记技术,并发现体内Spc24-Spc25拥有两个具差异动态性和功能性的储备库,其中核仁储备库侧重于在Hec1-Nuf2之前快速锚定动点,胞浆储备库则侧重于保障动点定位的饱和度。Ndc80的时序组装模型很好地诠释了其复合过渡型的功能特征,并将动点内层结构的缜密性衔接和外层空间的微管捕获特性高效地耦合。更多还原

【Abstract】 Loss or gain of whole chromosome,the form of chromosome instability commonly associated with cancers is thought to arise from aberrant chromosome segregation during cell division.Chromosome segregation in mitosis is orchestrated by the interaction of the kinetochore with spindle microtubules.Our recent study shows that Nek2A locates at the kinetochore in mitosis and possibly functions as a novel integrator of the spindle checkpoint signaling.Hecl is highlighted as a key component of Ndc80 complex as well as overexpressional hallmark of colorectal cancer.However,it remains unknown how Nek2A plays a role in kinetochore assembly and what triggers Hecl’s overexpression in cancer.Here,we reported that Nek2A-mediated phosphorylation of Hecl modulates the interaction of Hecl with CENP-H,an inner kinetochore component,in vitro and in vivo.We demonstrated that Spc24-Spc25 subcomplex attaches directly to CENP-H’s 1-105 aa,while Hec1 binds to CENP-H’s 106-148 aa via its C-terminal coiled coil and leucine zip rich region. Nek2A phosphorylates Hecl at Ser¹⁶⁵ during mitosis while such phosphorylation regulates Hec1-CENP-H interaction in vitro and in vivo.Interestingly,phosphory -lation of Ser¹⁶⁵ is not essential for the assembly of Hecl to kinetochore.However, there was a significant increase in syntelic attachments together with lagging chromosomes in Hec1^S165A-overexpressing cells,as well as chromosome bridge phenotype in anaphase.These findings revealed a key role for the Nek2A-mediated phosphor-regulation of Hec1 in microtubule affinity,as we validated by in vitro microtubule cosedimentaion assay.Taken together,we suggested a novel model based on Nek2A-mediated orientational change of Hec1’s globular domain,to trigger its interaction with CENP-H and enhance the affinity to microtubules simultaneously,by which Nek2A integrates KT-MT attachments into spindle checkpoint signaling.By SNAP-labeling,a novel approach for real time chasing,we made our latest progression on Ndc80 in vivo.We illustrated two pools of Spc24-Spc25 in nucleoli and cytoplasm portioned for primary targeting and saturating onto kinetochore prior to Hec1-Nuf2,respectively,and elucidated the scheduling assembly of Ndc80 as an optimized integration of kinetochore consolidation with microtubule association.更多还原