【作者】 朱梅；

【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】 真核生物细胞通过一系列复杂而有序的生化调控途径精确均等地把遗传信息以染色体的形式传递给子代细胞。有丝分裂期染色体的运动是由纺锤体微管和位于染色体着丝粒区域的一个特化的结构—动点之间程序化且动态的相互作用而协调完成的。我们以往的研究证实了有丝分裂驱动蛋白质CENP-E为纺锤体微管和动点连接的一个桥梁并且可以参与调节纺锤体检验点的级联反应。CENP-E是一个定位于动点的纤维冠状层并依赖于微管行走的马达蛋白质,它可以驱使动点与微管的正极端相结合引导染色体向赤道板排列,因此参与了由前中期向后期转换过程中的染色体的运动。为了进一步研究CENP-E调节有丝分裂进程的详尽分子机制,我们着手从CENP-E在有丝分裂期的作用网络出发,通过研究蛋白质作用的分子机制及动力学特征,从而阐明CENP-E功能可塑性及调节机制。本文聚焦在一个新发现的CENP-E作用分子septin 7(简称SEPT7),通过一系列生物化学及细胞生物学实验,我们的研究首次阐述了CENP-E与SEPT7的作用及其调控机制。Septin(SEPT)属于一类保守的细胞骨架蛋白质家族,它们具有类似的结构特征,septin在多种生物体内均可以形成异源多聚体的纤维状结构。SEPT纤维在有丝分裂期可以定位到纺锤体上。在我们研究SEPT7与CENP-E作用机制的同时,有研究报导表明SEPT2的敲除会导致染色体的错误排列以及影响CENP-E在动点上的定位。然而,CENP-E是否与SEPT有相互作用以及它们是如何协调染色体的排列与分离的机制目前仍不为所知。我们的研究结果发现了SEPT7对于稳定CENP-E的动点定位是需要的,SEPT7可以通过直接结合CENP-E的羧基端而将其稳定在动点上。我通过酵母共转以及体外结合试验证明了SEPT7是通过其羧基端与CENP-E相互作用的。免疫荧光的结果表明SEPT7沿着有丝分裂纺锤体分布并且可以终止于以CENP-E为标识的动点结构。当我用siRNA抑制SEPT7的表达会发现CENP-E不能够上动点而且会导致染色体排列的异常,这些表型都可以通过进一步外源转染SEPT7的rescue质粒而得到恢复。同时我还发现SEPT的敲除会导致双极定向的染色体之间的张力减弱以及纺锤体检验点的激活,SEPT7可能将CENP-E的动点定位与纺锤体检验点信号的级联反应相联系起来。这些发现揭示了SEPT7-CENP-E之间的相互作用对于稳定CENP-E的动点定位以及协调动点一微管之间的连接都是至关重要的。为了进一步去研究SEPT7的生物学特性以及它在SEPT2-SEPT6-SEPT7复合物组装过程中的作用,我对人的SEPT7进行了生化以及细胞生物学特性的分析。我发现SEPT7在体内和体外均可以组装形成纤维状的结构,其中包含GTPase的结构域以及氨基端的一小段富含碱性氨基酸序列的缺失突变体—SEPT7 N,对于维持SEPT7的纤维状定位,自身的结合以及与SEPT2和SEPT6的相互作用都是必需的。尽管GTPase的活性对于SEPT7的纤维组装是必不可少的,但它并不影响SEPT2/6/7复合物之间的相互作用。此外,SEPT7在体内还可以征募SEPT2和SEPT6从而形成纤维状结构的复合物。这些发现有助于我们进一步认识SEPT7的结构与功能的关系。综上所述,我们的研究揭示并阐述了SEPT7-CENP-E之间的相互作用对于稳定CENP-E的动点定位以及协调纺锤体微管与动点之间的连接是至关重要的。此外,我们对于SEPT7生化特性的初步分析有助于进一步地研究SEPT纤维的组装及功能。鉴于SEPT7及CENP-E在有丝分裂进程中的动态性,我们相信利用生物光子学技术与生化分析手段相结合,我们将会阐明septin动力学与可塑性在有丝分裂进程中的详尽功能及其纺锤体动力学的相互关联。由于septin功能的变异在肿瘤发生与发展中的潜在作用,对septin分子可塑性及动力学的解析将为校正细胞分裂的异常提供一个潜在的靶点。更多还原

【Abstract】 Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore,a multiprotein complex assembled onto centromeric DNA of the chromosome.CENP-E is a microtubule-based kinesin motor protein located on the outer kinetochore and is responsible for a stable microtubule-kinetochore attachment.Earlier studies from our laboratory demonstrate that CENP-E forms a link between chromosome attachment and spindle checkpoint. To delineate the molecular mechanisms underlying CENP-E function and regulation during mitosis,we conducted a systemic identification of proteins interacting with CENP-E using yeast genetic screen,in which more than a dozen of candidates were identified.One of such candidates is SEPT7,a GTPase composing a novel filamentous structure essential for cytokinesis.The first part of my research work focused on the characterization of CENP-E-septin interaction and functional relevance in mitosis.Septin(SEPT)belongs to a family of conserved cytoskeletal proteins,which share common structural domains and form heteropolymeric filaments in several organisms.SEPT filaments localized to the metaphase spindle during mitosis. Previous studies indicated that SEPT2 depletion results in chromosome misalignment correlated with loss of CENP-E from the kinetochores of congressing chromosomes. However,it has remained elusive as to whether CENP-E physically interacts with SEPT and how this interaction orchestrates chromosome segregation in mitosis.Here we show SEPT7 is required for a stable kinetochore localization of CENP-E.SEPT7 stabilizes the kinetochore association of CENP-E by directly interacting with its C-terminal domain.The region of SEPT7 associating with CENP-E was mapped to its C-terminal domain by GST pull-down and yeast two-hybrid assays. Immunoflourescence study shows that SEPT7 filaments distribute along the mitotic spindle and terminate at the kinetochore marked by CENP-E.Remarkably, suppression of synthesis of SEPT7 by siRNA abrogated the localization of CENP-E to the kinetochore and caused aberrant chromosome congression.These mitotic defects and kinetochore localization of CENP-E can be successfully rescued by introducing exogenous GFP-SEPT7 into the SEPT7-depleted cells.These SEPT7-suppressed cells display reduced tension at kinetochores of bi-orientated chromosomes and activate mitotic spindle checkpoint marked by Mad2 and BubR1 labelings on these misaligned chromosomes.We propose that SEPT7 forms a link between kinetochore localization of CENP-E and the mitotic spindle checkpoint.These findings reveal a key role for the SEPT7-CENP-E interaction in distribution of CENP-E to the kinetochore and mainteinance the kinetochore-microtubule attachment.To delineate the structure-functional relationship of SEPT7 molecule related to SEPT2-SEPT6-SEPT7 complex assembly,I carried out biochemical characterization of human SEPT7.My study shows that human SEPT7 can assemble into filaments in vitro and in vivo.The N-terminal SEPT7,including GTPase domain and a short region at the amino terminus,is required for its filaments localization,self-association and interaction with SEPT2 and SEPT6.Although GTPase activity is indispensable for SEPT7 filaments assembly,it barely affects the formation of hetero-polymeric septin complex and filaments.Furthermore,SEPT7 can bundle SEPT2 and SEPT6 to form an integral filaments structure in vivo.Thus,these observations provide insight into a better understanding of structure-functional relationship of SEPT7.Currently,I am carrying our biophotonic study to illustrate the molecular dynamics of SEPT7 molecule and its relation to chromosome segregation in mitosis.Taken together,my finding of SEPT7-CENP-E interaction provides novel insight into a better understanding of CENP-E molecular dynamics in mitosis.In addition,the molecular delineation of SEPT7 structure-functional relationship established here provides framework by which we illustrate the function and regulation of septin filaments during mitosis.Given the fact that aberrant spindle plasticity and dynamics are involved in pathogenesis of chromosome instability phenotype,better understandings of the molecular mechanisms of CENP-E-SEPT7 interaction will help to correct aberrant chromosome segregation and perhaps provide potential therapeutic strategies.更多还原