【作者】 史兴娟；

【导师】 周军；

【作者基本信息】 南开大学 ， 细胞生物学， 2012， 博士

【摘要】 中心体由一对互相垂直排列的中心粒及围绕在中心粒周围的无定形基质组成，是动物细胞主要的微管组织中心。中心体通过核化、组织微管骨架，调节许多细胞活动包括细胞形态的维持、细胞极性的建立、有丝分裂和细胞迁移等。Cep70是一个分子量为70kDa，含597个氨基酸的蛋白质，含有两个卷曲螺旋结构域。Cep70最初在人类中心体蛋白质组学的研究中发现，近几年Cep70在不同物种中的生物学功能逐渐被了解。斑马鱼的Cep70调节纤毛发生，从而影响胚胎发育。而且，Cep70家族的成员——衣藻前中心粒蛋白CRC70在中心粒前体的组装中发挥重要作用。但是，哺乳动物细胞中Cep70的定位和功能目前仍不清楚。在本研究中，我们通过免疫荧光实验确定Cep70是一个中心体蛋白。在细胞周期的各个时期，Cep70均定位在中心体上，它通过两个卷曲螺旋结构域与-微管蛋白相互作用。Cep70调节细胞内原有的和新生微管的组织，而且在体外实验中，Cep70促进微管组装。在有丝分裂期，Cep70表达降低使双极纺锤体的组织和定向异常。中心体蛋白不仅通过介导纺锤体的形成来调节有丝分裂，在其他细胞活动如细胞迁移中也发挥重要作用。本研究发现Cep70通过调节内皮细胞的迁移来影响血管新生。在小鼠体内血管新生实验中，Cep70小干扰RNA显著抑制血管新生。在体外实验中，Cep70表达下调阻碍了内皮细胞在基质胶上的成管能力和在胶原中的出芽生长。在对Cep70调节血管新生的机制研究中，发现Cep70通过介导内皮细胞的极化和迁移来调节血管新生。而且，Cep70表达下调显著降低了迁移细胞前端片状伪足区域膜皱褶的动态性。进一步研究发现，Rho GTP酶家族的Cdc42和Rac1的活化在Cep70调节血管新生中发挥作用。这些结果表明Cep70介导内皮细胞的极化和迁移，从而在血管新生中发挥作用。概括来说，本研究首次证实Cep70是一个中心体蛋白，通过结合-微管蛋白定位在中心体上。而且，Cep70能调节微管的组织和有丝分裂纺锤体的定向。另外，本研究发现Cep70通过介导血管内皮细胞的极化和迁移而影响血管新生。更多还原

【Abstract】 The centrosome, composed of a pair of barrel-shaped centrioles surrounded byelectron-dense pericentriolar material, is the major microtubule-organizing center inanimal cells. In addition to its role in the nucleation and organization of microtubules,the centrosome is also critical for the orchestration of cell cycle progression. Toensure bipolar organization and geometric precision of the mitotic spindle, thecentrosome serves as a cellular hub where several key regulators convene to mediatevital interactions that control spindle assembly. The centrosome may also serve toregulate other fundamental cellular functions, such as cell polarity and motility.Cep70, a candidate centrosomal protein of70kDa, was originally discovered ina proteomic study of the human centrosome. Recently, the biological function ofCep70in different species is beginning to be unraveled. The zebrafish homolog ofCep70has been shown to contribute to the assembly of cilia by determination of thelength of the axoneme. In addition, the Chlamydomonas procentriole protein CRC70,a member of the conserved Cep70family, acts as a scaffold for the assembly of thecentriole precursor. However, the localization and functions of Cep70in mammaliancells remain unknown.In this study, we report the molecular characterization and functional analysis ofa novel centrosomal protein, Cep70, in mammalian cells. Our data show that Cep70localizes to the centrosome throughout the cell cycle and binds to the key centrosomalcomponent,-tubulin, through the peptide fragments that contain the coiled-coildomains. Our data further reveal that the centrosomal localization pattern of Cep70isdependent on its interaction with-tubulin. Strikingly, Cep70plays a significant rolein the organization of both preexisting and nascent microtubules in interphase cells.Besides, Cep70promotes microtubule assembly in vitro by increasing microtubuleelongation. In addition, Cep70is necessary for the organization and orientation of thebipolar spindle during mitosis. These results thus report for the first time theidentification of Cep70as an important centrosomal protein that interacts with -tubulin and underscore its critical role in the regulation of mitotic spindle assembly.Centrosomal proteins are important for the organization of microtubules inanimal cells and regulate diverse microtubule-mediated cellular activities such as cellmigration, a critical step in angiogenesis. However, the participation of these proteinsin angiogenesis, which involves vascular endothelial cell migration from preexistingblood vessels, remains elusive. Here, we demonstrate that Cep70is necessary forangiogenic response in mice. This protein is also required for tube formation andcapillary sprouting in vitro from vascular endothelial cells. Wound healing andtranswell assays reveal that Cep70plays a significant role in endothelial cellmigration. Depletion of Cep70results in severe defects in membrane ruffle formationand centrosome reorientation, indicating a requirement for this protein in cellpolarization. In addition, Cep70is critically involved in microtubule rearrangement inresponse to the migratory stimulus. Our data further demonstrate that Cep70isimportant for Cdc42and Rac1activation to promote angiogenesis. These findingsthus establish Cep70as a critical regulator of the angiogenic process and emphasizethe significance of microtubules for vascular endothelial cell migration andangiogenesis.In conclusion, we describe the characterization of Cep70as a centrosomalprotein and demonstrate its localization to the centrosome throughout the cell cyclevia interaction with-tubulin. We also present evidence that Cep70is a criticalregulator for the organization of radial microtubule arrays and the spindle apparatus.In addition, Cep70is implicated in angiogenesis by regulating vascular endothelialpolarization and migration.更多还原