中心体结合蛋白Cep88在中心体复制及斑马鱼早期胚胎发育中的功能研究

项目名称： 中心体结合蛋白Cep88在中心体复制及斑马鱼早期胚胎发育中的功能研究

项目编号： No.31471260

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 生物科学

项目作者： 余娴文

作者单位： 厦门大学

项目金额： 80万元

中文摘要： Nek2是调控中心体复制的关键激酶，如何调控其激酶活性是当今的研究热点。我们在寻找与Nek2结合的蛋白中分离并鉴定了Cep88。在此基础上，我们制备了抗Cep88的抗体，建立了GFP-Cep88及CETN1-GFP两种细胞系；确定了內源的Cep88与Nek2的相互作用以及在细胞内的共定位；发现了Cep88可直接结合并抑制Nek2的激酶活性；确定了Cep88在细胞内的分布及中心体上的定位；初步研究了Cep88在调控中心体结构和功能上的作用。我们在斑马鱼中确立了Cep88在胚胎发育各个时期的表达谱，发现过量表达Cep88和敲低Nek2对斑马鱼胚胎发育的影响作用非常相似。在本项目中，我们将进一步研究Cep88与Nek2 协同调控中心体结构和功能的分子机制以及它们在斑马鱼早期胚胎发育中的作用。本项目的开展将揭示Cep88这个功能未知蛋白的生物学功能，必将有助于更好地阐明中心体复制及调控的分子机制。

中文关键词： 中心体复制；分子机制；斑马鱼；胚胎发育

英文摘要： Centrosome is the main microtubule organizer of mammalian cells, and is crucial for many biological processes such as formation of the mitotic spindle and cilia. Centrosome dysfunction or abnormal centrosome number is intimately implicated in tumorogenesis as well as certain types of develomental defects. Currently, over 200 new centrosomal proteins have been identified, however, most of their functions in centrosomal regulation are unknown. We have identified Cep88 （Centrosomal Protein 88 kDa） as an interacting protein of Nek2A （NIMA-related kinase 2A）, which is an important serine/threonine kinase responsible for the centrosome segregation in G2/M phase. In order to investigate this novel protein with unkown function, we have raised rabbit anti-Cep88 antibody,and have established two stable U2OS cell lines expressing GFP-Cep88 and CETN1-GFP。 We have also determined the centrosomal localization of Cep88. We have established the interaction between the endogenous Cep88 and Nek2, and have displayed their colocalization in the cells. Overexpressed Cep88 significantly reduces pericentriole materials and leads to the prematured centrosome separation, suggesting that Cep88 may be involved in the regulation of centrosome structure and funtion. We also found that Cep88 can directly bind to Nek2A and inhibit Nek2A kinase activity. We have found that zebrafish homologue zCep88 is expressed in the early -staged embryos. Elevation of zCep88 protein levels or reduction of zNek2 protein levels will cause similar developmental defects in the gastulation and segmentation processes in the zebrafish embryos, suggesting that these two protein are intimately cooperate to regulate the early development of zebrafish. We will further invesigate the regulatory roles of Cep88 in the structure and function of centrosomes in cell cycle, as well as in the early development of zebrafish. We believe that our studies will unveil the functions of this novel gene in centrosomal regulation as and embryogeneis.

英文关键词： centrosomal duplication;molecular mechanism;zebrafish;early developmenat