Mechanisms of the Unusual Cytokinesis in Trypanosomes

描述(申请人提供)：布鲁氏锥虫的胞质分裂是一种寄生原生动物，也是人类昏睡病的病原体，已知与其宿主的胞质分裂完全不同。锥体分裂中的裂解面不像酵母和动物那样由中心纺锤体定义，而是由新鞭毛和 鞭毛附着区(FAZ)。因此，锥虫体内的胞质分裂是从新的FAZ的前端开始的，分裂沟沿着长轴从细胞的前部向细胞的后端单向地进入。值得注意的是，锥虫体内没有形成肌动球蛋白收缩环，这表明锥虫卵裂沟可能具有一种特殊的结构和新的成分。然而，尽管有这些不同，Aurora样和Polo样激酶都与锥体胞质分裂的启动有关(Hammarton等人，2007；Li和Wang，2006；Tu等人，2006)。锥体中的极光激酶同源物TbAUK1形成一种独特的染色体乘客复合体(CPC)，在染色体分离和胞质分裂启动中发挥重要作用(Li等人，2008a)。在有丝分裂和胞质分裂过程中，CPC表现出动态的定位，在中后期从染色质迁移到中央纺锤体，在有丝分裂-胞质分裂过渡期间从中央纺锤体迁移到新FAZ的前端(Li等人，2008a；Li等人，200)。锥体中的Polo样激酶同源物TbPLK也集中在新的FAZ的前端，在那里它促进细胞质起始(De Graffenry等人，2008；Kumar和Wang，2006；Umeyama和Wang，2008)。位于新FAZ前端的CPC和TbPLK的下游因子尚未确定，锥体似乎缺乏CPC和Polo样激酶的大部分配对蛋白同源物，这表明锥体可能进化出了不同的CPC和PLK介导的胞质分裂途径。目前的建议是建立在TbAUK1和TbPLK在细胞质分裂中的基本作用的基础上，并旨在解决以下问题。(1)。TbAUK1和TbPLK是如何调控胞质分裂启动的？我们假设TbAUK1和TbPLK通过在新FAZ的前端形成一个复合体或调节一些共同的下游因子来共同调节胞质分裂的启动。我们的中心假设是，TbAUK1在完成其在有丝分裂中的基本功能后，迁移到新的FAZ的前端，在那里它与TbPLK和其他新的蛋白质合作，启动胞质分裂。(2)。TbAUK1活性和亚细胞定位在有丝分裂-胞质分裂转换和胞质分裂进程中是如何调节的？这主要是由于锥体中存在一种新的CPC，以及TbAUK1在有丝分裂和胞质分裂过程中独特的动态反式定位，这一点仍然知之甚少。(3)。CPC的结构-功能关系是什么？TbCPC1和TbCPC2这两个新的CPC成分的作用是什么？我们的假设是TbCPC1和/或TbCPC2是TbAUK1本地化的介体(S)，也可能是TbAUK1的激活子(S)。通过分子、细胞生物学、化学遗传学和生化手段，我们的总体目标是了解TbAUK1和TbPLK在胞质分裂启动中的机制作用，以及TbCPC1和TbCPC2对TbAUK1的调控。我的实验室的长期目标是描绘控制布氏毛滴虫有丝分裂和胞质分裂的调控网络，这将有助于我们基本理解有丝分裂、有丝分裂-胞质分裂协调和胞质分裂的分子基础，这些分子基础不同于通常认为的通过收缩肌球蛋白收缩环进行的细胞分裂。这些研究结果不仅具有重要的生物学意义，而且可能为抗锥虫病的化疗提供新的靶点。

DESCRIPTION (provided by applicant): Cytokinesis in Trypanosoma brucei, a parasitic protozoan and the causative agent of human sleeping sickness, is known to be totally different from that in its human host. The cleavage plane in a dividing trypanosome is not defined by the central spindle like in yeasts and animals, but rather by the position of the new flagellum and the flagellum attachment zone (FAZ). Consequently, cytokinesis in trypanosomes is initiated from the anterior tip of the new FAZ, and the cleavage furrow ingresses unidirectionally along the long axis from the anterior towards the posterior end of the cell. Strikingly, an actomyosin contractile ring is not formed in trypanosomes, indicating that trypanosome cleavage furrow may possess an unusual structure with novel components. Despite these differences, however, both Aurora-like and Polo-like kinases are implicated in cytokinesis initiation in trypanosomes (Hammarton et al., 2007; Li and Wang, 2006; Tu et al., 2006). The Aurora kinase homolog in trypanosomes, TbAUK1, forms a unique chromosomal passenger complex (CPC) that plays essential roles in chromosome segregation and cytokinesis initiation (Li et al., 2008a). The CPC exhibits a dynamic localization during mitosis and cytokinesis by migrating from chromatins to the central spindle during metaphase-anaphase transition and from the central spindle to the anterior tip of the new FAZ during mitosis-cytokinesis transition (Li et al., 2008a; Li et al., 200). The Polo-like kinase homolog in trypanosomes, TbPLK, is also concentrated at the anterior tip of the new FAZ where it promotes cytokinesis initiation (de Graffenried et al., 2008; Kumar and Wang, 2006; Umeyama and Wang, 2008). The downstream factors of the CPC and TbPLK at the anterior tip of the new FAZ are not identified, and trypanosomes appear to lack most of the partner protein homologs of the CPC and Polo-like kinase, suggesting that trypanosomes may have evolved distinct CPC- and PLK-mediated pathways for cytokinesis. The current proposal is built upon the essential roles of TbAUK1 and TbPLK in cytokinesis, and aims to address the following questions. (1). How is cytokinesis initiation regulated by TbAUK1 and TbPLK? We hypothesize that TbAUK1 and TbPLK cooperate to regulate cytokinesis initiation by forming a complex or regulating some common downstream factors at the anterior tip of the new FAZ. Our central hypothesis is that after fulfilling its essential function in mitosis TbAUK1 migrates to th anterior tip of the new FAZ where it cooperates with TbPLK and other novel proteins to initiate cytokinesis. (2). How is TbAUK1 activity and subcellular localization regulated during mitosis-cytokinesis transition and during cytokinesis progression? This is still poorly understood, mainly due to the presence of a novel CPC in trypanosomes and the unique dynamic trans-localization of TbAUK1 during mitosis and cytokinesis. (3). What is the structure-function relationship of the CPC and what are the roles of the two novel CPC components, TbCPC1 and TbCPC2? Our hypothesis is that TbCPC1 and/or TbCPC2 are the mediator(s) of TbAUK1 localization and are also likely the activator(s) of TbAUK1. Through molecular, cell biological, chemical genetic, and biochemical means, our overall goal in this proposal is to understand the mechanistic role of TbAUK1 and TbPLK in cytokinesis initiation, the regulation of TbAUK1 by its novel partners TbCPC1 and TbCPC2. The long-term goal of my laboratory is to delineate the regulatory networks that control mitosis and cytokinesis in T. brucei, which will facilitate our fundamental understanding of the molecular basis of mitosis, mitosis-cytokinesis coordination, and cytokinesis that is different from the commonly recognized cell division through the constriction of an actomyosin contractile ring. The outcome from these studies would not only have important biological significance, but also could provide novel targets for anti-trypanosomiasis chemotherapy.