Mechanisms of the Unusual Cytokinesis in Trypanosomes

描述（由申请人提供）：已知布氏锥虫（一种寄生原生动物和人类昏睡病的病原体）的胞质分裂与其人类宿主的胞质分裂完全不同。分裂锥虫的分裂平面不像酵母和动物那样由中心纺锤体限定，而是由新鞭毛的位置和 鞭毛附着区（FAZ）。因此，锥虫的胞质分裂从新FAZ的前端开始，卵裂沟沿长轴从细胞的前端向后端沿着单向进入。引人注目的是，在锥虫中没有形成肌动球蛋白收缩环，这表明锥虫卵裂沟可能具有不寻常的结构和新的成分。然而，尽管存在这些差异，Aurora样和Polo样激酶都涉及锥虫中的胞质分裂起始（Hammarton等人，2007; Li和Wang，2006; Tu等人，2006年）。锥虫中的Aurora激酶同系物TbAUK 1形成独特的染色体乘客复合物（CPC），其在染色体分离和胞质分裂起始中起重要作用（Li et al.，2008年a）。CPC在有丝分裂和胞质分裂期间通过在中期-后期转换期间从染色质迁移到中央纺锤体以及在有丝分裂-胞质分裂转换期间从中央纺锤体迁移到新FAZ的前端而表现出动态定位（Li et al.，2008 a; Li等人，200）。锥虫中的Polo样激酶同系物TbPLK也集中在新FAZ的前端，在那里它促进胞质分裂起始（de Graffenried et al.，2008年; Kumar和Wang，2006年; Umeyama和Wang，2008年）。CPC和TbPLK在新FAZ的前端的下游因子没有确定，锥虫似乎缺乏CPC和Polo样激酶的大部分伴侣蛋白同源物，这表明锥虫可能已经进化出不同的CPC和PLK介导的胞质分裂途径。 目前的建议是建立在TbAUK 1和TbPLK在胞质分裂中的重要作用，并旨在解决以下问题。（一）. TbAUK 1和TbPLK如何调控胞质分裂起始？我们推测TbAUK 1和TbPLK通过在新FAZ的前端形成复合物或调节一些共同的下游因子来合作调节胞质分裂起始。我们的中心假设是TbAUK 1在有丝分裂中完成其基本功能后迁移到新FAZ的前端，在那里它与TbPLK和其他新蛋白质合作启动胞质分裂。（二）.在有丝分裂-胞质分裂过渡期和胞质分裂进程中TbAUK 1活性和亚细胞定位是如何调节的？这仍然是知之甚少，主要是由于锥虫中存在一种新的CPC和独特的动态trans-localization的TbAUK 1在有丝分裂和胞质分裂。（三）、CPC的结构-功能关系是什么？两个新的CPC组分TbCPC 1和TbCPC 2的作用是什么？我们的假设是TbCPC 1和/或TbCPC 2是TbAUK 1定位的介体，也可能是TbAUK 1的激活剂。通过分子、细胞生物学、化学遗传学和生物化学手段，我们的总体目标是了解TbAUK 1和TbPLK在胞质分裂起始中的机制作用，以及TbAUK 1通过其新伙伴TbCPC 1和TbCPC 2的调节。 本实验室的长期目标是描绘T细胞有丝分裂和胞质分裂的调控网络。布氏杆菌，这将有助于我们从根本上了解有丝分裂的分子基础，有丝分裂-胞质分裂协调，胞质分裂，这是不同于通常公认的细胞分裂，通过收缩的肌动球蛋白收缩环。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.