Cytoskeleton scaffold assembly in Toxoplasma gondii

描述（由申请人提供）：由专性细胞内寄生虫刚地弓形虫引起的疾病与速殖子生命阶段的快速复制周期内在相关。速殖子分裂每分裂一轮产生两个子细胞，它们通过独特的“内部出芽”过程在母细胞内组装。外周细胞骨架的组装驱动出芽，并作为细胞器组装和分离的支架。细胞骨架由微管和两种与宿主不同的独特元素（中间丝与扁平囊泡的复合物称为内膜复合物或IMC）组成。本提案旨在描述复制机制的一个独特组成部分，即中间丝细胞骨架。迄今为止，已经研究了四种不同的灯丝基因（TgIMC1-4），但初步的基因组调查共鉴定了16个基因。对发育过程中几种纤维的初步研究发现了令人惊讶的行为谱，并暗示了迄今为止未被充分认识的中间细胞骨架的复杂性。因此，研究小组建议评估所有16个基因在寄生虫分裂过程中的定位、翻译后修饰和蛋白水解加工（目的1）。此外，初步数据表明，TgMORN1蛋白是两个丝结构（IMC和后杯）之间的中心组织者，它也为成熟过程中细胞骨架的收缩提供了一个支架。TgMORN1的关键作用将通过鉴定大TgMORN1复合体中的蛋白质进一步剖析（目的2）。最后，我们想构建一个过程的模型，其中分裂过程中的形态变化可以与生化事件联系起来（目的3）。完成本研究后，了解中间丝细胞骨架的组装、成熟、稳定性和周转的时间、地点和方式，将有助于预测如何最好地干扰分裂，为开发治疗弓形虫病的新疗法提供合理的基础。公共卫生相关性：有限的治疗方案可用于治疗由顶丛引起的疾病。我们建议研究弓形虫模型的内部出芽过程，特别是中间丝细胞骨架的贡献，并鉴定细胞分裂机制中的（独特的）蛋白质，提供潜在的新的特异性药物靶点。

DESCRIPTION (provided by applicant): Disease caused by the obligate intracellular parasite Toxoplasma gondii is inherently linked to fast replication cycles of the tachyzoite life stage. Tachyzoite division produces two daughters per division round, which are assembled within the mother cell through a unique 'internal budding' process. Assembly of the peripheral cytoskeleton drives budding and serves as a scaffold for organelle assemblage and segregation. The cytoskeleton is composed of microtubules together with two unique elements not shared with the host (intermediate filaments in complex with flattened vesicles together called the inner membrane complex or IMC). This proposal aims to characterize a unique component of the replication machinery, the intermediate filament cytoskeleton. To date, four different filament genes have been studied (TgIMC1-4), but a preliminary genomic survey identified 16 genes in total. Preliminary studies of several filaments throughout development identified a surprising spectrum in behavior and hint at a thus far underappreciated sophistication of the intermediate cytoskeleton. Therefore the research team proposes to assess the localization, post-translation modification, and proteolytic processing of all 16 genes throughout parasites division (Aim 1). Furthermore, preliminary data identifies protein TgMORN1 as a central organizer between two filament structures (the IMC and the posterior cup), which also provides a scaffold for the constriction of the cytoskeleton during maturation. The pivotal role of TgMORN1 will be further dissected by identifying the proteins in the large TgMORN1 complex (Aim 2). Finally, we want to construct a model of the process wherein morphological changes during the division process can be linked to biochemical events (Aim 3). Upon completion of this proposal, understanding on the who, when, where and how of the assembly, maturation, stability and turnover of the intermediate filament cytoskeleton will allow predictions on how to best interfere with division, providing a rational basis for development of new therapeutics to treat toxoplasmosis. PUBLIC HEALTH RELEVANCE: Limited treatment options are available to treat disease caused by Apicomplexa. We propose to study the internal budding process of the model parasite Toxoplasma gondii, in particular the contribution of the intermediate filament cytoskeleton, and identify the (unique) proteins in the cell division machinery providing potential new specific drug targets.