Cytoskeleton scaffold assembly in Toxoplasma gondii

描述(由申请人提供)：由专性细胞内寄生虫弓形虫引起的疾病固有地与速殖子生命阶段的快速复制周期有关。速殖子的分裂在每一轮分裂中产生两个子代，这些子代通过独特的内部萌芽过程在母细胞内组装。外周细胞骨架的组装驱动萌发，并作为细胞器组装和分离的支架。细胞骨架由微管和两个不与宿主共享的独特元素(中间丝与扁平的囊泡组成的复合体，称为内膜复合体或IMC)组成。这项提议旨在描述复制机制的一个独特组件--中间丝细胞骨架。到目前为止，已经研究了四个不同的丝状基因(TgIMC1-4)，但初步的基因组调查确定了总共16个基因。在整个发育过程中，对几个细丝的初步研究发现了令人惊讶的行为光谱，并暗示了到目前为止还没有得到重视的中间细胞骨架的复杂程度。因此，研究小组建议在整个寄生虫科(目标1)评估所有16个基因的定位、翻译后修饰和蛋白分解处理。此外，初步数据表明，TgMORN1蛋白是两个细丝结构(IMC和后杯)之间的中心组织者，也为成熟过程中细胞骨架的收缩提供了支架。通过鉴定大型TgMORN1复合体中的蛋白质(目标2)，将进一步剖析TgMORN1的关键作用。最后，我们想要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(目标3)。在这一建议完成后，了解中间丝细胞骨架的组装者、时间、地点和方式，成熟、稳定和周转，将能够预测如何最好地干预分裂，为开发治疗弓形虫病的新疗法提供合理的基础。与公共卫生相关：可用于治疗由蜂群引起的疾病的治疗选择有限。我们建议研究模式寄生虫弓形虫的内部萌发过程，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)蛋白质，为潜在的特异性药物靶点提供新的靶点。

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.