The unconventional Ark3 cluster in Toxoplasma gondii

摘要 专性细胞内寄生虫弓形虫可以在人类中引起严重的机会性感染， 特别针对未出生的儿童或艾滋病毒/艾滋病患者。宿主细胞的快速侵袭和细胞内 复制负责在寄生虫的溶解周期中观察到的组织损伤，并由 速殖子形态。虽然已经有了对抗弓形虫病的特定药物治疗，但严重的副作用 可能会产生影响，长期使用可能会导致过敏。 在裂解周期的下面是寄生虫特有的复制装置，它在体内形成两个新的子细胞 母体的胞浆(内生)。这一过程受到寄生虫特异的蛋白激酶的高度调控， 在很大程度上控制着细胞周期。三种极光相关的蛋白激酶(Ark1-3)已被发现 并以此为特征。在这些激酶中，Ark3的耗尽强烈地影响着形成的子代的生长， 但该激酶的详细作用仍不清楚。耐人寻味的是，该激酶有一个独特的亚细胞 本地化，在分割过程中形成线性结构，该结构从复制的 核远端的中心体。据报道，疟原虫也有类似的组装。 恶性疟原虫，在细胞末端检测到类似的PfArk3对裂殖子形成的动态 在红血球中循环。这种神秘的集会的性质在这两个国家中都没有得到进一步的调查 有机体。 这项建议的主要目标是剖析寄生虫中Ark3相关簇(AC)的性质。 为了迎合这一目标，我们最近基于邻近生物素化技术鉴定了两个新基因，它们的 产物在细胞分裂过程中表现出Ark3的动态变化。我们将它们命名为Ark3相关组件 (Arc)1和2。最值得注意的是，这些假想蛋白质显示出严重的适应性分数，并支持 前提是Ark3/Arc1/2组件正在执行至今未被识别的基本功能，以及 完全没有特征的组织结构。我们将通过以下两个具体目标来研究AC。 在Aim1下，我们将通过有条件的蛋白质降解来更详细地回顾Ark3的表型 并将其表型效应与条件耗尽的Arc1和ArC2的寄生虫进行比较。 超微结构扩展显微镜(U-EXM)的应用将进一步使我们能够研究表型 效果和功能定义了这个神秘的结构。为了进一步映射函数，在AIM2下，我们将 使用快速作用的生物素连接酶TurboID，将邻近生物素化作用相互作用于Arc1和Arc2。 在成功完成这项研究计划后，我们将对Ark3和Ark3有更深入的了解 它的非常规聚集性，并在弓形虫子代细胞形成的早期步骤中产生了新的内部。

Summary The obligate intracellular parasite Toxoplasma gondii can cause severe opportunistic infection in humans, specially targeting the unborn child or HIV/AIDS patients. Fast rounds of host cell invasion and intracellular replication are responsible for tissue lesions observed in the parasite’s lytic cycle and caused by the tachyzoite form. Although specific drug treatments to battle Toxoplasmosis are available, severe side effects can occur and long-term application might lead to hypersensitivity. Underlying the lytic cycle is a parasite-specific replication apparatus, which forms two new daughter cells in the cytosol of the mother (endodyogeny). This process is highly regulated by parasite-specific kinases, which largely control the cell cycle. Three aurora-related kinases (Ark1-3) have previously been identified and characterized. Among these kinases, Ark3 depletion strongly affects growth of the forming daughters, but detailed actions of the kinase remain unknown. Intriguingly, the kinase has a unique subcellular localization, forming a linear structure during the division process, which extends from the duplicated centrosomes distal towards the nucleus. A similar assembly has also been reported for Plasmodium falciparum, where a similar PfArk3 dynamic is detected towards merozoite formation at the end of the cell cycle in the red blood cell. The nature of this enigmatic assembly has not further been investigated in either organism. The main goal of this proposal is to dissect the nature of the Ark3-related cluster (AC) in the parasite. Catering to this goal is our recent proximity biotinylation-based identification of two new genes whose products exhibit Ark3 dynamics during the cell division. We have named them Ark3-related component (ARC) 1 and 2. Most notably, these hypothetical proteins display severe fitness scores and support the premise that the Ark3/ARC1/2 assembly is executing an essential function at a hereto unrecognized, and completely uncharacterized division structure. We will study the AC by the following two specific aims. Under Aim1 we will revisit the Ark3 phenotype in more detail by conditional protein degradation of the kinase and compare its phenotypic effects to parasites conditionally-depleted of ARC1 and ARC2. Application of ultrastructure expansion microscopy (U-ExM) will further allow us to investigate phenotypic effects and functionally define this mysterious structure. To further map the function, under Aim2 we will reciprocally apply proximity biotinylation to ARC1 and ARC2, using the fast acting biotin ligase TurboID. Upon successful completion of this research proposal we will have gained deep understanding of Ark3 and its unconventional cluster and generate novel insides into early steps of T. gondii daughter cell formation.