The unconventional Ark3 cluster in Toxoplasma gondii

总结 专性细胞内寄生虫弓形虫可引起人类严重的机会性感染， 专门针对未出生的婴儿或艾滋病毒/艾滋病患者。宿主细胞侵袭和细胞内 复制负责在寄生虫的溶解周期中观察到的组织病变， 速殖子型虽然有特定的药物治疗来对抗弓形虫病，但严重的 可能会产生影响，长期使用可能会导致过敏。 裂解周期的基础是寄生虫特异性的复制装置，它形成两个新的子细胞， 母亲的细胞质（内生）。这一过程受到寄生虫特异性激酶的高度调节， 主要控制细胞周期。三种极光相关激酶（Ark 1 -3）以前已被确定 和表征了在这些激酶中，Ark 3缺失强烈影响形成子体的生长， 但激酶的详细作用仍不清楚。有趣的是，激酶具有独特的亚细胞 本地化，在分裂过程中形成线性结构，从复制的 中心体远离细胞核。疟原虫也有类似的组装 恶性疟原虫，其中在细胞末端检测到类似的PfArk 3动力学朝向裂殖子形成 在红细胞中循环。这个神秘的集会的性质没有进一步调查，无论是在 有机体 该提案的主要目标是剖析寄生虫中Ark 3相关簇（AC）的性质。 迎合这一目标的是我们最近基于近距离生物素化的两个新基因的鉴定， 产物在细胞分裂期间表现出Ark 3动力学。我们将其命名为Ark 3相关组件 (ARC)1和2.最值得注意的是，这些假设的蛋白质显示出严重的适应性得分，并支持了 前提是Ark 3/ARC 1/2组件正在以迄今为止未被识别的方式执行基本功能，并且 完全没有特征的划分结构。我们将通过以下两个具体目标来研究AC。 在Aim 1中，我们将通过条件性蛋白质降解来更详细地回顾Ark 3表型。 激酶，并将其表型效应与ARC 1和ARC 2条件性耗竭的寄生虫进行比较。 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 影响和功能定义了这个神秘的结构。为了进一步映射函数，在Aim 2下，我们将 使用快速作用的生物素连接酶TurboID，将邻近生物素化应用于ARC 1和ARC 2。 在成功完成这项研究计划后，我们将对Ark 3有深入的了解， 它非传统的集群和产生新的内部到T的早期步骤。弓形虫子细胞形成。

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.