The Toxoplasma basal complex in cell division

摘要 弓形虫是一种专性的尖端复合体寄生虫，可引起严重的机会性感染。 目前的药物容易引起过敏反应，特别是长期使用时。在这项提议下， 将询问独特的细胞分裂过程，以确定可能的新药靶点。弓形虫除以 不同的内部发芽过程，两个子寄生虫聚集在一个母细胞内。这个 由扁平的肺泡泡组成的皮质膜骨架，由表型纤溶酶蛋白网络支撑 22个膜下微管在中心体上成核，并以顶端至基端的方式聚集 方向。在分裂的后半部分，子芽的后端(即基生复合体或BC) 在肌球蛋白J(MyoJ)的推动下开始逐渐变小。MyoJ的缺失只会对寄生虫的生存产生轻微的影响，即使它 使BC有些不受限制，符合细胞分裂抵抗肌动蛋白解聚的经典数据 探员们。然而，通过耗尽或过度表达BC支架来完全阻止BC的组装 MORN1蛋白导致寄生虫线粒体磨损，无法完成细胞分裂，并具有戏剧性的影响 关于生存能力。为了揭开这一有趣的过程，在R21的资助下，对BC进行了蛋白质组学解剖 8个BC组分上的邻近依赖生物素化(BioID)。这揭示了4-5种不同的蛋白质复合体 与超微结构一致。在这一提议下，进一步探讨了两个关键的观察结果：1.假定的MT 相关蛋白MAP1B-L1似乎组装在膜下MT的()端，是必不可少的 用于BC组装和寄生虫的生存能力；2.几种已鉴定的激酶和磷酸酶表明BC是 受差异磷酸化的调节。根据AIM 1 MAP1B-L1和另一个名为MAP1B的关键BC图- 在体外，将通过在寄生虫中产生缺失突变体来测试L2的MT结合能力 确定MT结合域，并通过外源表达在非洲爪哇轴突引导模型中为AS 与相关地图相关。在目标2下，我们将寻求在BioID方法中确定的另外四个候选者 具有可能的基本功能，它们都是在内部萌芽中狭隘保守的假想蛋白质 寄生虫和假定的粘附域。此外，我们将在BC组件上应用快速作用的TurboID 与像MAP1B-L1这样的组装BC瞬时关联，因为这些可能在当前 数据集，但定义了BC在细胞分裂中的必要步骤。在目标3下，我们将使用2个激活器和1个 利用全基因组CRISRP/Cas9文库进行磷酸酶合成致死性筛选。初步数据： 第一个测试的激酶已经证明了实验的可行性，并揭示了有趣的新见解。 蛋白质组和遗传数据集的结合有望为组装连线提供坚实的基础 公元前十年的示意图。在当前的工作模型中，BC首先在MT()-End上组装，然后 黏附蛋白的募集使MT末端保持在一起。总体而言，预计这将带来令人兴奋的新 对内部萌芽的洞察，它与精神分裂有何不同，并可能突出新的药物靶点。

Summary Toxoplasma gondii is an obligate intracellular apicomplexan parasite causing severe opportunistic infections. Current drugs are prone to induce hypersensitivity, especially upon long-term use. Under this proposal the unique cell division process will be interrogated to identify putative new drug targets. Toxoplasma divides by a distinct internal budding process whereby two daughter parasites are assembled within a mother cell. The cortical membrane skeleton composed of flattened alveolar vesicles supported by an epiplastin protein network and 22 subpellicular microtubules (MTs) is nucleated on the centrosomes and assembles in an apical to basal direction. In the second half of division the posterior end of the daughter buds (i.e. the basal complex or BC) starts to taper driven by Myosin J (MyoJ). Absence of MyoJ only modestly impact parasite viability, even while it leaves the BC somewhat unconstricted, fitting classic data on cell division resistance to actin depolymerizing agents. However, preventing assembly of the BC altogether by depleting or overexpressing the BC scaffolding protein MORN1 results in parasites with fraying MTs unable to complete cell division and has dramatic impact on viability. To unravel this intriguing process, under an R21 grant the BC was proteomically dissected through proximity dependent biotinylation (BioID) on 8 BC components. This revealed 4-5 different protein complexes aligning with the ultrastructure. Two key observations are further pursued under this proposal: 1. A putative MT Associated Protein, MAP1B-L1, appears to assemble on the (+)-ends of the subpellicular MTs and is essential for BC assembly and parasite viability; 2. Several kinases and phosphatases identified indicate the BC is regulated by differential phosphorylation. Under Aim 1 MAP1B-L1 and another critical BC MAP dubbed MAP1B- L2 will be tested for MT binding capacity by generating deletion mutants in the parasite, in vitro using the identified MT binding domains, and by exogenous expression in the Xenopus leavis axon guidance model as relevant to related MAPs. Under Aim 2 we will pursue four additional candidates identified in the BioID approach with a likely essential function, which are all hypothetical proteins narrowly conserved in internally budding parasites and harbor putative adhesion domains. In addition, we will apply fast acting TurboID on BC components transiently associating with the assembling BC like MAP1B-L1 as these were likely undersampled in the current dataset, yet define the essential step of the BC in cell division. Under Aim 3 we will subject 2 kinases and 1 phosphatase to synthetic lethality screening using the genome wide CRISRP/Cas9 library. Preliminary data of the first kinase tested already demonstrates experimental feasibility and revealed interesting new insights. Combining the proteomic and genetic data sets is expected to provide a solid basis to assemble the wiring diagram of the BC. In the current working model the BC is first assembled on the MT (+)-ends, followed by recruitment of adhesion proteins to keep the MT-ends together. Overall, this is expected to deliver exciting new insights into internal budding, how it differs from schizogony, and could highlight new drug targets.