The Toxoplasma basal complex in cell division

总结 弓形虫是一种专性细胞内顶复门寄生虫，可引起严重的机会性感染。 目前的药物容易引起过敏，尤其是长期使用时。根据该提案 将研究独特的细胞分裂过程，以确定假定的新药物靶标。弓形虫除以 独特的内部出芽过程，两个子寄生虫在母细胞内组装。的 由表塑蛋白网络支持的扁平肺泡囊泡组成的皮质膜骨架 22 个膜下微管 (MT) 在中心体上成核并从顶端到基底组装 方向。在分裂的后半部分，子芽的后端（即基部复合体或 BC） 开始由肌球蛋白 J (MyoJ) 驱动逐渐变细。 MyoJ 的缺失只会轻微影响寄生虫的生存能力，即使它 使 BC 稍微不受限制，符合细胞分裂对肌动蛋白解聚的抵抗力的经典数据 代理。然而，通过耗尽或过度表达 BC 支架来完全阻止 BC 的组装 MORN1 蛋白导致寄生虫的 MT 磨损，无法完成细胞分裂，并产生巨大影响 关于生存能力。为了解开这个有趣的过程，在 R21 资助下，通过蛋白质组学对 BC 进行了解剖 8 个 BC 成分上的邻近依赖性生物素化 (BioID)。这揭示了 4-5 种不同的蛋白质复合物 与超微结构对齐。该提案进一步探讨了两个关键观察结果： 1. 假定的 MT 相关蛋白 MAP1B-L1 似乎在膜下 MT 的 ( ) 端组装，并且是必需的 用于 BC 组装和寄生虫活力； 2. 鉴定出的几种激酶和磷酸酶表明 BC 是 受差异磷酸化调节。在目标 1 MAP1B-L1 和另一个被称为 MAP1B- 的关键 BC MAP 下 L2 将通过在寄生虫中产生缺失突变体来测试 MT 结合能力，在体外使用 确定了 MT 结合域，并通过 Xenopus leavis 轴突引导模型中的外源表达为 与相关 MAP 相关。在目标 2 下，我们将寻求 BioID 方法中确定的另外四个候选者 具有可能的基本功能，这些都是在内芽中严格保守的假设蛋白质 寄生虫和窝藏假定的粘附域。此外，我们将在 BC 组件上应用快速作用的 TurboID 与 MAP1B-L1 等组装 BC 短暂关联，因为这些在当前情况下可能采样不足 数据集，但定义了 BC 在细胞分裂中的基本步骤。在目标 3 下，我们将研究 2 种激酶和 1 使用全基因组 CRISRP/Cas9 文库进行磷酸酶合成致死筛选。初步数据 第一个测试的激酶已经证明了实验可行性并揭示了有趣的新见解。 结合蛋白质组和遗传数据集有望为组装线路提供坚实的基础 BC 的示意图。在当前的工作模型中，BC 首先组装在 MT ( ) 端，然后是 招募粘附蛋白以将 MT 末端保持在一起。总体而言，这预计将带来令人兴奋的新 深入了解内部萌芽，它与分裂生殖有何不同，并可以突出新的药物靶标。

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.