Functional Analysis of Novel Components of the Toxoplasma Inner Membrane Complex

弓形虫内膜复合物新成分的功能分析

• 批准号: 9384311
• 负责人: Peter John Bradley
• 金额: $ 37.89万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9384311
• 关键词: Animals; Apical; Biology; Biotin; Biotinylation; CRISPR screen; Cell membrane; Cells; Cellular biology; Characteristics; Coccidiosis; Complex; Cone; Cytoskeletal Filaments; Cytoskeleton; Daughter; Disease; Eimeria tenella; Elements; Event; Falciparum Malaria; Family; Human; Immobilization; In Vitro; Infection; Invaded; Knock-out; Knowledge; Lead; Ligase; Mass Spectrum Analysis; Mediating; Medical; Membrane; Methods; Microtubules; Morphology; Motor; Names; Neospora caninum; Organelles; Parasites; Peripheral; Play; Process; Proteins; Proteome; Reporting; Role; Series; Shapes; Streptavidin; Surgical sutures; System; TimeLine; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Vesicle; Virulence; Xenopus oocyte; cell motility; choline transporter; cohort; daughter cell; design; experimental study; fitness; genome-wide; in vivo; insight; member; membrane biogenesis; novel; novel therapeutics; obligate intracellular parasite; parasite invasion; pathogen; protein complex; protein function; scaffold

PROJECT SUMMARY Toxoplasma gondii and related apicomplexan parasites contain a specialized organelle called the inner membrane complex (IMC) that plays essential roles in host cell invasion and daughter cell formation. Determining the precise functions of the IMC has been hampered by a limited understanding of its protein constituents, which have predominantly focused on the motor components of the glideosome that powers parasite motility and the family of alveolins that are embedded within the IMC cytoskeletal network underlying the flattened membrane vesicles of the organelle. We have recently overcome this gap in our knowledge using in vivo biotinylation (BioID) to identify over forty new IMC components, more than doubling the known IMC proteome. These new proteins have revealed a surprising level of compartmentalization within the organelle, with distinct components segregating to the cytoskeletal or membrane subregions of the apical cap, IMC body, or the recently discovered IMC sutures, which organize the IMC membranes into their characteristic rectangular plates. Analyses of a few of these proteins have revealed new important roles in controlling parasite shape, cellular division, and invasion. In this proposal, we will leverage our new cohort of IMC proteins to first provide a temporal and spatial understanding of how these components participate in the dynamic process of internal budding known as endodyogeny. This will reveal groups of proteins that function to assemble the IMC at specific steps of endodyogeny and also provide insight into the dynamic process of distinguishing maternal and daughter IMCs during division. We will then explore how the key IMC suture proteins ISC3 and ISC6 regulate proper IMC membrane biogenesis, parasite morphology and replication. Finally, we exploit the recent genome-wide CRISPR screen in Toxoplasma to focus on a subset of our new IMC proteins that are likely to play crucial roles within distinct suborganellar compartments of the organelle. Together, this project will fundamentally transform our understanding of the organization and function of the Toxoplasma IMC. As this organelle is parasite-specific and is not present in its human host, elucidating critical IMC components and their respective functions promises to reveal ideal targets for the design of novel therapies against T. gondii and other apicomplexan parasites.

项目摘要 弓形虫和相关的顶复门寄生虫含有一个专门的细胞器称为内 膜复合物（IMC）在宿主细胞入侵和子细胞形成中起重要作用。 确定IMC的精确功能受到对其蛋白质的有限理解的阻碍 成分，主要集中在动力的滑体的运动组件， 寄生虫运动和肺泡蛋白家族，嵌入IMC细胞骨架网络， 细胞器的扁平膜囊泡。我们最近已经克服了我们知识上的这一差距， 体内生物素化（BioID），以确定40多个新的IMC成分，是已知IMC的两倍多 蛋白质组这些新的蛋白质揭示了细胞器内令人惊讶的区室化水平， 不同的组分分离到顶帽，IMC体， 或最近发现的IMC缝合线，其将IMC膜组织成其特征性的 矩形板对其中一些蛋白质的分析揭示了在控制 寄生虫的形状、细胞分裂和侵入。在这项提案中，我们将利用我们新的IMC蛋白质组， 首先提供这些组件如何参与动态的时间和空间的理解， 称为endodysis的内部出芽过程。这将揭示蛋白质组的功能， 在endodynamic的特定步骤组装IMC，也提供了对 在分裂过程中区分母亲和女儿的IMC。然后我们将探讨关键的IMC缝合 蛋白质ISC 3和ISC 6调节适当的IMC膜生物发生、寄生虫形态和复制。 最后，我们利用最近在弓形虫中的全基因组CRISPR筛选，重点关注我们新发现的一个子集。 IMC蛋白可能在细胞器的不同亚细胞器区室中发挥关键作用。 总之，这个项目将从根本上改变我们对组织和功能的理解， 弓形虫IMC。由于这种细胞器是寄生虫特异性的，并且不存在于其人类宿主中，因此阐明了关键的 内模控制元件及其各自的功能，为设计新颖的内模控制系统提供了理想的目标 抗T.弓形虫和其他顶复门寄生虫。