Cytoskeletal organization in apicomplexan parasites

顶端复门寄生虫的细胞骨架组织

• 批准号: 6708619
• 负责人: HEIDE SCHATTEN
• 金额: $ 7.32万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6708619
• 关键词: Apicomplexa; Toxoplasma gondii; actins; cell cycle; cell nucleus; centrosome; cytoskeleton; electron microscopy; microfilaments; parasitism; pellicle; western blottings

DESCRIPTION (provided by the applicant): The purpose of this research is to understand cytoskeletal organization and functions during cell division of the intracellular protozoan parasite Toxoplasma gondii, a model for harmful apicomplexan parasites that include Plasmodium falciparum and Cryptosporidium parvum. Despite the importance of the cytoskeleton for cell division in these unicellular organisms the mechanisms that power the separation of the genome remain uncertain, largely because only limited data exist about the organization and composition of the cytoskeleton. Recent data suggest an unconventional division machinery that divides the nucleus and separates the apicoplast, a single non-photosynthetic plastid that is essential for parasite survival. We hypothesize (1) that the parasite's unconventional division apparatus is based on a complex organization of microtubules, their organizing center (the centriole-centrosome complex) and perhaps other cytoskeletal components yet to be determined. Fibers of the division apparatus may associate with the pellicle (the parasite's outer membrane system) to separate the genome and the apicoplast during cell division in T. gondii; we hypothesize (2) that the centriole-centrosome complex plays an important role in the organization of the cytoskeleton and may contain proteins that differ from mammalian cells. Our specific aims are to (1) analyze the parasite's cytoskeleton during cell division with focus on the interactions with the pellicle, the apicoplast and the nucleus, and (2) to characterize the centriole-centrosome complex with focus on proteins that may differ from mammalian cells and may serve as targets to inhibit cell division in apicomplexan parasites. The novel approach of the proposed experiments is to use high-resolution field emission scanning electron microscopy (HRFESEM) on thick-sectioned resin de-embedded material. This method used in preliminary experiments allowed for the first time viewing of actin-like filaments and intermediate-like filaments which had not been possible with any other method available so far. We will further isolate the division apparatus of T. gondii and generate antibodies to determine parasite-specific centrosome proteins. This research will address unresolved questions regarding the cytoskeleton and the centriole-centrosome complex during cell division in T. gondii and, perhaps, the existence of other cytoskeletal fibers. This research will advance our understanding of cell division in apicomplexan parasites and may lead to the identification of targets to control replication of harmful apicomplexan parasites. Pilot data from this RO3 research will be used to apply for funding through the RO1 mechanism.

描述（由申请人提供）：本研究的目的是了解细胞内原生动物寄生虫弓形虫的细胞分裂过程中的细胞骨架组织和功能，弓形虫是有害的顶复门寄生虫的模型，包括恶性疟原虫和小隐孢子虫。尽管细胞骨架对于这些单细胞生物中的细胞分裂很重要，但驱动基因组分离的机制仍然不确定，很大程度上是因为关于细胞骨架的组织和组成的数据有限。最近的数据表明，一种非常规的分裂机制可以分裂细胞核并分离顶端质体，顶端质体是寄生虫生存所必需的单一非光合质体。我们假设（1）寄生虫的非常规分裂装置基于微管的复杂组织、其组织中心（中心粒-中心体复合体）以及可能还有其他尚未确定的细胞骨架成分。弓形虫细胞分裂过程中，分裂装置的纤维可能与表膜（寄生虫的外膜系统）结合以将基因组和顶质体分开；我们假设（2）中心粒-中心体复合物在细胞骨架的组织中起着重要作用，并且可能含有与哺乳动物细胞不同的蛋白质。我们的具体目标是（1）分析细胞分裂过程中寄生虫的细胞骨架，重点关注与外皮、顶质体和细胞核的相互作用，以及（2）表征中心粒-中心体复合物，重点关注可能不同于哺乳动物细胞的蛋白质，这些蛋白质可能作为抑制顶复合体寄生虫细胞分裂的靶标。所提出的实验的新颖方法是在厚截面树脂去嵌入材料上使用高分辨率场发射扫描电子显微镜（HRFESEM）。初步实验中使用的这种方法首次允许观察肌动蛋白样丝和中间样丝，这是迄今为止任何其他可用方法所不可能实现的。我们将进一步分离弓形虫的分裂装置并产生抗体来确定寄生虫特异性中心体蛋白。这项研究将解决有关弓形虫细胞分裂过程中的细胞骨架和中心粒-中心体复合体的未解决问题，或许还有其他细胞骨架纤维的存在。这项研究将增进我们对顶复门寄生虫细胞分裂的理解，并可能导致识别控制有害顶复门寄生虫复制的靶标。 RO3 研究的试点数据将用于通过 RO1 机制申请资金。