Cytoskeletal organization in apicomplexan parasites

描述（由申请方提供）：本研究的目的是了解细胞内原生动物寄生虫刚地弓形虫（一种包括恶性疟原虫和隐孢子虫在内的有害顶复门寄生虫的模型）细胞分裂期间的细胞骨架组织和功能。尽管细胞骨架对这些单细胞生物的细胞分裂很重要，但驱动基因组分离的机制仍然不确定，主要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，一个非常规的分裂机制，分裂细胞核和分离顶质体，一个单一的非光合质体，是寄生虫生存所必需的。我们假设（1）寄生虫的非常规分裂装置是基于微管的复杂组织，它们的组织中心（中心粒-中心体复合体）和其他尚待确定的细胞骨架成分。分裂器的纤维可能与表膜（寄生虫的外膜系统）结合，在T.弓形虫;我们假设（2）中心粒-中心体复合体在细胞骨架的组织中起重要作用，并且可能含有不同于哺乳动物细胞的蛋白质。我们的具体目标是（1）分析寄生虫的细胞分裂过程中的细胞骨架，重点是与表膜，顶质体和细胞核的相互作用，和（2）表征中心粒-中心体复合物，重点是蛋白质，可能不同于哺乳动物细胞，并可能作为目标，以抑制细胞分裂的顶复体寄生虫。该实验的新方法是使用高分辨率场发射扫描电子显微镜（HRFESEM）对厚切片树脂脱嵌材料。在初步实验中使用的这种方法允许第一次观察肌动蛋白样丝和中间体样丝，这是迄今为止使用任何其他方法都不可能实现的。我们将进一步分离T.并产生抗体以确定寄生虫特异性中心体蛋白。本研究将解决尚未解决的问题，有关细胞骨架和中心粒-中心体复合体在细胞分裂T。弓形虫，也许还有其他细胞骨架纤维的存在。这项研究将促进我们对顶复门寄生虫细胞分裂的理解，并可能导致确定控制有害顶复门寄生虫复制的靶点。从RO 3研究中获得的试验数据将用于通过RO 1机制申请资金。

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.