Molecular Dissection of the Leishmania Paraflagellar Rod

利什曼原虫副鞭毛杆的分子解剖

• 批准号: 9724105
• 负责人: Jonathan LeBowitz
• 金额: $ 12.97万
• 依托单位: Purdue Research Foundation
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9724105&HistoricalAwards=false
• 关键词: Molecular; Dissection; Leishmania; Paraflagellar; Rod

9724105 LeBowitz The paraflagellar rod (PFR) is essential for the normal motility of Leishmania promastigotes, yet the molecular structure, assembly and function of this unique cytoskeletal structure are not well understood. The PFR extends the length of the axoneme after it emerges from the flagellar pocket and is built up from discrete filaments organized in lattice-like arrays. The major protein components of the PFR do not reveal significant homology to other cytoskeletal proteins. This lack of homology together with the elaborate and unusual structure of the PFR suggests a novel type of cytoskeletal filament and organization. Based on the characterization of L. mexicana lines lacking the major PFR proteins, PFR-1 and PFR-2, a simple model for the organization of the PFR is proposed that has a number of testable features. First, PFR-1 protein is an essential constituent of the proximal domain of the PFR and PFR-2 is absent from the proximal domain. Second, PFR-1 protein can assemble into filaments in the absence of PFR-2 either forming a homopolymer or polymerizing with as yet unidentified partners. Third, PFR-2 protein is a primary constituent of the distal domain of the PFR. Fourth, assembly of the distal domain of the PFR requires prior assembly of the proximal domain of the PFR whose assembly in turn is coordinated with assembly of the axoneme. This ordering of PFR assembly explains the observed inability of PFR-2 protein to assemble into a stable structure in the absence of PFR-1, and it explains the presence of a properly positioned structure that contains PFR-1 in the absence of PFR-2. Furthermore, it accounts for the puzzling observation that in dividing cells examined by immunofluorescence with anti-PFR antibodies, the daughter flagellum often stains more weakly than the parental flagellum. This would be expected if the PFR was assembled stepwise from the proximal to the distal zone. Genetic and cytological experiments are proposed t o test and extend this model of the organization of the PFR in Leishmania mexicana. These specific objectives will be accomplished: 1. Ultrastructural characterization of PFR-1 and PFR-1/PFR-2 null mutants. The objective of this set of experiments is to determine whether or not substructures of the PFR are retained in the mutant lines that lack PFR-1 2. Determination of the location within the PFR of the PFR-1 and PFR-2 proteins. The objective of this set of experiments is to separately localize PFR-1 and PFR-2 proteins by immunogold labeling of thin sections and whole mount cytoskeletons. 3. Test of the order of assembly of the domains of the PFR. The objective of these experiments is to determine whether the nascent daughter flagella of dividing L. mexicana assemble the proximal domain of the PFR prior to the distal domain. Among unicellular organisms there is an immense diversity of differences in physiology and structure that reflects the vast experimentation in form and function that has occurred during evolution. The means by which unicellular organisms move using flagella and cilia as basis has been elaborated and altered as among diverse unicellular organisms. In this award Dr. LeBowitz will study the function and form of the paraflagellar rod of Leishmania. Understanding how the paraflagellar rod is assembled will yield important information on the diversity of motility mechanisms of unicellular organisms. Members of Leishmania species include pathogens that are causitive agents of human disease. Potential applications of this information includes finding compounds that impede the formation of paraflagellar rod preventing motility and spread of the organisms. ***

小行星9724105 旁鞭毛杆（PFR）是利什曼原虫前鞭毛体正常运动所必需的，但 这种独特的细胞骨架结构的分子结构、组装和功能还没有被很好地理解。 PFR在轴丝从鞭毛袋中出来后延伸轴丝的长度，并由 以格子状阵列组织的离散细丝。 PFR的主要蛋白质成分没有揭示 与其他细胞骨架蛋白具有显著的同源性。 这种同源性的缺乏， PFR的不寻常结构表明了一种新型的细胞骨架丝和组织。基于 L.的特征墨西哥品系缺乏主要的PFR蛋白，PFR-1和PFR-2， 组织的PFR提出了具有一些可测试的功能。 首先，PFR-1蛋白是 PFR和PFR-2的近端结构域的必需成分在近端结构域中缺失， 域 第二，PFR-1蛋白可以在PFR-2不存在的情况下组装成细丝， 均聚物或与尚未鉴定的伙伴聚合。第三，PFR-2蛋白是一种主要的 PFR的远端结构域的组成部分。 第四，PFR远端结构域的组装需要 PFR的近端结构域的预先组装，其组装又与PFR的近端结构域的组装协调， 轴丝 PFR组装的这种顺序解释了观察到的PFR-2蛋白不能组装 在没有PFR-1的情况下，它变成了一个稳定的结构，它解释了正确定位的 在不存在PFR-2的情况下含有PFR-1的结构。 此外，它解释了令人困惑的 观察到在用抗PFR抗体通过免疫荧光检查的分裂细胞中， 子鞭毛通常比亲本鞭毛染色更弱。 如果 PFR从近端到远端区域逐步组装。遗传学和细胞学实验 提出测试和扩展这种模式的组织PFR在墨西哥利什曼原虫。 这些 具体目标是：一， PFR-1和PFR-1/PFR-2的超微结构表征 无效突变体这组实验的目的是确定是否亚结构的 PFR保留在缺乏PFR-12的突变株系中。 确定PFR内的位置 PFR-1和PFR-2蛋白。 这组实验的目的是分别定位PFR-1和 PFR-2蛋白的免疫金标记的薄切片和整个安装细胞骨架。3. 试验 PFR结构域的组装顺序。 这些实验的目的是确定 分裂L. mexicana组装PFR的近端结构域之前， 远端域。 在单细胞生物中，生理学和结构上存在巨大的差异， 反映了在进化过程中发生的形式和功能的大量实验。的手段 单细胞生物以鞭毛和纤毛为基础移动的原理已经被详细阐述和改变， 不同的单细胞生物。在这个奖项中，LeBowitz博士将研究 利什曼原虫的鞭毛旁杆。了解副鞭毛杆是如何组装的， 关于单细胞生物运动机制多样性的重要信息。成员 利什曼原虫属物种包括作为人类疾病的致病因子的病原体。的潜在应用 这些信息包括发现阻止副鞭毛杆形成的化合物， 生物体的运动和扩散。 ***