Molecular Dissection of the Leishmania Paraflagellar Rod

鞭毛旁杆（PFR）对promastigotes利什曼原虫的正常运动至关重要，但这种独特的细胞骨架结构的分子结构、组装和功能尚不清楚。PFR从鞭毛囊中出现后延长轴突的长度，并由以晶格状阵列组织的离散细丝组成。PFR的主要蛋白质成分与其他细胞骨架蛋白没有明显的同源性。这种同源性的缺乏加上PFR的复杂和不寻常的结构表明了一种新型的细胞骨架丝和组织。基于墨西哥L.墨西哥菌株缺乏主要PFR蛋白（PFR-1和PFR-2）的特征，提出了一个简单的PFR组织模型，该模型具有许多可测试的特征。首先，PFR-1蛋白是PFR近端结构域的重要组成部分，而PFR-2在近端结构域缺失。其次，在缺乏PFR-2的情况下，PFR-1蛋白可以组装成细丝，形成均聚物或与尚未确定的伙伴聚合。第三，PFR-2蛋白是PFR远端结构域的主要组成部分。第四，PFR远端结构域的组装需要PFR近端结构域的预先组装，而PFR近端结构域的组装又与轴突的组装协调。PFR组装的这种顺序解释了在缺乏PFR-1的情况下，观察到的PFR-2蛋白无法组装成稳定的结构，也解释了在缺乏PFR-2的情况下，存在一个包含PFR-1的正确定位结构。此外，它还解释了一个令人困惑的现象，即在用抗pfr抗体进行免疫荧光检测的分裂细胞中，子代鞭毛的染色往往比亲代鞭毛更弱。如果PFR从近端到远端区逐步组装，这是可以预期的。提出了遗传学和细胞学实验来检验和扩展墨西哥利什曼原虫PFR组织的这种模型。这些具体目标将得到实现：PFR-1和PFR-1/PFR-2零突变体的超微结构特征。这组实验的目的是确定PFR的亚结构是否保留在缺乏PFR-1 - 2的突变系中。确定PFR-1和PFR-2蛋白在PFR中的位置。本实验的目的是通过免疫金标记薄切片和全载细胞骨架分别定位PFR-1和PFR-2蛋白。3. PFR结构域组装顺序的测试。这些实验的目的是确定分裂L. mexicana的新生子鞭毛是否在PFR的近端区域之前组装远端区域。在单细胞生物中，生理和结构上存在着巨大的多样性差异，这反映了在进化过程中发生的形式和功能上的大量实验。单细胞生物以鞭毛和纤毛为基础运动的方式在不同的单细胞生物中得到了阐述和改变。在这个奖项中，LeBowitz博士将研究利什曼原虫鞭毛旁杆的功能和形式。了解鞭毛旁杆是如何组装的，将对单细胞生物运动机制的多样性产生重要的信息。利什曼原虫种类的成员包括病原体，是人类疾病的病原体。这些信息的潜在应用包括发现阻碍鞭毛旁杆形成的化合物，防止生物体的运动和传播。＊＊＊

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. ***