Molecular Dissection of the Leishmania Paraflagellar Rod

9724105利什曼原虫的前鞭毛体旁杆状结构(PfR)是利什曼原虫正常运动所必需的，但这种独特的细胞骨架结构的分子结构、组装和功能尚不清楚。PFR在轴丝从鞭毛袋中出现后延长其长度，并由排列成格子状阵列的离散细丝组成。PFR的主要蛋白质组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复杂而不寻常的结构，表明了一种新型的细胞骨架细丝和组织。基于缺乏主要PFR蛋白PFR-1和PFR-2的墨西哥菜品系的特征，提出了一个简单的PFR组织模型，该模型具有许多可检验的特征。首先，PFR-1蛋白是PFR近端结构域的重要组成部分，而PFR-2在近端结构域中缺失。其次，在没有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。这种PFR组装的顺序解释了为什么在没有PFR-1的情况下PFR-2蛋白无法组装成稳定的结构，也解释了为什么在没有PFR-2的情况下存在含有PFR-1的正确定位的结构。此外，它解释了令人费解的观察到，在用抗PFR抗体进行免疫荧光检查的细胞分裂时，子鞭毛常比亲本鞭毛染色更弱。如果PFR是从近端到远端逐步组装的，这是可以预期的。建议进行遗传学和细胞学实验，以测试和扩展墨西哥利什曼原虫PFR的组织模式。这些特定的目标将被完成：1.Pfr-1和Pfr-1/Pfr-2缺失突变体的超微结构特征。这组实验的目的是确定在缺乏PFR-1的突变系中是否保留了PFR的亚结构。2.确定PFR-1和PFR-2蛋白在PFR中的位置。这组实验的目的是通过免疫金标记法分别定位PFR-1和PFR-2蛋白。3.测试PFR的域的组装顺序。这些实验的目的是确定分裂墨西哥斑潜蝇的新生子鞭毛先组装Pfr的近端区域，然后组装远端区域。在单细胞生物体中，在生理和结构上存在着巨大的差异，这反映了在进化过程中在形式和功能上进行的大量实验。单细胞生物体以鞭毛和纤毛为基础的运动方式已被详细阐述，并在不同的单细胞生物体中发生了变化。在这个奖项中，Lebowitz博士将研究利什曼原虫纤毛旁杆的功能和形态。了解椎旁杆是如何组装的，将为了解单细胞生物体运动机制的多样性提供重要信息。利什曼原虫的成员包括可导致人类疾病的病原体。这些信息的潜在应用包括寻找化合物，这些化合物可以阻止牙周杆状结构的形成，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. ***