RUI: PTL-1, A Tau-Like Microtubule Binding Protein in C. elegans

9604180 Aamodt 技术摘要：哺乳动物 tau 微管相关蛋白与轴突生长、微管间距和微管成束有关。 鉴定并表征了与 tau 重复区域同源的秀丽隐杆线虫基因。 该基因被命名为 ptl-1（具有 Tau 样重复序列的蛋白质）。 ptl-1 转录本与哺乳动物 tau 转录本一样，通过选择性剪接产生编码具有可变重复次数的蛋白质的信息。 预测的 ptl-1 产物在重复区域与 tau 具有很强的序列同源性，并且在大小、氨基酸含量、电荷分布、预测的二级结构、疏水性和灵活性方面与 tau 相似。 体外细菌表达 PTL-1 与微管结合。 这些结果表明，tau 样蛋白进化得很早，并表明它们可能存在于许多不同的门中。 线虫是一个强大的系统，适合遗传、分子和细胞分析，可用于研究 tau 类蛋白质的功能。 将解决三个主要问题：PTL-1 是否以类似于哺乳动物 tau 的方式促进微管蛋白聚合？ 基因在哪里表达？ 基因产物的功能是什么？ 将测试重组 PTL-1，以确定它是否像 tau 一样在体外增加微管成核和组装。 将产生针对 PTL-1 的抗体，并制备 ptl-1::lacZ 融合基因以确定 ptl-1 表达的时间和地点。最后，将产生不表达 ptl-1 的线虫菌株。 如果ptl-1的缺失诱发表型，哺乳动物的tau基因就会表达出来，以确定是否可以挽救。 这些研究将提供有关 ptl-1 在秀丽隐杆线虫中的作用以及 PTL-1 和 tau 之间关系的信息。 该项目的相关性源于它对我们理解基本细胞和发育机制的贡献。 摘要：微管是真核细胞中极其重要的结构。 它们在细胞和细胞内运动中发挥作用，作为纤毛和鞭毛跳动的手段，染色体在细胞分裂过程中分配给子细胞，细胞延伸适当地成形和定向，以及细胞内成分从细胞的一个部分移动到另一部分。 最后两个功能对于具有极长轴突的神经元细胞尤其重要（高等动物的一些轴突可能有几英尺长！）。 轴突需要将细胞体连接到身体其他地方的正确端点，并且神经兴奋剂和其他物质需要从细胞体中的合成位点运输到轴突的末端，以使神经元正常发挥作用。 已知许多辅助蛋白与微管结合，并以某种方式参与其正确的形成和/或功能；这些统称为微管相关蛋白（MAP）。 神经元细胞中的一种主要 MAP 被称为 tau，尽管它已在哺乳动物系统中得到了广泛研究，但关于 tau 在细胞中的功能仍有许多基本问题尚未得到解答。 在该项目中，将利用最近在简单模型生物体——秀丽隐杆线虫（一种线虫）中发现的 tau 样蛋白。 关于线虫的简单且完善的背景信息，加上遗传学的力量，将使人们对这种 tau 蛋白在线虫和其他动物中的功能有新的发现。 ***

9604180 Aamodt Technical Abstract: The mammalian tau microtubule-associated proteins have been implicated in axonal outgrowth, microtubule spacing and microtubule bundling. A caenorhabditis elegans gene with homology to the repeat region of tau was identified and characterized. This gene was named ptl-1 for Protein with Tau-Like repeats. The ptl-1 transcript, like mammalian tau transcripts, is alternatively spliced to produce messages that encode proteins with variable numbers of repeats. The predicted ptl-1 products have strong sequence homology to tau over the repeat region and are similar to tau in size, amino acid content, charge distribution, predicted secondary structure, hydrophobicity , and flexibility Bacterially expression PTL-1 bound to microtubules in vitro. These results show that tau-like proteins evolved early and suggests that they may be present in many different phyla. C. elegans is a powerful system amenable to genetic, molecular and cellular analysis in which to study the functions of the tau class of proteins. Three major questions will be addressed: Does PTL-1 promote tubulin polymerization in a manner similar to that of mammalian tau? Where is the gene expressed? And what is the function of the gene product? Recombinant PTL-1 will be tested to determine whether it increases microtubule nucleation and assembly in vitro as tau does. Antibodies will be produced against PTL-1 and ptl-1::lacZ fusion genes will be made to determine when and where ptl-1 is expressed. Finally, a strain of C. elegans that does not express ptl-1 will be made. If absence of ptl-1 induces a phenotype, the mammalian tau gene will be expressed to determine whether it can rescue. These studies will provide information about the role of ptl-1 in C. elegans and the relationship between PTL-1 and tau. The relevant of this project derives from its contribution to our understanding of basic cellular and developmental mechanisms. Lay Abstract: Microtubules are fundamentally important structures in eukaryotic cells. They function in cellular and intracellular motility, serving as the means whereby cilia and flagella beat, chromosomes are distributed to daughter cells during cell division, cellular extensions are properly shaped and oriented, and intracellular components are moved from one part of the cell to another. These last two functions are notably critical in neuronal cells that bear extremely long axons (some axons in higher animals may be several feet in length!). The axons need to connect the cell bodies to the right terminal point elsewhere in the body, and neuroexcitatory and other materials need to be transported from their site of synthesis in the cell body to the terminal end of the axon in order for the neuron to function properly. Many accessory proteins are known that bind to microtubules and are somehow involved in their proper formation and/or function; these are known collectively as Microtubule-Associated Proteins, or MAPs. One of the predominant MAPs in neuronal cells is called tau, and although it has been studied extensively in mammalian systems, there are still many fundamental questions about tau's function in the cell that remain unanswered. In this project, the recent discovery of a tau-like protein in the simple model organism, C. elegans (a nematode worm), will be exploited. The simplicity and well-established background information about the worm, together with the power of genetics, will allow new discoveries about the function of this tau-like protein in both the worm and other animals. ***