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、微管间距和微管束有关。鉴定并鉴定了一个与tau基因重复区同源的秀丽线虫基因。这个基因被命名为PTL-1，意思是带有类似Tau重复序列的蛋白质。PTL-1转录本和哺乳动物tau转录本一样，可以交替剪接产生编码重复次数可变的蛋白质的信息。预测的PTL-1产物在重复区域与tau有很强的序列同源性，在大小、氨基酸含量、电荷分布、预测的二级结构、疏水性以及体外细菌结合微管的柔性表达方面与tau相似。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，可用于遗传、分子和细胞分析，在其中研究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？将对重组的PTL-1进行测试，以确定它是否像tau一样在体外增加微管的成核和组装。将产生针对PTL-1和PTL-1的抗体：将产生LacZ融合基因，以确定PTL-1何时何地表达。最后，将产生一株不表达PTL-1的线虫。如果PTL-1的缺失诱导了一种表型，哺乳动物tau基因就会被表达，以确定它是否可以挽救。这些研究将提供有关PTL-1在线虫中的作用以及PTL-1和tau之间的关系的信息。这个项目的相关性源于它对我们理解基本的细胞和发育机制的贡献。摘要：微管是真核细胞的重要结构。它们在细胞和细胞内的运动中发挥作用，作为纤毛和鞭毛跳动的手段，染色体在细胞分裂期间被分配给子细胞，细胞伸展被适当地塑造和定向，以及细胞内成分从细胞的一个部分移动到另一个部分。后两种功能对于具有超长轴突(高等动物的一些轴突可能有几英尺长！)的神经细胞来说是非常关键的。轴突需要将细胞体连接到身体其他地方的正确终点处，神经兴奋性物质和其他物质需要从细胞体的合成部位运输到轴突的末端，才能使神经元正常工作。已知许多辅助蛋白与微管结合，并以某种方式参与微管的正确形成和/或功能；这些统称为微管相关蛋白，或MAP。神经细胞中最主要的图谱之一被称为tau，尽管它在哺乳动物系统中已经得到了广泛的研究，但关于tau在细胞中的功能仍然有许多基本的问题没有答案。在这个项目中，将利用最近在简单模式生物线虫(线虫)中发现的类似tau的蛋白质。关于蠕虫的简单性和完善的背景信息，再加上遗传学的力量，将允许对这种类似tau的蛋白质在蠕虫和其他动物中的功能进行新的发现。***

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. ***