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

小行星9604180 技术摘要：哺乳动物tau微管相关蛋白与轴突生长、微管间距和微管集束有关。 一个秀丽隐杆线虫基因的同源性的重复区域的tau蛋白的鉴定和表征。 该基因被命名为ptl-1，即具有Tau样重复的蛋白质。 ptl-1转录本，像哺乳动物tau转录本一样，被选择性剪接以产生编码具有可变重复数目的蛋白质的信息。 预测的ptl-1产物在重复区域上与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、疏水性和柔性方面与tau相似。 这些结果表明，tau样蛋白进化较早，并表明它们可能存在于许多不同的门。 C. elegans是一个强大的系统，适合于遗传、分子和细胞分析，在其中研究tau类蛋白的功能。 三个主要问题将得到解决：PTL-1促进微管蛋白聚合的方式类似于哺乳动物的tau蛋白？ 基因在哪里表达？ 基因产物的功能是什么？ 将测试重组PTL-1以确定其是否像tau那样在体外增加微管成核和组装。 将产生针对PTL-1的抗体，并将制备ptl-1：：lacZ融合基因以确定ptl-1表达的时间和位置。最后，一株C.将制备不表达PTL-1的秀丽隐杆线虫。 如果ptl-1的缺失诱导表型，则哺乳动物tau基因将被表达以确定其是否可以拯救。 这些研究将提供ptl-1在C.以及PTL-1与tau蛋白的关系。 这个项目的相关性来自于它对我们理解基本细胞和发育机制的贡献。 摘要：微管是真核细胞中非常重要的结构。 它们在细胞和细胞内运动中起作用，作为纤毛和鞭毛跳动的手段，染色体在细胞分裂期间分布到子细胞，细胞延伸正确成形和定向，细胞内组分从细胞的一个部分移动到另一个部分。 后两种功能在具有极长轴突的神经元细胞中尤其重要（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部位的正确终点，神经兴奋性和其他物质需要从细胞体中的合成部位运输到轴突的末端，以便神经元正常发挥功能。 已知许多辅助蛋白与微管结合，并以某种方式参与其正常形成和/或功能;这些被统称为微管相关蛋白或MAP。 神经元细胞中的主要MAP之一称为tau，尽管它已在哺乳动物系统中被广泛研究，但关于tau在细胞中的功能仍有许多基本问题尚未解决。 在这个项目中，最近在简单模式生物C。elegans（一种线虫）将被利用。 关于蠕虫的简单性和完善的背景信息，以及遗传学的力量，将允许关于这种tau样蛋白在蠕虫和其他动物中的功能的新发现。 ***

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. ***