Characterization of Ribosomal Plaque Domains in Axons

9604841科尼格建议的研究集中在轴突外边界间歇性分布的新的、专门化的区域，称为“轴浆周围斑块”。这些斑块是在金鱼中枢神经系统中Mauthner神经元的分离轴浆中发现的，现在已经证实它们存在于兔脊神经根的轴突中；因此，它们很可能是一般长轴突的共同特化。斑块结构域中核糖体的鉴定表明，后者是潜在的与轴浆蛋白局部周转有关的蛋白质合成的局部中心。这一前提与关于轴突的活力和质量如何保持稳定状态的主流观点大相径庭。一个主要的实验目标是在电子显微镜水平上分析金鱼和兔轴突斑块结构域中的主要结构成分，以确定斑块域的空间组织，并表征较老的和较新进化的脊椎动物轴突的共同特征和显著差异。另一个主要的实验目的是验证这一假设，即斑块结构域代表蛋白质合成的局部中心，是从神经元细胞体运输RNA的靶向终端。为此，将利用免疫染色和分子生物学技术分别评估金鱼和兔轴突中延伸因子1a的定位与斑块结构域的关系，延伸因子1a是蛋白质合成的重要辅助因子，以及编码特定基因产物的信使RNA的定位。标记的RNA构建物也将被显微注射到Mauthner细胞中，以评估RNA的靶向性和从细胞体到斑块结构域的运输。最后，还将用放射自显影技术评估金鱼和兔轴突中代谢标记的从头合成蛋白质的位置，以确定新生多肽链与斑块结构域之间的空间关系。这项拟议研究的信息应该有助于极大地改善对轴浆蛋白和长轴突活力是如何长期保持的理解。

9604841 Koenig The proposed study centers on novel, specialized domains located at intermittent intervals in the outer boundary of axons, called "periaxoplasmic plaques." The plaques were discovered in isolated axoplasm from the Mauthner neuron located in the goldfish central nervous system, and their presence has now been confirmed in axons of rabbit spinal nerve roots; therefore, they are likely to be specializations common to long axons in general. Identification of ribosomes in plaque domains suggests that the latter are potential local centers of protein synthesis concerned with local turnover of axoplasmic proteins. This premise is a significant departure from prevailing views on how the axon's vitality and mass are maintained in a steady state. One major experimental aim is to analyze the principal structural components located within plaque domains of goldfish and rabbit axons at an electron microscopic level in order to define the spatial organization of the plaque domain, and to characterize common features and distinctive differences between older and more recently evolved vertebrate axons. Another major experimental aim is to test the hypothesis that plaque domains represent local centers of protein synthesis, and are targeted endstations for RNA trafficking from the neuronal cell body. For this purpose, immunostaining, and molecular biological techniques will be used to evaluate localization in relationship to plaque domains in goldfish and rabbit axons of elongation factor 1a, an essential cofactor of protein synthesis, and localization of messenger RNA that codes for specific gene products, respectively. Labeled RNA constructs will also be microinjectied into the Mauthner cell to evaluate targeting and transport of RNA to plaque domains from the cell body. Finally, sites of de novo protein synthesis, assayed by metabolic radiolabeling in goldfish and rabbit axons, will also be evaluated by autoradiography in order to determ ine the spatial relationship between the localization of nascent peptide chains to plaque domains. Information from the proposed study should contribute to a greatly improved understanding of how axoplasmic proteins, and vitality of long axons are maintained on a long term basis.