Characterization of Ribosomal Plaque Domains in Axons

轴突核糖体斑块结构域的表征

• 批准号: 0118368
• 负责人: Edward Koenig
• 金额: $ 30万
• 依托单位: SUNY at Buffalo
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-15 至 2005-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0118368&HistoricalAwards=false
• 关键词: Characterization; Ribosomal; Plaque; Domains; Axons

The axon serves as a long line of communication that makes up the 'hard wiring' of the nervous system. Its long term maintenance and potential for structural plasticity depends on a continuous supply of proteins. It was generally assumed that all axoplasmic proteins were synthesized in the cell body and transported at slow rates to the axon. The reasoning was based on an apparent lack of axoplasmic ribosomes, the ultrastructural correlates of a protein synthesizing machinery, despite metabolic and biochemical evidence to the contrary. Recently, periodic small discrete ribosome-containing domains were documented in the periphery of axoplasm isolated from myelinated axons. The research proposal is concerned with testing the hypothesis that "periaxoplasmic ribosomal domains" serve as centers of protein synthesis, which supply immediate surrounding regions with essential proteins, such as actin. Experiments will be performed in the Mauthner neuron, which is a large identifiable cell in the goldfish brain stem that projects its myelinated axon along the full length of spinal cord. They will determine (1) whether a cDNA construct that codes for a fusion protein, comprised of actin and green fluorescent protein (GFP), in which the latter serves as a reporter, will result in the transport of the mRNA from the cell body to the axon, (2) localization of the mRNA in periaxoplasmic ribosomal domains and (3) synthesis and subsequent distribution of the fusion protein product to surrounding axoplasm. Additional experiments will test the hypothesis in rabbit myelinated motor axons, in which sites of initial metabolic uptake of [35S]methionine along axons will be analyzed and related to the localization of periaxoplasmic ribosomal domains. Subsequent time-dependent distribution of radioactive proteins to surrounding axoplasm will be mapped, and analysis of radiolabeled proteins will also be undertaken. A complementary issue is whether any membrane proteins are also synthesized in the axon compartment, for which signal recognition particles (SRPs) would be required. Experiments are proposed to analyze (1) for the occurrence of SRPs, (2) for the presence and localization of SNAP-25 mRNA, which codes for a membrane protein involved in transmitter release, and (3) for glycosylation, a process of cotranslationally adding sugars to newly synthesized membrane proteins. Specifically, the experiments will address an important question as to whether periaxoplasmic ribosomal domains are metabolically active in synthesizing proteins for local utilization in the axon compartment. More generally, however, the findings will add substantially to a better fundamental understanding of the complex biology of the axon.

轴突作为一条长长的通讯线路，构成了神经系统的“硬接线”。它的长期维持和结构可塑性的潜力取决于蛋白质的持续供应。一般认为，所有的轴浆蛋白在细胞体中合成，并以缓慢的速率运输到轴突。这种推理是基于轴浆核糖体的明显缺乏，轴浆核糖体是蛋白质合成机制的超微结构相关物，尽管代谢和生物化学证据与此相反。最近，周期性的小离散核糖体含有域被记录在外周的轴浆分离有髓轴突。该研究计划涉及测试假设，即“轴浆周核糖体结构域”作为蛋白质合成的中心，为周围区域提供必需的蛋白质，如肌动蛋白。实验将在Mauthner神经元中进行，Mauthner神经元是金鱼脑干中的一个大的可识别细胞，它沿着脊髓的全长投射其有髓轴突。他们将确定（1）编码融合蛋白的cDNA构建体（由肌动蛋白和绿色荧光蛋白（GFP）组成，其中后者作为报告基因）是否会导致mRNA从细胞体转运到轴突，（2）mRNA在轴浆周核糖体结构域中的定位，以及（3）融合蛋白产物的合成和随后分布到周围的轴浆。另外的实验将在兔有髓鞘运动轴突中检验该假设，其中将分析[35 S]蛋氨酸沿着轴突的初始代谢摄取位点，并将其与轴浆周核糖体结构域的定位相关。随后的时间依赖性分布的放射性蛋白质周围轴质将被映射，放射性标记的蛋白质的分析也将进行。另一个补充问题是，轴突隔室中是否也合成了任何膜蛋白，这需要信号识别颗粒（SRPs）。实验拟分析（1）SRPs的出现，（2）SNAP-25 mRNA的存在和定位，其编码参与递质释放的膜蛋白，和（3）糖基化，将糖共价地添加到新合成的膜蛋白的过程。具体而言，实验将解决一个重要的问题，即轴浆周围核糖体结构域是否在轴突隔室中局部利用的蛋白质合成中具有代谢活性。然而，更普遍的是，这些发现将大大增加对轴突复杂生物学的更好的基本理解。