Characterization of Ribosomal Plaque Domains in Axons

轴突是一条长长的通讯线路，构成神经系统的“硬连线”。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，所有轴浆蛋白都是在细胞体中合成的，并以缓慢的速度运输到轴突。这一推论是基于明显缺乏轴浆核糖体，这是蛋白质合成机制的超微结构关联，尽管代谢和生化证据与此相反。最近，在从有髓轴突分离的轴浆的外围发现了周期性的小的离散核糖体结构域。这项研究计划是为了验证“轴浆周围核糖体结构域”作为蛋白质合成中心的假设，它为邻近的区域提供必要的蛋白质，如肌动蛋白。实验将在莫特纳神经元中进行，这是金鱼脑干中的一种大型可识别细胞，它沿着整个脊髓投射有髓轴突。他们将确定(1)编码由肌动蛋白和绿色荧光蛋白(GFP)组成的融合蛋白的cDNA结构，后者作为报告蛋白是否会导致mRNA从细胞体运输到轴突，(2)mRNA在轴浆核糖体周围区域的定位，以及(3)融合蛋白产物的合成和随后分布到周围的轴浆。其他实验将在兔有髓运动轴突中验证这一假说，其中将分析轴突上初始代谢摄取[35S]蛋氨酸的位置，并与轴浆周围核糖体区域的定位有关。随后将绘制放射性蛋白质到周围轴浆的随时间变化的分布图，还将进行放射性标记蛋白质的分析。一个补充问题是，是否有任何膜蛋白也在轴突间隔中合成，这需要信号识别颗粒(SRP)。实验被用来分析(1)SRPs的发生，(2)SNAP-25mRNA的存在和定位，它编码了参与递质释放的膜蛋白，以及(3)糖基化，这是一个向新合成的膜蛋白共翻译添加糖的过程。具体地说，这些实验将解决一个重要的问题，即轴浆周围核糖体结构域在合成蛋白质以供轴突室内局部使用的过程中是否具有新陈代谢活性。然而，更广泛地说，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。

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.