Local Synthesis in the Mauthner Axon

轴质位于神经细胞的轴突，由细胞骨架蛋白和细胞质蛋白组成，它们通过一种鲜为人知的缓慢运输过程供应给轴突。由于所有蛋白质都表现出半衰期的特征，因此在轴突的缓慢运输过程中，蛋白质很可能通过降解经历了显著的生物衰变。由于生物衰变造成的大量损失，为了维持轴突蛋白质组成和质量的稳定状态，需要局部补充蛋白质的机制。该研究项目将检查选定的细胞骨架蛋白制造系统的定位和分布，并确定哪些蛋白质在轴突局部合成。尽管现有证据表明，髓鞘轴突的局部合成能力有限，而且在短半衰期内对新合成蛋白质的局部需求可能不大，但整个轴突的合成量可能很大。实验将在毛特纳神经元（m -轴突）的细胞水平上进行，m -轴突是金鱼中枢神经系统中一个可识别的细胞，有一个很大的髓鞘轴突延伸到脊髓的长度。使用m -轴突制备的一个主要优点是髓鞘与胶质细胞体分离，使轴突与髓鞘分离成为可能，而不受污染，用于细胞体和轴突的分析。将使用Koenig博士实验室设计和开发的专门方法来研究超纯显微样品。研究者将利用原位杂交技术定位m轴突中的核糖体RNA，并评估其密度和分布。信使rna将定位于m轴突的肌动蛋白和/或微管蛋白，这些重要的细胞骨架蛋白通过缓慢的运输从细胞体提供给轴突。在体外制备中，将对m轴突合成的轴突多肽进行分析，以确定哪些蛋白质是在短半衰期内局部合成的，并确定标记的蛋白质是由于从头合成的。将m细胞轴突体内合成的蛋白质与体外合成的蛋白质进行比较，以鉴定在生理条件下合成的蛋白质和半衰期较长的蛋白质。这项研究的结果将解决一个重要的未解决的问题，即成熟的轴突如何补偿其外源供应蛋白质的生物衰变。

Axoplasm, located in the axons of nerve cells, is composed of cytoskeletal and cytosolic proteins which are supplied to the axon by a poorly understood process of slow transport. Since all proteins exhibit a characteristic of half-life, it is likely that during slow transport in the axon, proteins undergo significant biological decay through degradation. In order to maintain the steady state of protein composition and mass of the axon due to significant losses through biological decay, local mechanisms for the replenishment of proteins are necessary. This research project will examine the localization and distribution of selected cytoskeletal protein manufacturing systems and ascertain what proteins are synthesized locally in the axon. Although the evidence available indicates that myelinated axons have a limited capacity for local synthesis and that local requirements for newly synthesized proteins in a short half-life period are probably modest, the amount of synthesis occurring over the whole axon is probably significant. Experiments will be conducted at the cellular level on the Mauthner neuron, the M-axon, an identifiable cell in the CNS of the goldfish with a large myelinated axon extending the length of the spinal cord. A major advantage in using the M-axon preparation is that the myelin sheath is separated from glial cell bodies, making it possible to separate the axon from the myelin sheath uncontaminated for the analysis of the cell body and the axon. Specialized methods, designed and developed in Dr. Koenig's laboratory, for studying ultrapure microscopic samples will be used. The investigator will utilize in situ hybridization techniques to localize ribosomal RNA in the M-axon and to assess its density and distribution. Messenger RNAs will be localized in the M-axon for actin and/or tubulin, important cytoskeletal proteins that are supplied to the axon from the cell body by slow transport. Analysis of axonal polypeptides synthesized in the M-axon, in an in vitro preparation, will be performed in order to characterize which proteins are synthesized locally in the short half-life period and to establish that labeled proteins are due to de novo synthesis. Proteins synthesized in the M-cell axon in vivo will be compared with those synthesized in vitro in order to identify proteins synthesized under physiological conditions, and proteins with a longer half-life. The results of this study will address an important unresolved issue of how the mature axon compensates for biological decay of its exogenously supplied proteins.