Local Synthesis in the Mauthner Axon

轴浆位于神经细胞的轴突中，由细胞骨架和胞浆蛋白组成，通过一个鲜为人知的缓慢运输过程提供给轴突。由于所有蛋白质都表现出半衰期的特征，很可能在轴突的缓慢运输过程中，蛋白质通过降解经历了显著的生物衰退。为了维持蛋白质组成和轴突质量的稳定状态，由于生物衰退而造成的重大损失，蛋白质的局部补充机制是必要的。这项研究项目将检查选定的细胞骨架蛋白质制造系统的定位和分布，并确定在轴突中局部合成哪些蛋白质。尽管现有证据表明有髓轴突的局部合成能力有限，而且在短半衰期内局部对新合成蛋白质的需求可能不大，但整个轴突的合成量可能是相当大的。实验将在细胞水平上对莫特纳神经元进行，即M轴突，这是金鱼中枢神经系统中的一种可识别细胞，有一个巨大的有髓轴突延伸到脊髓长度。使用M-轴突制剂的一个主要优点是髓鞘与神经胶质细胞体分离，使轴突从未受污染的髓鞘中分离出来，用于细胞体和轴突的分析。将使用由凯尼格博士的实验室设计和开发的专门方法来研究超纯显微样本。研究人员将利用原位杂交技术定位核糖体RNA在M轴突中的位置，并评估其密度和分布。信使RNA将定位于M轴突中的肌动蛋白和/或微管蛋白，这是一种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。将对体外制备的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。将体内M细胞轴突合成的蛋白质与体外合成的蛋白质进行比较，以确定在生理条件下合成的蛋白质，以及半衰期更长的蛋白质。这项研究的结果将解决一个重要的悬而未决的问题，即成熟的轴突如何补偿其外源提供的蛋白质的生物衰退。

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.