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.
