Maintenance and synthesis of proteins for an anucleate axon

Abstract

The anucleate (distal) segment of a crayfish medial giant axon (MGA) remains intact for months in vivo after severing the axon from its cell body, a phenomenon referred to as long-term survival (LTS). We collected axoplasm from chronic anucleate MGAs by perfusing 2-cm lengths of axons with an intracellular saline. This axoperfusate was analyzed by SDS-PAGE and silver stained. Axoperfusate proteins from intact MGAs and from chronic anucleate MGAs exhibiting LTS for up to 6 months were the same. Furthermore, immunoreactive levels of actin and β-tubulin were similar in axoperfusates from intact and chronic anucleate MGAs. This maintenance of proteins in chronic anucleate MGAs must be due to a lack of protein degradation and/or to local protein synthesis by a source other than the cell body. To investigate local protein synthesis in vitro, we added [ 35S]-methionine to the extracellular saline surrounding intact and chronic anucleate MGAs. After 4- to 6-h incubations, radiolabelled proteins were detected in axoperfusates analyzed by SDS-PAGE and fluorography. The similarity between radiolabelled proteins in axoperfusates and MGA glial sheaths indicated a glial origin for the radiolabelled axoperfusate proteins. Various observations and control experiments suggested that glial-axonal protein transfer occurred by a physiological process. Glial-axonal protein transfer may contribute to the maintenance of proteins during LTS of chronic anucleate MGAs.