Ribosomes and polyribosomes are present in the squid giant axon: an immunocytochemical study.

Ribosomes and polyribosomes were detected by immuno-electron microscopy in the giant axon and small axons of the squid using a polyclonal antibody against rat brain ribosomes. The ribosomal fraction used as antigen was purified by ultracentrifugation on a sucrose density gradient and shown to contain ribosomal RNAs and native ribosomes. The polyclonal antibody raised in rabbits reacted with at least ten proteins on immunoblots of purified rat brain ribosomes as well as with a set of multiple ribosomal proteins prepared from the squid giant fiber lobe. Immunoreactions were performed on cryostat sections of the stellate nerve cut at a distance of more than 3 cm from the stellate ganglion, using pre-embedding techniques. Ribosomes and polyribosomes were identified within the giant axon and small axons using electron microscopic methods, following binding of peroxidase-conjugated anti-rabbit IgG secondary antibody. Polysomes were more frequently localized in peripheral axoplasm, including the cortical layer of the giant axon, and were generally associated with unidentified cytoskeletal filaments or with dense matrix material. The immunochemical demonstration of ribosomes and polyribosomes in the giant axon and small axons of the squid confirms similar observations in the squid and the goldfish obtained with the method of electron spectroscopic imaging, and strongly supports the view that a local system of protein synthesis is present in axons. The immunochemical method here described offers an alternative tool for the selective identification of ribosomes, and is likely to prove of value in the analyses of other axonal systems.

Local radiolabeling of the 68 kDa neurofilament protein in rat sciatic nerves.

Rat sciatic nerve segments, 4.5 cm distal to the dorsal root ganglion (DRG), were incubated in vivo with [35S]methionine. Fluorography of 2-D polyacrylamide gels of the nerve proteins demonstrated the labeling of the 68-kDa neurofilament protein, which was identified by immunoblotting. This experimental design excludes the dorsal root ganglion as the source of the radiolabeled neurofilament protein and suggests that this neuron-specific protein may be synthesized in axons.

Axoplasmic incorporation of amino acids in a myelinated fiber exceeds that of its soma: a radioautographic study.

The axoplasmic incorporation of amino acids was studied in the giant Mauthner axon to determine its magnitude, to estimate the half-life of the resulting material, and to establish whether or not it undergoes transport. The incorporation of labeled leucine, lysine, and proline was followed by radioautography. The tracers were injected away from the cell body, either in the floor of the IV ventricle or into the spinal cord. Radioautographic reaction on the Mauthner somata was barely discernible. However, at the site of injection, for survivals to 5.6 days, a peak of reaction was observed on the Mauthner axoplasm, with a sharp and symmetric decrement in both central and distal directions; the intensity of the peak decreased with survival (14% at 5.6 days). In intact fibers, the intensity of the reaction was 1.4% compared with that over the Nissl substance of neighboring somata; by contrast, in severed fibers whose axoplasm had been directly exposed to the tracers the reaction rose to 4.1%. At early times, the intensity of the response of the axoplasm as a function of survival of the fish did not have a lag time. In high-resolution radioautograms, most of the grains overlay the ground axoplasm. Cycloheximide depressed the reaction over the fiber open to the extracellular space by 55% whereas only by 20% over the intact fiber. Adsorption of free amino acids or their binding to tRNA were ruled out as a cause of the radioautographic response. Our results indicated that incorporation of amino acids into macromolecules of the Mauthner fiber occurred locally, and its magnitude over the whole axoplasm was estimated to be one order of magnitude larger than that occurring in the soma. The bulk of the material resulting from this incorporation did not undergo transport and survived some days. We suggest that the amino acids were incorporated by a ribosomal mechanism.