Transfer of Newly Synthesized Proteins from Schwann Cells to the Squid Giant Axon

Abstract

The squid giant axon is presented as a model for the study of macromolecular interaction be- teins synthesized in the Schwann cells surrounding the axon are subsequently transferred into the axoplasm. MATERIALS AND METHODSProtein synthesis was studied in squid giant axons obtained from live squid which were kept in a sea tank and used within 48 hr of capture. The giant axons were obtained by decapitating the squid and dissecting the axons under a stream of running sea water. The axons, 4-6 cm long, were tied with thread at both ends, removed from the mantle, and cleaned of adhering connective tissue in a petri dish filled with sea water under observation with a binocular dissecting microscope. Axons were incubated for 0-4 hr at 18-20°in 1 ml of Millipore-filtered (MPF) sea water containing either 10 or 100 1,Ci of L-[4,5-3H]leucine. The incubation was terminated by rinsing the axon in a large volume of MIPF sea water at 40 for 5-10 min. The axoplasm was immediately extruded from the sheath by a method described previously (8) except that a length of polyethylene tubing was substituted for the rubber roller. All of the data presented below were obtained by this method. However, alternate methods of extruding the axoplasm with a roller or by compression with fine forceps produced comparable results. The sheath and axoplasm were homogenized in 50 mM Tris-HCL buffer, pH 7.4. Aliquots of the homogenate were taken for the following analysis: total radioactivity; hot (950 for 30 min) trichloroacetic acid precipitable radioactivity measured on filters; protein measured by the method of Lowry et al. (9); and in some cases sodium dodecyl sulfate, gel electrophoresis (10) or isoelectric focusing (11) on polyacrylamide gels. In other experiments the giant axons were prepared for radioautography at the end of the incubation. Table 1 compares the amount of trichloroacetic acid-precipitable radioactivity found in axoplasm and sheath after 120 min of incubation. The axoplasm contains 5-24% of the labeled proteins. The variation in the relative labeling of the axoplasm is not completely understood; however, it may be a result of variations in the amount of connective tissue which remained attached to the sheath after dissection. Puromycin and acetoxycycloheximide (AXM) inhibited the incorporation into both axoplasm and sheath by 80% or more, whereas chloramphenicol had little effect on incorporation. The inhibition produced by puromycin and AXM did not result from reduced precursor entry into the axon because these drugs had no demonstrable effect upon the solu-1188 RESULTS AND DISCUSSION

Cell-to-cell transfer of glial proteins to the squid giant axon: The glia- neuron protein transfer hypothesis

“…The results of the experiments with chloramphenicol and puromycin reinforce our previous results and those of others indicating that the incorporation of [3H]leucine into proteins of the squid giant fiber occurs via protein synthesis on eukaryotic ribosomes. In contrast to our previous results on unpaired axons (42), the paired control paradigm indicates that cycloheximide is not a completely effective inhibitor of protein synthesis in the squid giant fiber, since 0.1 mg/ml cycloheximide reduced protein synthesis in the sheath by only 50% when compared with paired controls. Cycloheximide does not completely inhibit protein synthesis in Aplysia ganglia (57), suggesting that this protein synthesis inhibitor may not be effective on intact molluscan neurons.…”

“…These studies confirmed and extended previous observations (20) indicating that squid giant fibers separated from their neuron cell bodies are capable of incorporating labeled amino acids into proteins, some of which appear in the axon (42). The appearance of these labeled proteins in the axon raised an interesting question regarding the site at which these proteins were synthesized.…”

“…The appearance of labeled proteins in the axoplasm of the squid giant axon has been demonstrated by autoradiography, and the ratio of labeled protein found in the axoplasm versus the sheath (axoplasm/sheath ratio) was similar in the autoradiographic and extrusion experiments (42). Furthermore, the number of silver grains was reduced to background by puromycin but not by chloramphenicol.…”

“…Our previous studies indicated that the protein synthesis inhibitors puromycin and cycloheximide reduced the incorporation of amino acids into proteins of the squid giant fiber (42). No significant difference was found between axons incubated in chloramphenicol and their contralateral controls (Table I).…”

“…Therefore, the data were corrected for the size of the aliquot and expressed as hot TCA-precipitable cpm or TCA-soluble cpm (total cpmhot TCA-precipitable cpm). In our earlier work (42) we expressed the incorporated radioactivity relative to the proteins in the sample. We have found that expression of the data relative to a unit length of giant fiber compares favorably with the expression relative to protein content.…”