Active polysomes in the axoplasm of the squid giant axon

Abstract

Axons and axon terminals are widely believed to lack the capacity to synthesize proteins, relying instead on the delivery of proteins made in the perikaryon. In agreement with this view, axoplasmic proteins synthesized by the isolated giant axon of the squid are believed to derive entirely from periaxonal glial cells. However, squid axoplasm is known to contain the requisite components of an extra-mitochondrial protein synthetic system, including protein factors, tRNAs, rRNAs, and a heterogeneous family of mRNAs. Hence, the giant axon could, in principle, maintain an endogenous protein synthetic capacity. Here, we report that the squid giant axon also contains active polysomes and mRNA, which hybridizes to a riboprobe encoding murine neurofilament protein. Taken together, these findings provide direct evidence that proteins (including the putative neuron-specific neurofilament protein) are also synthesized de novo in the axonal compartment.

The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons

“…In this regard, we lysed distal axons growing in the side compartments of Campenot chambers and purified axonal polysomes by sedimentation through 2 M sucrose. This technique has been used previously to isolate translationally active axonal polysomes (Giuditta et al 1991;Crispino et al 1997). The finding derived from qRT-PCR analysis clearly established that TH mRNA is present in axonal polysomes.…”

“…To ensure that the amplicons generated by qRT-PCR were indeed derived from polysomal RNA, we purified polysomes with an isolation buffer containing EDTA, a Mg 2+ chelating agent known to dissociate polysomes. Under these isolation conditions, the dissociated polysomes are unable to sediment through heavy sucrose (Giuditta et al 1991;Crispino et al 1993). The qRT-PCR tdTomato mRNA levels in the cell bodies of SCG neurons determined by qRT-PCR 72 h after DNA transfection.…”

“…Addition of ethylenediaminetetraacetic acid (EDTA), a chelating agent known to dissociate polysomes, markedly diminished the PCR-generated signals. Under these experimental conditions, the dissociated polysomes are incapable of sedimenting through 2.0 M sucrose and hence were eliminated from the polysome fraction (Giuditta et al 1991;Crispino et al 1993Crispino et al , 1997. Less than 2% of total TH RNA co-sedimented with the polysome fraction in the presence of EDTA.…”

“…In some experiments, polysomes were disaggregated prior to sedimentation by replacing the Mg 2+ in the isolation buffers with the chelating agent, EDTA (20 mM). Under these conditions, polysomes are known to disassemble into the component ribosomal subunits, releasing their mRNA and nascent polypeptides (for example, see Giuditta et al 1991). EDTA-treated polysomes are unable to sediment through the dense sucrose cushion.…”

Medium weight neurofilament mRNA in goldfish Mauthner axoplasm

“…However, when axons are severed from their cell bodies in invertebrates [2] or lower vertebrates such as the goldfish [18], the distal axonal segment often survives for months to years. Proteins in an axon isolated from its cell body could be maintained only through some combination of (1) Although often regarded as a particularly unlikely mechanism to maintain axonai proteins [2,16], axoplasmic protein synthesis has been reported for squid giant axons [7] and goldfish Mauthner axons (M-axons) [14]. The M-axon is a particularly advantageous preparation because more basic components of the translational machinery (transfer RNA (tRNA), ribosomal RNA (rRNA), and ribosomes) have been identified in the M-axon [13][14][15] compared to any other vertebrate axon.…”