The Giant Axon of the Squid: A Simple System for Axonal Transport Studies.

The squid giant axon has a long history of being a superb experimental system in which to investigate a wide range of questions concerning intracellular transport. In this protocol we describe the method used for dissecting the axon to preserve its viability in vitro, and the technique for injecting exogenous materials into the living axon. Now that the squid genome is emerging, and the CRISPR/cas9 system has been successfully applied to knock-out squid genes, the giant axon will resume its place in the scientific pantheon of powerful experimental systems in which to address biological questions pertaining to all eukaryotes.

A peptide zipcode sufficient for anterograde transport within amyloid precursor protein.

Fast anterograde transport of membrane-bound organelles delivers molecules synthesized in the neuronal cell body outward to distant synapses. Identification of the molecular "zipcodes" on organelles that mediate attachment and activation of microtubule-based motors for this directed transport is a major area of inquiry. Here we identify a short peptide sequence (15 aa) from the cytoplasmic C terminus of amyloid precursor protein (APP-C) sufficient to mediate the anterograde transport of peptide-conjugated beads in the squid giant axon. APP-C beads travel at fast axonal transport rates (0.53 mum/s average velocity, 0.9 mum/s maximal velocity) whereas beads coupled to other peptides coinjected into the same axon remain stationary at the injection site. This transport appears physiologic, because it mimics behavior of endogenous squid organelles and of beads conjugated to C99, a polypeptide containing the full-length cytoplasmic domain of amyloid precursor protein (APP). Beads conjugated to APP lacking the APP-C domain are not transported. Coinjection of APP-C peptide reduces C99 bead motility by 75% and abolishes APP-C bead motility, suggesting that the soluble peptide competes with protein-conjugated beads for axoplasmic motor(s). The APP-C domain is conserved (13/15 aa) from squid to human, and peptides from either squid or human APP behave similarly. Thus, we have identified a conserved peptide zipcode sufficient to direct anterograde transport of exogenous cargo and suggest that one of APP's roles may be to recruit and activate axonal machinery for endogenous cargo transport.