The growth cone is responsible for axonal growth, where membrane expansion is most likely to occur. Several recent reports have suggested that presynaptic proteins are involved in this process; however, the molecular mechanism details are unclear. We suggest that by cleaving a presynaptic protein syntaxin, which is essential in targeting synaptic vesicles as a target SNAP receptor (t-SNARE), neurotoxin C1 of Clostridium botulinum causes growth cone collapse and inhibits axonal growth. Video-enhanced microscopic studies showed (a) that neurotoxin C1 selectively blocked the activity of the central domain (the vesicle-rich region) at the initial stage, but not the lamellipodia in the growth cone; and (b) that large vacuole formation occurred probably through the fusion of smaller vesicles from the central domain to the most distal segments of the neurite. The total surface area of the accumulated vacuoles could explain the membrane expansion of normal neurite growth. The gradual disappearance of the surface labeling by FITC-WGA on the normal growth cone, suggesting membrane addition, was inhibited by neurotoxin C1. The experiments using the peptides derived from syntaxin, essential for interaction with VAMP or alpha-SNAP, supported the results using neurotoxin C1. Our results demonstrate that syntaxin is involved in axonal growth and indicate that syntaxin may participate directly in the membrane expansion that occurs in the central domain of the growth cone, probably through association with VAMP and SNAPs, in a SNARE-like way.

Polypeptides in the dorsal root ganglion (L5) of the adult rat were radioactively labeled, and components slowly migrating in the sciatic nerve (peripheral axons) and dorsal root (central axons) were analyzed, using SDS-polyacrylamide slab gel electrophoresis and fluorography. In particular, the transport rates and amounts of six major polypeptides, i.e., the triplet (reference 15; with mol wts of 200,000, 160,000, and 68,000 daltons), alpha- and beta-tubulins and actin were compared between the two axon branches. In peripheral axons, fronts of the triplet, tubulins, and actin migrate at 2-3 mm/d, 9-13 mm/d and approximately 19 mm/d, respectively. The corresponding values in central axons are 1-2 mm/d, 3-4 mm/d, and approximately 4 mm/d, indicating an obvious asymmetry in the transport rate between the two branches of bifurcating axons. A greater amount of labeled triplet, tubulins, and actin each is found to migrate in peripheral than in central axons. Another striking aspect of asymmetry between the two branches relates to the tubulins/triplet ratio which is significantly higher in the peripheral branch. Considerable proportions of radioactivities associated with tubulins and actin in the ganglion are nonmigratory, which are thought to derive mostly from periaxonal satellite cells. In contrast, most if not all of the labeled triplet is migratory, suggesting a virtual absence of triplet polypeptides in satellite cells. The possible significance of peripheral-central inequalities in slow axoplasmic transport is discussed from the viewpoints of axon volume and axonal outgrowth.