Colchicine blocks exclusively and completely the intra-axonal transport of tubulin

Colchicine injected into the L4 dorsal root ganglion of the rat 10 min before injecting L[35 S]methionine completely blocked the transport of tubulin in the sciatic nerve and the dorsal root. The labelled protein seen in the tubulin region on SDS gel electrophoresis of the colchicine-treated nerve was a neurofilament-associated protein (NAP) distinct from tubulin. The transport of other major cytoskeletal components, 1'. e. the neurofilament proteins and actin was not affected by colchicine, indicating that these components can be transported independently of tubulin. In contrast to the fast axonal transport which is almost completely arrested by colchicine, the slow transport seems to be only partially inhibited by this agent (for review, see reference 4). In the sensory fibres in the sciatic nerve, tubulin transport is preferentially blocked by injecting colchicine into the dorsal root ganglion (6). The transport of other cytoskeletal components, Le. the neurofilament proteins and actin (5, 8, 9), remains unaffected. The blockage of tubulin transport, however, was not complete and a portion of tubulin appeared to migrate with the neurofilament triplet (200K, 160K and 68K in molecular weight). This has been attributed mainly to the fact that this system consists of a mixture of myelinated and unmyelinated fibres (ll, 12) which may differ in their sensitivities towards the drug (6). Also, a portion of tubulin may be present in a form resistant to colchicine (6). We have recently described the presence of two populations of transported tubulin which differ not only in their rates of transport but also in their solubility and subunit composition (13, 14). The faster portion is tubulin migrating independently of the neurofilament triplet, whereas the slower portion seems to migrate in association with the triplet. The latter portion contains, in addition to tubulin and the triplet, a unique polypeptide ‘NAP’ which is not found in the faster portion. NAP proved to be a neurof1lament-associated protein distinct from tubulin, though its molecular weight and isoelectric point are within the range for tubulin subspecies. In this report, we have reexamined the effect of colchicine on the tubulin transport, taking the presence of NAP into consideration. Male albino rats of the Wistar strain, 7 weeks of age, were used throughout the experiment. L[35S]Methionine (80 ,uCi in 0.4 pl) was injected into the L4 dorsal root ganglion as previously described (6). In the experiments indicated, colchicine (4 pg in 0.2 pl) was injected into the ganglion 10 min before injecting the radioisotope. Nine days or 2 weeks after the injection, the dorsal root, L4 ganglion and the sciatic nerve were dissected out, cut into consecutive 3 mm segments and analysed by one-dimensional (7) or two-dimensional (10) gel electrophoresis coupled with visualisation by fluorography (2). A considerable amount of radiolabelled tubulin migrated ahead of the neurofilament triplet in the control nerve (Fig. la; cf 14). Colchicine completely blocked the appearance of this faster portion of tubulin (Fig. lb). Though weakened in intensity, some radioactivity in the tubulin region was seen to migrate with the neurofilament proteins even in the colchicine382 T. TASHIRO, Y. KOMIYA and M. KUROKAWA