Delivery of newly synthesized tubulin to rapidly growing distal axons of sympathetic neurons in compartmented cultures.

Abstract

Growing axons receive a substantial supply of tubulin and other proteins delivered from sites of synthesis in the cell body by slow axonal transport. To investigate the mechanism of tubulin transport most previous studies have used in vitro models in which the transport of microtubules can be visualized during brief periods of growth. To investigate total tubulin transport in neurons displaying substantial growth over longer periods, we used rat sympathetic neurons in compartmented cultures. Tubulin synthesized during pulses of [35S]methionine was separated from other proteins by immunoprecipitation with monoclonal antibodies to alpha and beta tubulin, further separated on SDS-PAGE, and quantified by phosphorimaging. Results showed that 90% of newly synthesized tubulin moved into the distal axons within 2 d. Furthermore, the leading edge of tubulin was transported at a velocity faster than 4 mm/d, more than four times the rate of axon elongation. This velocity did not diminish with distance from the cell body, suggesting that the transport system is capable of distributing newly synthesized tubulin to growth cones throughout the axonal tree. Neither diffusion nor the an mass transport of axonal microtubules can account for the velocity and magnitude of tubulin transport that was observed. Thus, it is likely that most of the newly synthesized tubulin was supplied to the growing axonal tree in subunit form such as a heterodimer or an oligomer considerably smaller than a microtubule.