Microtubule reassembly from nucleating fragments during the regrowth of amputated neurites

Abstract

We have proposed that stable microtubule (MT) fragments that resist depolymerization may serve as nucleating elements for the local control of MT dynamics in the axon (Heidemann, S.R., M.A. Hamborg, S.J. Thomas, B. Song, S. Lindley, and D. Chu, 1984, J. Cell Biol., 99:1289-1295). Here we report evidence that supports this proposal in studies on the role of MTs in the regrowth of neurites from the distal segments of amputated chick sensory neurites. Amputated neurites collapse to 'beads' of axoplasm that rapidly regrow (Shaw, G., and D. Bray, 1977, Exp. Cell Res., 104:55-62). We examined both unarrested regrowth and regrowth after MT disassembly by either cold (-5°C for 2 h) or nocodazole (0.1 μg/ml for 15-20 min). In all these cases regrowth occurred at 3.5-4.5 μm/min with no delay times other than the times to reach 37°C or rinse out the nocodazole. Electron micrographs of untreated beads show many MTs of varying lengths, while those of cold- and nocodazole-treated beads show markedly shorter MTs. The robust regrowth of neurites from beads containing only very short MTs argues unfurling of intact MTs from the bead into the growing neurite. Electron micrographs of cold-treated beads lysed under conditions that cause substantial MT depolymerization in untreated intact neurites show persistent MT fragments similar to those in unlysed cold-treated beads. We interpret this as evidence that the MT fragments in cold-treated beads are somehow distinct from the majority of the MT mass that had depolymerized. Collapsed neurites treated with a higher dose of nocodazole (1.0 μg/ml for 15-20 min) were completely devoid of MTs and regrew only after a 15-20 min delay in two cases but never regrew in 11 other cases. We found that MTs did not return in beads treated with 1.0 μg/ml nocodazole even 30 min after removal of the drug. It was unlikely that the inability of these beads to reassemble MTs was due to incomplete removal of nocodazole in that a much higher dose (20 μg/ml nocodazole) could be quickly rinsed from intact neurites. Beads treated with 1.0 μg/ml nocodazole could, however, be stimulated to reassemble MTs and regrow neurites by treatment wih taxol. We conclude that the immmediate, robust regrowth of neurites from collapsed beads of axoplasm requires MT nucleation sites to support MT reassembly. Our data suggest that tubulin within the bean can elongate existing MTs normally but has a limited capacity for self-nucleation relative to brain tubulin in vitro.