Individual microtubules in the axon consist of domains that differ in both composition and stability

Abstract

We have explored the composition and stability properties of individual microtubules (MTs) in the axons of cultured sympathetic neurons. Using morphometric means to quantify the MT mass remaining n axons after various times in 2 μg/ml nocodazole, we observed that ∼48% of the MT mass in the axon is labile, depolymerizing with a t1\2 of ∼5 min, whereas the remaining 52% of the MT mass is stable, depolymerizing with a t1\2. of ∼240 min. Immunofluorescence analyses show that the labile MTs in the axon are rich in tyrosinated α-tubulin, whereas the stable MTs contain little or no tyrosinated α-tubulin and are instead rich in posttranslationally detyrosinated and acetylated a-tubulin. These results were confirmed quantitatively by immunoelectron microscopic analyses of the distribution of tyrosinated a-tubulin among axonal MTs. Individual MT profiles were typically either uniformly labeled for tyrosinated a-tubulin all along their length, or were completely unlabeled. Roughly 48% of the MT mass was tyrosinated, ∼52% was detyrosinated, and ∼85% of the tyrosinated MTs were depleted within 15 min of nocodazole treatment. Thus, the proportion of MT profiles that were either tyrosinated or detyrosinated corresponded precisely with the proportion of MTs that were either labile or stable respectively. We also observed MT profiles that were densely labeled for tyrosinated α-tubulin at one end but completely unlabeled at the other end. In all of these latter cases, the tyrosinated, and therefore labile domain, was situated at the plus end of the MT, whereas the detyrosinated, and therefore stable domain was situated at the minus end of the MT, and in each case there was an abrupt transition between the two domains. Based on the frequency with which these latter MT profiles were observed, we estimate that minimally 40% of the MTs in the axon are composite, consisting of a stable detyrosinated domain in direct continuity with a labile tyrosinated domain. The extreme drug sensitivity of the labile domains suggests that they are very dynamic, turning over rapidly within the axon. The direct continuity between the labile and stable domains indicates that labile MTs assemble directly from stable MTs. We propose that stable MTs act as MT nucleating structures that spatially regulate MT dynamics in the axon.