Selective stabilization of microtubules within the proximal region of developing axonal neurites

Abstract

This study examined the distribution of labile and stable microtubules (MTs) during axonal neurite elaboration in NB2a/d1 cells using immunocytochemical markers of unmodified (tyrosinated; Tyr), modified (detyrosinated [Glu] and acetylated [Acet]) and total tubulin. Prominent total and Tyr tubulin immunoreactivity was relatively evenly distributed throughout axonal neurites. By contrast, Acet or Glu immunoreactivity was relatively concentrated within the proximal region of the neurite. Ultrastructural analyses demonstrated an array of longitudinal MTs that apparently span the entire neurite length. The observed differential localization of modified tubulin subunits in axonal neurites of these cells may therefore derive from selective stabilization of proximal regions of full-length axonal MTs. This was substantiated by the observation of Acet immunoreactivity on 30–50% of MTs within the most proximal axonal region, along with a proximal-distal decline to ≤5% of Acet-immunoreactive MTs, in immunoelectron microscopy (immuno-EM) analyses. Microinjected biotinylated subunits were initially detected in assembled form within soma and proximal neurites, indicative of ongoing tubulin subunit incorporation into MTs within, and/or MT translocation into, proximal neurites. Because acetylation and detyrosination are functions of MT age, their concentration in this region despite deposition and/or transport of biotinylated tubulin suggests that a subset of axonal MTs undergoes subunit turnover and/or translocation at rates vastly slower than that of the majority of axonal MTs. Selective stabilization of the proximal region of a subset of axonal MTs may serve to construct a relatively stationary scaffold against which other axonal elements could translocate to more distal axonal regions for continued axonal outgrowth.