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

This study examined the distribution of labile and stable microtubules (MTs ) during axonal neurite elaboration in NB2a/d1 cells using immunocytochemic al markers of unmodified (tyrosinated; Tyr), modified (detyrosinated [Glu] and acetylated [Acet]) and total tubulin, Prominent total and Tyr tubulin i mmunoreactivity was relatively evenly distributed throughout axonal neurite s. By contrast, Acet or Glu immunoreactivity was relatively concentrated wi thin the proximal region of the neurite. Ultrastructural analyses demonstra ted an array of longitudinal MTs that apparently span the entire neurite le ngth. The observed differential localization of modified tubulin subunits i n axonal neurites of these cells may therefore derive from selective stabil ization of proximal regions of full-length axonal MTs. This was substantiat ed by the observation of Acet immunoreactivity on 30-50% of MTs within the most proximal axonal region, along with a proximal-distal decline to less t han or equal to 5% of Acet-immunoreactive MTs, in immunoelectron microscopy (immuno-EM) analyses. Microinjected biotinylated subunits were initially d etected in assembled form within soma and proximal neurites, indicative of ongoing tubulin subunit incorporation into MTs within, and/or MT translocat ion into, proximal neurites. Because acetylation and detyrosination are fun ctions 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 proxima l region of a subset of axonal MTs may serve to construct a relatively stat ionary scaffold against which other axonal elements could translocate to mo re distal axonal regions for continued axonal outgrowth. (C) 1999 Elsevier Science Inc.