Nickel (Ni2+) enhancement of alpha-tubulin acetylation in cultured 3T3 cells

Abstract

Stable microtubules (MTs) are well known to contain acetylated alpha-tubulin. Ni2+-induced MT bundling may be accompanied with such tubulin post-translational modification. To explore this possibility as a mechanism of Ni2+-induced cytoskeletal injury, we have examined acetylated alpha-tubulin levels in cultured 3T3 cells by both immunoprecipitation assays and fluorescent staining of MTs using a monoclonal antibody (clone 6-11B-1) specific for acetylated artubulin. Cell extracts prepared from [35S]methionine labeled cultures in the presence or absence of Ni2+ were immunoprecipitated and analyzed by SDS-PAGE followed by autoradiography. A predominant protein band (molecular mass 55 kDa), representing acetylated alpha-tubulin, appeared in Ni2+-treated cells in a dose and time-dependent manner whereas the corresponding labeled protein band was only barely detectable in control cells. Consistent with the immunoblot findings, MTs in control 3T3 cells in the absence of Ni2+ were not labeled by the 6-11B-1 antibody except for some short, discontinuous segments localized in the cell center or the perinuclear MT organizing center area. In contrast, treatment of cells with NiCl2 (2 mM for 20 hr) resulted in, as expected, the formation of MT bundles that were intensely stained by the 6-11B-1 monoclonal antibody specific for acetylated alpha-tubulin. Furthermore, MTs containing acetylated alpha-tubulin in Ni2+-treated cells were resistant to disassembly induced by nocodazole, and at least partially resistant to cold temperature (0 degrees C), which also depolymerizes MTs. Since acetylated alpha-tubulin serves as a marker for the presence of stable MTs, the marked enhancement of alpha-tubulin acetylation in Ni2+-treated cells indicates that stabilization of MTs may be an important mechanism by which Ni2+ induces cell injury since stabilized MTs in turn should favor MT bundling, an unusual form of cytoskeletal perturbation in response to Ni2+ exposure.