Microtubule dynamics during early neural differentiation of P19 embryonal carcinoma cells

Altered microtubule (MT) stability prior to neurite extension was examined in P19 embryonal carcinoma cells induced to differentiate along the neural pathway by retinoic acid. In undifferentiated P19 cells few acetylated MTs were detected by immunofluorescence staining and these were resistant to disassembly by colchicine at 1 $\mu$g/mL for 45 minutes. Twenty-four hours after neural induction there was an increase in the number of acetylated MTs and in the number of MTs that were resistant to colchicine treatment. This suggested that the MT array in undifferentiated cells is initially dynamic and becomes less dynamic during early neural differentiation prior to morphogenesis. Quantitative immunoblotting showed no change in the levels of total tubulin, but a two-fold increase in the relative level of acetylated tubulin was detected. MT turnover in P19 cells was then examined directly by microinjecting biotin-conjugated bovine brain tubulin. Computer-aided silicon intensified target camera (SIT) imaging of biotin-tubulin incorporation into the MT array showed that by 30 minutes, 92.4 $\pm$ 3.02% of total MTs turned over in undifferentiated cells. In contrast, in the retinoic acid-induced cells 52.8 $\pm$ 19.37% of the MT array turned over in 30 minutes. In conclusion, there are changes in colchicine stability and in tubulin acetylation that are correlated with changes in MT dynamics occurring prior to neurite outgrowth.