Dynamic post-translational modification of tubulin in rat cerebral cortical neurons extending neurites in culture: effects of taxol.
Dissociated embryonic (E18-E20) rat cortical neurons were grown in culture and double-labelled by immunofluorescence with antibodies directed against tyrosinated (YL 1/2), detyrosinated (SUP GLU), and acetylated (6-11B-1) alpha-tubulin. Within 90 min of plating, neurons extended growth cones that were YL 1/2+ but SUP GLU- and 6-11B-1-. The neurite that forms behind the advancing growth cone is also, initially, YL 1/2+ and SUP GLU-/6-11B-1-. However, when it has attained a length of about half the cell body diameter, it becomes SUP GLU+ and 6-11B-1+. The effects of the microtubule polymerizing agent taxol (15 microM) on growth cone and neurite alpha-tubulins was investigated. Taxol, as reported previously, caused the formation of microtubule loops in the central domain of the growth cone, a loss of filopodia, and the collapse of the growth cone onto the loops. The taxol effects peaked at 60 min, when over 85% of neurites showed microtubule loops, and declined thereafter, so that at 420 min in taxol, only about 23% of neurites had microtubule loops. Over this period there was an inverse correlation between the presence of microtubule loops and growth cones. Taxol had striking effects on the intensity of SUP GLU and 6-11B-1 staining in neurons. In 48 h cultures, a 30 min exposure to taxol enhanced the SUP GLU and 6-11B-1 staining of dendrites and axons and produced a loss of YL 1/2 staining in axons. Immunoblotting experiments confirmed that there was an overall reduction in YL 1/2 immunoreactivity and an increase in SUP GLU immunoreactivity. These observations support previous suggestions that the neurite microtubules are assembled in the growth cone and post-translationally modified in the neurite and, in addition, imply that growth cones can overcome the effects of taxol in the continued presence of the compound.
Mansfield
S G
SG
Division of Biomedical Sciences, King's College London, Strand, UK.
Gordon-Weeks
P R
PR
D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D004622:Embryo, Mammalian; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D007700:Kinetics; D008870:Microtubules / Q000648:ultrastructure; D016501:Neurites / Q000648:ultrastructure*; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D017239:Paclitaxel; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D051381:Rats; D014404:Tubulin / Q000378:metabolism*
D000470:Alkaloids; D014404:Tubulin; D017239:Paclitaxel