Ž . Developmental Brain Research 100 1997 133–138 Short communication Profile of glutamylated tubulin expression during cerebellar granule cell development in vitro S.A. Przyborski, M.A. Cambray-Deakin ) Department of Biomedical Science, UniÕersity of Sheffield, Western Bank, Sheffield, S10 2TN, UK Accepted 4 February 1997 Abstract The developmental regulation of tubulin and several of its posttranslational modifications was examined during the differentiation of rat cerebellar granule cells in vitro. In particular, we have noted that the glutamylation of a- and b-tubulin subunits varies during development and becomes more prominent in differentiating neuronal processes. These results indicate that glutamylation of tubulin may be important in the stabilization of microtubules during the maturation of the neuronal cytoskeleton. Keywords: Tubulin; Glutamylation; Acetylation; Tyrosination; Cerebellar granule cell; Neuritogenesis; In vitro Neurons possess varied morphologies that are character- ized by the presence of long elaborate cytoplasmic exten- sions, a fundamental feature of intricate neural networks. The formation and maintenance of these axonal and den- dritic processes is dependent upon the plasticity of a Ž w x. complex array of cytoskeletal elements review 32 . Cy- toskeletal diversity may be generated by, for example, the assembly of microtubules with different associated pro- Ž . teins MAPs or by differences in the forms of a- and b-tubulin subunits within the microtubule polymer. No- tably, neuronal microtubules possess much higher tubulin heterogeneity than any other cell type. Indeed, several studies have noted the existence of numerous isoforms that are predominantly or specifically expressed in nerve cells w x 9,11,13,15,17,24,33 . This multiplicity of a- and b-tubu- lins results from the expression of several tubulin genes Ž . and the posttranslational modification PTM of the result- Ž w x. ing tubulin isotypes reviews 20,23,25 . Such PTMs in- wx w x clude the detyrosination 3 and acetylation 26 of a- w x tubulin and the phosphorylation of class III b-tubulin 14 . In addition, previous reports have described a glutamyla- tion of a- and b-tubulin entailing the addition of 1–6 glutamyl units on the side chain of an encoded glutamate w x near the carboxy terminus 4,12,30,31,34 . We have previously noted the distribution of acetylated ) Ž . Ž . Corresponding author. Fax: 44 114 2765413. E-mail: M.Cambray-Deakin@Sheffield.ac.uk and tyrosinated isoforms of a-tubulin during the develop- wx ment of neurons in vitro 8 and have also recorded their wx localization in brain sections 7 . As part of an ongoing study to characterize the plasticity of the developing neu- w x ronal cytoskeleton 27–29 , we report here the expression of glutamylated a- and b-tubulin during the differentiation of cultured cerebellar granule cells. Our results show sig- nificant regulation in the abundance and heterogeneity of glutamylated a- and b-tubulin concurrent with neuronal development. Dissociated cultures of granule cell neurons were pre- pared from the cerebella of 7 day old neonate rats as wx previously described 6 . The cells were maintained in Ž . Minimum Essential Medium Eagle’s modified plus Ž . Earle’s salts supplemented with 10% vrv fetal calf serum, 25 mM KCl, 290 mgrml glutamine, 33 mM glucose, 25 Urml penicillin and 25 mgrml streptomycin. Cultures were incubated at 378C in 5% CO and sampled at various 2 time points over a 21 day period. Protein samples from cultures at different stages of Ž development were separated by SDS-PAGE 12% poly- . w x acrylamide according to the method of Laemmli 22 and subsequently transferred onto nitrocellulose membranes using the Biorad Mini-protean System. For two dimen- sional PAGE, specimens were initially resolved by isoelec- Ž tric focusing Biorad Mini-protean 2-D cell; 1.6% pH 5–7 . and 0.4% pH 3–10 ampholytes followed by separation on 15% SDS gels and transfer onto nitrocellulose membrane. Success of transfer was determined by Ponceau S staining 0165-3806r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. Ž . PII S0165-3806 97 00031-X