The cytoskeleton of eukaryotic cells comprises an interconnected network of microtubules, microfilaments, and intermediate filaments. Microtubules are dynamic structures that play a major role in a wide range of processes, including cell morphogenesis, cell division, intracellular transport and signaling. Microtubules are formed by heterodimers of áâ-tubulin. The á-tubulin gene is highly conserved in eukaryotes. Its C-terminal amino acid is tyrosine which can be removed by tubulin carboxypeptidase and reincorporated by tubulin tyrosine ligase. This post-translational reaction is called the tyrosination/detyrosination cycle, and its physiological role is still unknown. Site-directed mutagenesis was performed on commercial vector pEGFPtub (Clontech), changing the C-terminal tyrosine codifying codon by another one that codifies for arginine, glycine or aspartic acid. These mutants are not substrate for tubulin carboxypeptidase. Obtained vectors were transfected into NIH 3T3 cells to analyze, by immunofluorescence, the expression of each mutant tubulin, its capacity to assemble into microtubules, and the localization of other cytoskeleton proteins.
