Polyglutamylation and polyglycylation of alpha- and beta-tubulins during in vitro ciliated cell differentiation of human respiratory epithelial cells.

摘要

Tubulins are the major proteins within centriolar and axonemal structures. In all cell types studied so far, numerous alpha- and beta-tubulin isoforms are generated both by expression of a multigenic family and various post-translational modifications. We have developed a primary culture of human nasal epithelial cells where the ciliated cell differentiation process has been observed and quantified. We have used this system to study several properties concerning polyglutamylation and polyglycylation of tubulin. GT335, a monoclonal antibody directed against glutamylated tubulins, stained the centriole/basal bodies and the axonemes of ciliated cells, and the centrioles of non-ciliated cells. By contrast, axonemal but not centriolar tubulins were polyglycylated. Several polyglutamylated and polyglycylated tubulin isotypes were detected by two-dimensional electrophoresis, using GT335 and a specific monoclonal antibody (TAP952) directed against short polyglycyl chains. Immunoelectron microscopy experiments revealed that polyglycylation only affected axonemal tubulin. Using the same technical approach, polyglutamylation was shown to be an early event in the centriole assembly process, as gold particles were detected in fibrogranular material corresponding to the first cytoplasmic structures involved in centriologenesis. In a functional assay, GT335 and TAP952 had a dose-dependent inhibitory effect on ciliary beat frequency. TAP952 had only a weak effect while GT335 treatment led to a total arrest of beating. These results strongly suggest that in human ciliated epithelial cells, tubulin polyglycylation has only a structural role in cilia axonemes, while polyglutamylation may have a function both in centriole assembly and in cilia activity.