Microtubule diversity in ciliated cells: evidence for its generation by post-translational modification in the axonemes of Paramecium and quail oviduct cells.
The diversity of microtubular networks was analyzed in quail oviduct and in Paramecium cells using conventional and confocal immunofluorescence as well as pre- and post-embedding EM immunocytochemistry with a variety of anti-tubulin antibodies. The 6-11B-1 monoclonal antibody, specific for the post-translational acetylation of Lys 40 of alpha-tubulin, and a polyclonal antibody raised against Paramecium axonemal tubulin (anti-PA tubulin antibody) both decorated stable microtubular arrays in Paramecium ie ciliary axonemes and a set of microtubular bundles associated with the cortex, suggesting that the two antibodies may be directed against the same epitope. However, several differences in the immunocytological patterns yielded by each antibody on the two cell types were evident. For example, in quail, as in all other Metazoa, the anti-PA tubulin antibody only decorated axonemes enclosed in normal ciliary membrane while it was unreactive on cytoplasmic tubulins. Immunoblotting of peptide maps of axonemal tubulins demonstrated that the epitopes of the two antibodies were indeed completely different. Double immunolabelling of dividing paramecia using a universal anti-tubulin antibody and the anti-PA tubulin one revealed that all newly assembled microtubular arrays were first detected by the universal antibody and, only shortly afterwards, by the anti-PA tubulin one. This provided a strong indication that the anti-PA tubulin antibody is directed against a post-translational modification taking place on already assembled microtubules (MTs) (as previously known to be the case for acetylation and detyrosination). In taxol-treated quail cells undergoing ciliogenesis, massive assembly of MTs and even axonemes occurred in the cytoplasm. These MTs were not decorated by the anti-PA tubulin antibody however, suggesting that in Metazoa the post-translational modification can only take place within the ciliary lumen. The present work provides one further mechanism for generating MT immunological and biochemical diversity post-translationally; this may account for the high multiplicity of tubulin isoforms observed in ciliates which contain very little if any genetic diversity of tubulin genes.
Adoutte
A
A
Laboratoire de Biologie Cellulaire, Université Paris-Sud, Orsay, France.
Delgado
P
P
Fleury
A
A
Levilliers
N
N
Lainé
M C
MC
Marty
M C
MC
Boisvieux-Ulrich
E
E
Sandoz
D
D
D000818:Animals; D000906:Antibodies; D002923:Cilia / Q000648:ultrastructure*; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D007150:Immunohistochemistry; D008870:Microtubules / Q000648:ultrastructure*; D010057:Oviducts / Q000166:cytology / Q000648:ultrastructure*; D010247:Paramecium / Q000648:ultrastructure*; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D011784:Quail; D014404:Tubulin / Q000032:analysis* / Q000276:immunology
D000906:Antibodies; D000939:Epitopes; D014404:Tubulin