Expression of tubulin polyglycylation in Giardia lamblia.
By means of immunofluorescence, immunoelectron microscopy and immunoblotting, we show that polyglycylation, a posttranslational modification of tubulin widely spread among eukaryotes, is present in the diplomonad, Giardia lamblia, a putative ancestral cell possessing a highly developed microtubular cytoskeleton. This modification was recently discovered in the ciliated protist, Paramecium, and was not found in the Euglenozoa, a lineage considered as ancient. We used two monoclonal antibodies (mAbs), TAP 952 and AXO 49, specifically recognizing mono- and polyglycylated tubulin isoforms, to detect this modification in Giardia extracts and to localize it in the different classes of microtubules within the cell. The alpha- and beta-tubulin subunits were recognized by the two mAbs, indicating that both tubulin subunits are glycylated, in agreement with lately reported mass spectrometry results. Noticeably, Giardia tubulin was much more reactive with AXO 49 than with TAP 952. In situ, AXO 49 intensely labeled the microtubules present in the four pairs of flagella and the median body, and lightly decorated the microtubules from the adhesive disc. In contrast, TAP 952 intensely labeled only the microtubules of the median body. The results indicate a differential expression of glycylated isoforms within various microtubular structures of Giardia lamblia. They also suggest that the complete set of enzymes required for polyglycylation is expressed in very divergent eukaryotes.
Campanati
L
L
Universidade do Estado do Rio de Janeiro, Departamento de Histologia e Embriologia, Brazil.
Bré
M H
MH
Levilliers
N
N
de Souza
W
W
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002478:Cells, Cultured; D016829:Giardia lamblia / Q000378:metabolism* / Q000648:ultrastructure; D006031:Glycosylation; D015151:Immunoblotting; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D010247:Paramecium / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D014404:Tubulin