Immunocytochemical differentiation of microtubules in the cytoskeleton of Giardia lamblia using monoclonal antibodies to alpha-tubulin and polyclonal antibodies to associated low molecular weight proteins

Abstract

In interphase trophozoites of Giardia lamblia, separate populations of microtubules constitute the four parts of the mastigont apparatus: flagella, ventral disc, funis and median body. Antigenic differences between the tubules have been investigated by light and electron immunocytochemistry after labelling with two monoclonal antibodies to alpha-tubulin (YL 1/2 and YOL 1/34 clones), and with polyclonal antibodies to Giardia tubule-associated proteins. Both anti-tubulins stained all tubules after isolated structures were fixed in formaldehyde, but different patterns of reactivity were shown by unfixed tubules. YL 1/2 antibodies labelled flagellar axonemes and basal bodies, funis and median body tubules. Disc microtubules were mostly unlabelled, but the antibody bound strongly to the outer edge of the disc where the ends of tubules are embedded. YOL 1/34 antibodies stained disc tubules uniformly, and cross-reacted with the median body but not with tubules of axonemes, basal bodies or funis. Antibodies to giardins 14A and 14B (approximately 30 000 Mr filament-forming proteins) localized these proteins in the microribbons attached to disc microtubules. The median body was also labelled by anti-giardins, indicating an ontogenetic relationship between this organelle and the ventral disc. A second set of approximately 30 000 Mr proteins with no immunoreactivity to anti-giardin was found in flagella purified without removing flagellar membranes. These polypeptides were Triton-soluble and therefore probably originated from an extra-axonemal site. A rabbit antiserum to the labile flagellar proteins specifically stained the two ventral flagella, but not the other six flagella on this cell.