Definition of individual components within the cytoskeleton of <i>Trypanosoma brucei</i> by a library of monoclonal antibodies

TLDR

The detergent‑insoluble cytoskeleton of *Trypanosoma brucei* contains multiple morphologically distinct regions and organelles, many of which are only visible by electron microscopy. Monoclonal antibodies were generated by hybridoma cloning from mice immunized with cytoskeletal proteins or salt extracts, and their antigens were characterized using immunofluorescence, immunogold electron microscopy, and Western blotting. The antibody panel delineates all major cytoskeletal structures, including tubulin isotypes of the axoneme and sub‑pellicular array, the flagellum attachment zone, the paraflagellar rod, basal bodies, and a detergent‑insoluble nuclear component.

Abstract

ABSTRACT The detergent-insoluble T. brucei cytoskeleton consists of several morphologically distinct regions and organelles, many of which are detectable only by electron microscopy. We have produced a set of monoclonal antibodies that define each structural component of this highly ordered cytoskeleton. The monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof. Four antibodies define particular tubulin isotypes and locate the microtubules of the axoneme and sub-pellicular array; two antibodies recognize the flagellum attachment zone; one recognizes the paraflagellar rod and another the basal bodies. Finally, one antibody defines a detergent-insoluble component of the nucleus. The antigens detected by each monoclonal antibody have been analysed by immunofluorescence microscopy, immunogold electron microscopy and Western blotting.