Subcellular sequestration of an antigenically unique beta-tubulin

Abstract

Tubulin from Trypanosoma brucei was characterized by Western blotting using well defined monoclonal antibodies reacting with alpha- or beta-tubulin and a new monoclonal antibody, 1B41, raised against a microtubule-enriched fraction of T. brucei, which specifically reacts with the beta-subunit of tubulin from either T. brucei or rat brain. This antibody has been used to examine the subcellular distribution of the corresponding antigen in T. brucei by indirect immunofluorescence. The epitope recognized by 1B41 is restricted to a thin line extending from the basal body region to the anterior end of the cell body. To determine the relationship between the immunoreactive zone and the flagellum, double-label immunofluorescence was performed in both interphase and mitotic cells with 1B41 and a flagellar marker, the monoclonal antibody 5E9, specific for the paraflagellar rod polypeptides of trypanosomes. These experiments revealed that the immunoreactive tubulin was contained in a part of the subpellicular cytoskeleton that remained in a constant spatial correspondence with the flagellum throughout the cell division cycle. The beta-tubulin recognized by 1B41 may be segregated into the microtubular structures associated with a cisterna of the endoplasmic reticulum forming the subflagellar microtubule quartet (SFMQ). These results suggest that the presence of an antigenically unique beta-tubulin defines a subpopulation of microtubules possessing specific dynamic properties that may be involved in the morphogenesis of daughter cells during the division of T. brucei.