Accessory tubules and axonemal microtubules of Apis mellifera sperm flagellum differ in their tubulin isoform content

In the insect sperm flagellum, an extra set of nine additional microtubules , named accessory tubules, is present surrounding the axoneme. Using a sarc osyl/urea extraction, we were able to fractionate the microtubular cytoskel eton of the sperm flagellum of the insect Apis mellifera resulting in the d issociation of the axonemal microtubule protein components and the accessor y tubules. This has allowed us to compare the tubulin isoform content of ax onemal microtubules and accessory tubules by immunoelectron microscopy and immunoblotting using a panel of monoclonal antibodies directed against diff erent tubulin post-translational modifications (PTMs). All the PTMs occurri ng in axonemal tubulin are also present in accessory tubules, which indicat es the close relativeness of accessory tubules to axonemal rather than to c ytoplasmic microtubules. However, our results demonstrate the presence of s ignificant differences in the tubulin isoform content of axonemal microtubu les and accessory tubules. First, the tubulin tyrosination extent of access ory tubules is far lower than that of axonemal microtubules, thus confirmin g at the molecular level their morphogenetic origin as outgrowths from the B-subtubule of each microtubular doublet. Second, although polyglycylation seems to occurr at the same extent in both microtubular systems, alpha-tubu lin exhibits a larger amount of monoglycylated sites in axonemal microtubul es than in accessory tubules. Third, a greater amount of P-tubulin molecule s is glutamylated in axonemal microtubules than in accessory tubules. Moreo ver, highly acidic isoforms, likely molecules with longer polyglutamate sid e chains, are present only in axonemal microtubules. Taken together, our da ta are indicative of a higher level of tubulin heterogeneity in axonemal mi crotubules than in accessory tubules. They also show a segregation of post- translationally modified isoforms between accessory tubules and axonemal mi crotubules and suggest the implication of PTMs in the functional specializa tion of the two microtubular systems. (C) 2000 Wiley-Liss, Inc.