Tubulin orientation in microtubules probed with domain-specific antibodies

A panel of monoclonal antibodies specific to alpha- or beta-tubulin subunits was used to study the location of tubulin molecules in microtubules. Limited proteolysis of tubulin with trypsin and chymotrypsin followed by immunoblotting demonstrated that the antibodies discriminated between structural domains of tubulin subunits. Antibodies against N-terminal domains were tested for their ability to interfere with the formation of microtubules in vitro. Although the antibodies exhibited similar association constants when tested on immobilized tubulin, they differed in their inhibitory effect on microtubule assembly. The sedimentation assay using microtubules prepared from purified tubulin showed an almost undetectable binding of the antibodies with the strongest inhibitory power to the microtubules. Immunofluorescence staining of unfixed detergent-extracted cells revealed that antibodies to determinants on C-terminal domains labelled microtubules, but these were not decorated with antibodies against N-terminal domains. The same results were obtained after a microinjection of antibodies into living cells. The data indicate that while parts of C-terminal domains of both subunits are exposed on the exterior of microtubules, considerable regions of the N-terminal domains are not. The surface regions of N-terminal domains appear to be involved in the formation of microtubules.