Differences in the exposure of C- and N-terminal tubulin domains in cytoplasmic microtubules detected with domain-specific monoclonal antibodies.
A panel of 11 monoclonal antibodies specific to alpha- or beta-tubulin subunits was used to study the location of tubulin molecules in cytoplasmic microtubules. Specificity of antibodies was confirmed by immunoblotting and immunofluorescence experiments on fixed cells. The limited proteolysis of tubulin with trypsin and chymotrypsin followed by immunoblotting demonstrated that the antibodies discriminated between structural domains of both subunits. Epitope mapping of isolated alpha-tubulin revealed that a set of antibodies against the N-terminal domain of the alpha-subunit (TU-01, TU-02, TU-03, TU-09, 6-11B-1) recognized at least four different antigenic determinants. Immunofluorescence staining of unfixed detergent-extracted cells showed that antibodies to determinants on C-terminal domains labelled microtubules, but these were not decorated with antibodies to N-terminal domains. The same results were obtained after microinjection of antibodies into living cells. The unchanged distribution of microtubules in injected cells was confirmed by double-label immunofluorescence with polyclonal antibodies. The data indicate that while parts of C-terminal domains of both subunits are exposed on the exterior of the microtubules, considerable regions of the N-terminal domains are either not exposed on the surface of cytoplasmic microtubules, or are masked by interacting proteins.
Dráber
P
P
Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Prague.
Dráberová
E
E
Linhartová
I
I
Viklický
V
V
D000818:Animals; D000911:Antibodies, Monoclonal; D000939:Epitopes / Q000032:analysis; D005455:Fluorescent Antibody Technique; D008870:Microtubules / Q000032:analysis*; D013552:Swine; D014404:Tubulin / Q000032:analysis*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D014404:Tubulin