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.

Institute of Molecular Genetics Czechoslovak Academy of Sciences Prague

Citace poskytuje Crossref.org

Target antigens for Hs-14 monoclonal antibody and their various expression in normozoospermic and asthenozoospermic men

In vivo kinetics of U4/U6·U5 tri-snRNP formation in Cajal bodies

Exposure of beta-tubulin regions defined by antibodies on an Arabidopsis thaliana microtubule protofilament model and in the cells

Distinct localization of a beta-tubulin epitope in the Tetrahymena thermophila and Paramecium caudatum cortex

Plant gamma-tubulin interacts with alphabeta-tubulin dimers and forms membrane-associated complexes

Association of brain gamma-tubulins with alpha beta-tubulin dimers

Nuclear gamma-tubulin during acentriolar plant mitosis

Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L. cells

Expression of phosphorylated high molecular weight neurofilament protein (NF-H) and vimentin in human developing dorsal root ganglia and spinal cord

Immunological discrimination of beta-tubulin isoforms in developing mouse brain. Post-translational modification of non-class-III beta-tubulins

Immunostaining of human spermatozoa with tubulin domain-specific monoclonal antibodies. Recognition of a unique beta-tubulin epitope in the sperm head