Probing of Microtubule Structures with Monoclonal Antibodies and Immunofluorescence Microscopy

Microtubules are filamentous polymers that combine stability of structure with an ability to organize rapidly in response to internal and external signals. Microtubules are assembled from αβ-tubulin heterodimers and a collection of microtubule-associated proteins. γ-Tubulin is a relatively new member of the tubulin family that is restricted to the microtubule organizing centers. It was hypothesized that γ-tubulin plays a crucial role in nucleation and anchorage of microtubules1. In vertebrates α- and β-tubulin subunits exhibit high heterogeneity, and more than 20 tubulin charge variants (isotubulins) can be distinguished by horizontal high-resolution isoelectric focusing2. Some of the isoforms result from the expression of multiple tubulin genes. Gene products differ primarily in the C-terminal variable domain consisting of 15 amino acids. On the basis of these variable domains and cell type distribution several evolutionary conserved isotypic classes were identified3. The tubulin heterogeneity is further increased by post-translational modifications. The following variations have been described for brain tubulin: reversible removal of C-terminal tyrosine, acetylation, polyglutamylation, phosphorylation and removal of penultimate glutamyl at the C-terminal end leading to Δ2-tubulin. Except for acetylation, all of these modifications occur in the C-terminal, hypervariable regions of both subunits4. Although the structure of microtubules is known from electron microscopy combined with image reconstruction, X-ray scattering and video microscopy, information on tubulin structure is indirect and derived mostly from biochemical experiments.