Microtubule Assembly of Isotypically Purified Tubulin and Its Mixtures

Abstract

Numerous isotypes of the structural protein tubulin have now been characterized in various organisms and their expression offers a plausible explanation for observed differences affecting microtubule function in vivo. While this is an attractive hypothesis, there are only a handful of studies demonstrating a direct influence of tubulin isotype composition on the dynamic properties of microtubules. Here, we present the results of experimental assays on the assembly of microtubules from bovine brain tubulin using purified isotypes at various controlled relative concentrations. A novel data analysis is developed using recursive maps which are shown to be related to the master equation formalism. We have found striking similarities between the three isotypes of bovine tubulin studied in regard to their dynamic instability properties, except for subtle differences in their catastrophe frequencies. When mixtures of tubulin isotypes are analyzed, their nonlinear concentration dependence is modeled and interpreted in terms of lower affinities of tubulin dimers belonging to the same isotype than those that represent different isotypes indicating hitherto unsuspected influences of tubulin dimers on each other within a microtubule. Finally, we investigate the fluctuations in microtubule assembly and disassembly rates and conclude that the inherent rate variability may signify differences in the guanosine-5'-triphosphate composition of the growing and shortening microtubule tips. It is the main objective of this article to develop a quantitative model of tubulin polymerization for individual isotypes and their mixtures. The possible biological significance of the observed differences is addressed.

Nucleocytoplasmic shuttling of soluble tubulin in mammalian cells

“…Thus, mammals appear to have six forms of α and seven forms of β tubulin that differ in their C-terminal or N-terminal regions, and further categorized in distinct sub-classes (Sullivan, 1988). The relative composition of α-and β-tubulin isoforms affects the structure and function of microtubules (Lu and Luduena, 1994;Rezania et al, 2008). Tubulin mutations and various posttranslational modifications, such as acetylation, palmitoylation, phosphorylation, polyglutamylation, polyglycylation and detyrosination also affect microtubule structure under a variety of normal or pathological conditions (Burkhart et al, 2001;Luduena, 1998;McKean et al, 2001;Sullivan, 1988).…”

A first principle (3+1)-dimensional model for microtubule polymerization

“…Experimental panels in Figs. 1 and 2 represent the experimental data published by Rezania et al [37]. Simulation panels show the numerical results of the normalized vortex length as a function of time for the given set of parameters.…”

Targeting Toxoplasma Tubules: Tubulin, Microtubules, and Associated Proteins in a Human Pathogen

“…Isotypes have small variations in their amino acid sequences and consequently exhibit subtly different biochemical properties. In multicellular organisms, specific cell types can modify properties such as the microtubule assembly rate by changing the relative expression levels of individual isotypes that contribute to the pool of available subunits (4,5,18,19). Moreover, specific isotypes can be required for specialized structures, such as the flagellar axoneme (20).…”

“…These polymers are constructed by assembly of ␣-␤-tubulin heterodimers and represent an important target for small molecules that are used to treat cancers and helminth infections (1)(2)(3). Specialized properties of distinct microtubule populations can be mediated by small differences in the sequences of specific ␣-and ␤-tubulin isotypes, posttranslational modifications to both ␣-and ␤-tubulin subunits, and interactions with specific microtubuleassociated proteins (MAPs) (4)(5)(6)(7)(8). These factors permit microtubules to coordinate diverse and essential roles in mitosis and meiosis, cytoplasmic architecture, and motility.…”