TAPping into the treasures of tubulin using novel protein production methods

Abstract

Microtubules are cytoskeletal elements with important cellular functions, whose dynamic behaviour and properties are in part regulated by microtubule-associated proteins (MAPs). The building block of microtubules is tubulin, a heterodimer of α- and β-tubulin subunits. Longitudinal interactions between tubulin dimers facilitate a head-to-tail arrangement of dimers into protofilaments, while lateral interactions allow the formation of a hollow microtubule tube that mostly contains 13 protofilaments. Highly homologous α- and β-tubulin isotypes exist, which are encoded by multi-gene families. In vitro studies on microtubules and MAPs have largely relied on brain-derived tubulin preparations. However, these consist of an unknown mix of tubulin isotypes with undefined post-translational modifications. This has blocked studies on the functions of tubulin isotypes and the effects of tubulin mutations found in human neurological disorders. Fortunately, various methodologies to produce recombinant mammalian tubulins have become available in the last years, allowing researchers to overcome this barrier. In addition, affinity-based purification of tagged tubulins and identification of tubulin-associated proteins (TAPs) by mass spectrometry has revealed the 'tubulome' of mammalian cells. Future experiments with recombinant tubulins should allow a detailed description of how tubulin isotype influences basic microtubule behaviour, and how MAPs and TAPs impinge on tubulin isotypes and microtubule-based processes in different cell types.

Keywords: microtubule; protein-protein interactions; tubulins.

Figure 1. Schematic outline of different classes of MAPs and TAPs

α- and β-tubulin isotypes are generated as monomers (α-tubulin: red balls, β-tubulin: green balls). These subunits fold and then they interact to form functional, assembly-competent tubulin dimers with the help of various types of chaperones, which are a prime example of a class of TAPs. Other TAPs are also present in the cellular cytoplasm and bind tubulins for various reasons. Tubulins assemble into MTs. One growing MT is schematically indicated, with its plus (+) and minus (-) ends. MAPs regulate MT behaviour and are often categorized by their mode of binding. Indicated are ‘classic’ MAPs that bind along the MT lattice, +TIPs and -TIPs, which accumulate at growing plus and minus ends, respectively, and motor proteins, which transport cargo along MTs.

Figure 2. Schematic representation of tubulin expression constructs

Constructs named ‘dual’ express two tubulins from one mRNA by virtue of the P2A system. Constructs named ‘single’ express only one tubulin subunit. The right hand of the figure indicates use of the constructs in the mass spectrometry experiments aimed at identifying TAPs.

Figure 3. Comparison of mass spectrometry experiments

We pulled down biotinylated tubulins from HEK293T cells expressing either ‘single’ or ‘dual’ tubulin constructs. TAPs were co-precipitated and identified using mass spectrometry. The ‘single’ approach yielded 42 potential TAPs, whereas the ‘dual’ approach yielded 328 candidate TAPs (note that these numbers were obtained after filtering of the original mass spectrometry data). Here, we compare co-precipitation of different classes of proteins (V: various proteins of this class are present in the dataset, +/-: only few proteins of this class are present in the dataset, - : no proteins of this class are present in the dataset).