Microtubule posttranslational modifications provide unique recognition patterns for associated proteins

Abstract

Microtubules are key components of the eukaryotic cytoskeleton involved in vital functions in virtually every cell. Among the emerging molecular mechanisms to adapt microtubules to their diverse functions is the biochemical diversification of tubulin molecules by posttranslational modifications (PTMs) and differential gene expression, a concept known as the ‘tubulin code’. A key question remains whether the tubulin code has the potential to selectively control microtubule interactions of different microtubule-associated proteins (MAPs) to act as a specific signalling system. To answer this question, we used a medium-throughput in vitro approach to screen 46 proteins for their binding preferences to microtubules with altered PTM or isotype composition. We demonstrate that subsets of these MAPs have unique sensitivities to PTMs, while other proteins are not affected. As a result, PTMs, or combinations of them, differentially attract or repulse individual MAPs to microtubules. Our findings offer mechanistic proof for a key hypothesis of the tubulin code—the capacity to selectively and differentially regulate MAP–microtubule interactions. Biochemical diversity of tubulin, generated by its posttranslational modifications or tubulin isotypes, endows microtubules with distinct properties and functions, a concept known as the ‘tubulin code’. This study reveals differential regulation of a broad variety of microtubule-associated proteins (MAPs) by the tubulin code. A medium-throughput screen using lysates of mammalian cells expressing fluorescently tagged MAPs allows quantification of the impact of tubulin posttranslational modifications and isotypes on MAP-microtubule interactions. Some MAPs show a specific recognition pattern for one or several tubulin modifications, while others are insensitive. Differential splicing of the mitochondria adaptor protein syntaphilin changes its sensitivity to tubulin polyglutamylation. Members of the conserved echinoderm microtubule-associated protein-like (EML) family differ in their sensitivity to tubulin tyrosination and polyglutamylation, illustrating evolutionary diversification of tubulin-code readers. A medium-throughput screen using lysates of mammalian cells expressing fluorescently tagged MAPs allows quantification of the impact of tubulin posttranslational modifications and isotypes on MAP-microtubule interactions. Some MAPs show a specific recognition pattern for one or several tubulin modifications, while others are insensitive. Differential splicing of the mitochondria adaptor protein syntaphilin changes its sensitivity to tubulin polyglutamylation. Members of the conserved echinoderm microtubule-associated protein-like (EML) family differ in their sensitivity to tubulin tyrosination and polyglutamylation, illustrating evolutionary diversification of tubulin-code readers. A screen encompassing 46 microtubule-associated proteins reveals their distinct affinities to tubulin posttranslational modifications.