Unraveling Microtubule Complexity through the Control of Post-Translational Modifications and Ionic Environment

Microtubules are fundamental components of the cytoskeleton of the cell, formed by the assembly of αβ-tubulin heterodimers. The microtubule network is crucial for maintaining cell structure, enabling intracellular transport, and facilitating cell division. To face such diverse biological processes, cell must continuously reorganize their network by modulating dynamic features of microtubules. The "Tubulin Code" is a term that regroups the complex interplay of cellular factors influencing microtubule properties. Among them, post-translational modifications (PTMs) ensure the specialization of the network by regulating interactions with microtubule-associated proteins (MAPs). Due to the absence of adequate tools for exploring the regulation and functionality of these PTMs, the mechanisms governing the establishment of PTMs along the microtubule remain elusive. In the first part of this thesis, I introduce an innovative approach known as semi-synthetic tubulin, designed to decipher the molecular mechanisms underlying microtubule PTMs. With this novel technology, I reveal that a crosstalk exists between two microtubule PTMs, polyglutamylation and detyrosination. Moreover, I demonstrate that the detyrosinase activity of the tubulin tyrosine carboxypeptidase (vasohibin/SVBP) is enhanced by the extent of glutamylated chains present within the same microtubule structure. Microtubule dynamics are also subjected to influences from their surrounding environment. Indeed, ions play a significant role in determining the fate of the polymerization reaction. However, the precise effect of certain monovalent ions on microtubule dynamics has not been explored yet. In the second part of this thesis, I show the divergent effect of sodium (Na+) and acetate (Ac-) on microtubule dynamics and integrity. While Na+ destabilizes the microtubule structure, Ac- promotes the maintenance of the microtubule architecture over time. Taken together, these results provide a more detailed insight into the complexity of microtubule biochemistry. The semi-synthetic tubulin approach gives an innovative angle into the molecular investigation of the tubulin code, whereas the study of the ionic effect on microtubule assembly offers new insights on microtubule dynamics and integrity. </p

Two-color in vitro assay to visualize and quantify microtubule shaft dynamics

Microtubules are dynamic polymers where tubulin exchanges not only at the ends but also all along the microtubule shaft. In vitro reconstitutions are a vital approach to study microtubule tip dynamics, while direct observation of shaft dynamics is challenging. Here, we describe a dual-color in vitro assay to visualize microtubule shaft dynamics using purified, labeled bovine brain tubulin. With this assay, we can quantitatively address how proteins or small molecules impact the dynamics at the microtubule shaft. For complete details on the use and execution of this protocol, please refer to Andreu-Carbó et al. (2022)

Tubulin engineering by semi-synthesis reveals that polyglutamylation directs detyrosination

Microtubules, a critical component of the cytoskeleton, carry post-translational modifications (PTMs) that are important for the regulation of key cellular processes. Long-lived microtubules, in neurons particularly, exhibit both detyrosination of α-tubulin and polyglutamylation. Dysregulation of these PTMs can result in developmental defects and neurodegeneration. Owing to a lack of tools to study the regulation and function of these PTMs, the mechanisms that govern such PTM patterns are not well understood. Here we produce fully functional tubulin carrying precisely defined PTMs within its C-terminal tail. We ligate synthetic α-tubulin tails—which are site-specifically glutamylated—to recombinant human tubulin heterodimers by applying a sortase- and intein-mediated tandem transamidation strategy. Using microtubules reconstituted with these designer tubulins, we find that α-tubulin polyglutamylation promotes its detyrosination by enhancing the activity of the tubulin tyrosine carboxypeptidase vasohibin/small vasohibin-binding protein in a manner dependent on the length of polyglutamyl chains. We also find that modulating polyglutamylation levels in cells results in corresponding changes in detyrosination, corroborating the link between the detyrosination cycle to polyglutamylation.</p