The regulation of tubulin isotypes in tubulin tyrosination, microtubule stability and polarized growth

Abstract

Microtubules play a crucial role in cell division, cellular cargo transportation, and cell motility in eukaryotes. They are dynamic structures composed of α/β-tubulin heterodimers, which assemble into microtubule arrays in a head-to-tail manner and determine the microtubule polarity. The composition of tubulin subunits can vary significantly due to the presence of different tubulin isotypes and post-translational modifications (PTMs). One PTM is tyrosination/detyrosination, which involves the cyclic removal and religation of the carboxy-terminus tyrosine residue of α-tubulin. This evolutionary conserved modification serves as a widely used marker for distinguishing dynamic and stable microtubules and regulates microtubule structures and functions in cells. Multiple tubulin isotypes function as substrates of tubulin tyrosination in vivo. However, the role of human tubulin isotypes in tyrosination and microtubule dynamic instability has not been explored, mainly due to the lack of homogeneous recombinant human tubulins for in vitro reconstitution studies. This study aims to investigate the effect of tubulin isotypes in the regulation of tubulin tyrosination and microtubule stability. Firstly, isotypically pure recombinant human tubulin with controlled tyrosination or detyrosination states is generated. Additionally, a strategy is developed to label recombinant tubulin with one biotin or fluorophore for the immobilization and visualization of single microtubules under total internal reflection fluorescence (TIRF) microscopy. Through enzymatic tyrosination comparison of detyrosinated α1B- and α4A-tubulin, it is discovered that human α4A-tubulin is a poor substrate for tubulin tyrosine ligase. The difference in tyrosination rates is attributed to the primary sequence variation in the unstructured C-terminal tail of the isotypes, rather than the structured tubulin core. This study further demonstrates that the incorporation of α4A-tubulin not only increases the intrinsic stability of microtubules but also provides resistance to depolymerization stimulated by mitotic centromere-associated kinesin (MCAK), a kinesin-13 microtubule depolymerase. Our single-molecule TIRF-based assay reveals that the composition of α-tubulin isotypes and their tyrosination/detyrosination states provide graded control over the microtubule-binding and depolymerizing activities of MCAK. To explore the regulation of microtubule dynamics and polarized growth by tubulin isotypes, the in vitro reconstitution dynamics assay is established. By using isotypically pure human recombinant tubulin, this study determines that human α-tubulins can control the microtubule dynamics. Particularly, this work uncovers dynamic α4A/β3-microtubules display a predominant minus-end-outgrowth behavior, revealing the first microtubule minus-end preferential growth driven by its intrinsic subunits. Collectively, these findings illustrate the integrated role of α-tubulin isotypes in regulating the activities of enzymes involved in tubulin tyrosination and microtubule dynamic instability.