The role of microtubule allostery and mimicry in the regulation of tubulin post-translational modifications

ABSTRACT Microtubules, assembled from αβ-tubulin dimers, play pivotal roles in various cellular processes ranging from cell migration to cell division. The functionality of microtubules is diversified through post-translational modifications (PTMs) that are “written” onto the C-terminal tails (CTTs) of tubulin. PTMs on tubulin CTTs affect microtubule function by altering the on-rate of microtubule-associated proteins (MAPs) to the microtubule lattice. Despite the widespread acceptance of this “Tubulin Code” theory, the mechanisms by which microtubules are selected for modification remain poorly understood. This proposal will fill this gap using biosensors we have developed that allow real-time visualization of microtubule PTMs both in vitro and in cells. Our preliminary data have revealed that one microtubule PTM, detyrosination, occurs on MTs that are in a GTP-bound, or “expanded”, state; GDP-bound, or “compacted” MTs are remain tyrosinated. These data have led us to hypothesize that the α-tubulin CTT (αCTT) on microtubules switches between a docked state on compacted MTs and an undocked state on expanded microtubules. In this model, the accessibility of the αCTT to PTM writers and/or αCTT-binding MAPs is regulated by the conformational state of the microtubule lattice. We will test the significance of this concept in Aim 1. In Aim 2, we will investigate a second mechanism that may regulate “undocking” of αCTTs. We propose that a subset of MAPs contain αCTT-mimicking motifs that compete for the αCTT docking site on the microtubule surface, thereby keeping αCTTs undocked. Collectively, our project aims to deepen our understanding of microtubule PTMs and potentially reshape the Tubulin Code theory by adding αCTT accessibility as another layer of regulation. This work will place tubulin in line with other GTP-binding proteins that act as molecular switches by representing the GTP-bound/expanded lattice conformation and the GDP- bound/compacted lattice conformation as the “on” and “off” states for the MAP binding and PTM writing, which in turn regulates the fate of cellular microtubules.