Microtubules are dynamic cytoskeletal polymers whose mechanical properties and functions are partly regulated by post-translational modifications. Acetylation of lysine 40 on α-tubulin occurs within the microtubule lumen and is associated with long-lived, stable microtubules. This modification is catalyzed by the acetyltransferase αTAT1 and reversed by the deacetylase HDAC6, yet regulation within the confined lumen and the influence of lattice integrity on enzymatic activity remain poorly understood.

In this work, we show that microtubule lattice conformation and integrity directly control luminal acetylation. Using in vitro reconstitution, we show that αTAT1 efficiently acetylates expanded lattices, while compacted or damaged lattices reduce its activity. To directly measure lattice integrity, we developed MT-DS (Microtubule Damage Sensor), a fluorescent probe that labels lattice openings with high spatial and temporal resolution. It binds to microtubules via taxanes and has a multivalent architecture that promotes its accumulation at lattice openings. Using MT-DS, we visualized intrinsic lattice defects enriched at annealing sites, showing that imperfections are inherent to microtubule assembly. We further demonstrate that the kinesin-1Δ6 motor mutant induces de novo lattice damage, establishing lattice integrity as an experimentally accessible parameter.

In cells, we show that kinesin-1–induced lattice damage perturbs the characteristic perinuclear-to-periphery exponential acetylation gradient. Lattice openings facilitate HDAC6 entry into the lumen, thereby promoting deacetylation. Extending MT-DS to cells provided first insights into microtubule lattice heterogeneity in the complex cellular environment.

Together, this work demonstrates that microtubule lattice integrity is not only structural but acts as a dynamic regulator of luminal acetylation. By developing a tool to directly monitor lattice damage and linking structural changes to enzymatic activity, this thesis provides a framework for understanding how mechanical and biochemical signals cooperate to control microtubule function in vitro and in cells.